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.

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.

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.

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.

Persistent near-tropical warmth on the Antarctic continent during the early Eocene epoch.

The warmest global climates of the past 65 million years occurred during the early Eocene epoch (about 55 to 48 million years ago), when the Equator-to-pole temperature gradients were much smaller than today and atmospheric carbon dioxide levels were in excess of one thousand parts per million by volume. Recently the early Eocene has received considerable interest because it may provide insight into the response of Earth's climate and biosphere to the high atmospheric carbon dioxide levels that are expected in the near future as a consequence of unabated anthropogenic carbon emissions. Climatic conditions of the early Eocene 'greenhouse world', however, are poorly constrained in critical regions, particularly Antarctica. Here we present a well-dated record of early Eocene climate on Antarctica from an ocean sediment core recovered off the Wilkes Land coast of East Antarctica. The information from biotic climate proxies (pollen and spores) and independent organic geochemical climate proxies (indices based on branched tetraether lipids) yields quantitative, seasonal temperature reconstructions for the early Eocene greenhouse world on Antarctica. We show that the climate in lowland settings along the Wilkes Land coast (at a palaeolatitude of about 70° south) supported the growth of highly diverse, near-tropical forests characterized by mesothermal to megathermal floral elements including palms and Bombacoideae. Notably, winters were extremely mild (warmer than 10 °C) and essentially frost-free despite polar darkness, which provides a critical new constraint for the validation of climate models and for understanding the response of high-latitude terrestrial ecosystems to increased carbon dioxide forcing.

Holocene Southern Ocean surface temperature variability west of the Antarctic Peninsula.

The disintegration of ice shelves, reduced sea-ice and glacier extent, and shifting ecological zones observed around Antarctica highlight the impact of recent atmospheric and oceanic warming on the cryosphere. Observations and models suggest that oceanic and atmospheric temperature variations at Antarctica's margins affect global cryosphere stability, ocean circulation, sea levels and carbon cycling. In particular, recent climate changes on the Antarctic Peninsula have been dramatic, yet the Holocene climate variability of this region is largely unknown, limiting our ability to evaluate ongoing changes within the context of historical variability and underlying forcing mechanisms. Here we show that surface ocean temperatures at the continental margin of the western Antarctic Peninsula cooled by 3-4 °C over the past 12,000 years, tracking the Holocene decline of local (65° S) spring insolation. Our results, based on TEX(86) sea surface temperature (SST) proxy evidence from a marine sediment core, indicate the importance of regional summer duration as a driver of Antarctic seasonal sea-ice fluctuations. On millennial timescales, abrupt SST fluctuations of 2-4 °C coincide with globally recognized climate variability. Similarities between our SSTs, Southern Hemisphere westerly wind reconstructions and El Niño/Southern Oscillation variability indicate that present climate teleconnections between the tropical Pacific Ocean and the western Antarctic Peninsula strengthened late in the Holocene epoch. We conclude that during the Holocene, Southern Ocean temperatures at the western Antarctic Peninsula margin were tied to changes in the position of the westerlies, which have a critical role in global carbon cycling.

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.

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.

Rapid Detection of Viable Bacillus anthracis Spores in Environmental Samples by Using Engineered Reporter Phages.

Bacillus anthracis, the causative agent of anthrax, was utilized as a bioterrorism agent in 2001 when spores were distributed via the U.S. postal system. In responding to this event, the Federal Bureau of Investigation used traditional bacterial culture viability assays to ascertain the extent of contamination of the postal facilities within 24 to 48 h of environmental sample acquisition. Here, we describe a low-complexity, second-generation reporter phage assay for the rapid detection of viableB. anthracis spores in environmental samples. The assay uses an engineered B. anthracis reporter phage (Wß::luxAB-2) which transduces bioluminescence to infected cells. To facilitate low-level environmental detection and maximize the signal response, expression of luxABin an earlier version of the reporter phage (Wß::luxAB-1) was optimized. These alterations prolonged signal kinetics, increased light output, and improved assay sensitivity. Using Wß::luxAB-2, detection of B. anthracis spores was 1 CFU in 8 h from pure cultures and as low as 10 CFU/g in sterile soil but increased to 10(5)CFU/g in unprocessed soil due to an unstable signal and the presence of competing bacteria. Inclusion of semiselective medium, mediated by a phage-expressed antibiotic resistance gene, maintained signal stability and enabled the detection of 10(4)CFU/g in 6 h. The assay does not require spore extraction and relies on the phage infecting germinating cells directly in the soil sample. This reporter phage displays promise for the rapid detection of low levels of spores on clean surfaces and also in grossly contaminated environmental samples from complex matrices such as soils.

Ultrastructure and ssrRNA sequencing of Myxidium amazonense n. sp. a myxosporean parasite of Corydoras melini from the Rio Negro river, Amazonas state, Brazil.

In a survey of myxozoan parasites of ornamental freshwater fish from the Rio Negro river, it was found that seven of 30 (23.3 %) Corydoras melini specimens examined had plasmodia of a new Myxidium species (Myxidium amazonense n. sp.) in the gallbladder. The fish were caught in the Rio Negro river, in the municipality of Santa Isabel do Rio Negro, in the state of Amazonas, Brazil. The plasmodia had a tubular shape, which was organized as a spiral spring with several turns in the gallbladder. The development of the myxospores was asynchronic, with disporic pansporoblasts. Mature myxospores were elongated, with 17.0 ± 0.9 (16.1-17.9) µm in length and 3.7 ± 0.7 (3.0-4.4) µm in width, and lightly arcuate from the valval view, with their bodies tapering slowly until ending in rounded extremities. The valval surface had nine to ten grooves in each valve. The polar capsules, one at either end of the spore, had a length of 5.4 ± 0.5 (4.9-5.9) µm and a width of 3.4 ± 0.6 (2.8-4.0) µm. Ultrastructural analysis showed that the wall of the plasmodia had numerous microvilli-like structures, pinocytotic canals, and cytoplasmic bridges connecting the pansporoblasts to each other and to the ectoplasm zone. Phylogenetic analysis, based on a small subunit ribosomal RNA (ssrRNA), identified the new species as a sister species of Myxidiumceccarelli, the unique South American Myxidium species whose ssrRNA sequence is available in the NCBI database. This study is the first description of Myxidium species in ornamental freshwater fish from Amazon.

A new isolate of Amoeboaphelidium protococcarum, and Amoeboaphelidium occidentale, a new species in phylum Aphelida (Opisthosporidia).

Microalgae used in the production of biofuels represents an alternative to fossil fuels. One problem in the production of algae for biofuels is attacks by algal parasitoids that can cause population crashes when algae are cultivated in outdoor ponds (Greenwell et al. 2010). Integrated solutions are being sought to mitigate this problem, and an initial step is pest identification. We isolated an algal parasitoid from an open pond of Scenedesmus dimorphus used for biofuel production in New Mexico and examined its morphology, ultrastructure and molecular phylogeny. A phylogenetic analysis placed this organism in Aphelida as conspecific with Amoeboaphelidium protococcarum sensu Karpov et al. 2013. As a result we re-evaluated the taxonomy of Amoeboaphelidium protococcarum sensu Letcher et al. 2013 and here designate it as a new species, Amoeboaphelidium occidentale.

Intact cell/intact spore mass spectrometry (IC/ISMS) on polymer-based, nano-coated disposable targets.

Identification and differentiation of microorganisms has and still is a long arduous task, involving culturing of the organism in question on different growth media. This procedure, which is still commonly applied, is an established method, but takes a lot of time, up to several days or even longer. It has thus been a great achievement when other analytical tools like matrix-assisted laser desorption/ionization (MALDI) mass spectrometry were introduced for faster analysis based on the surface protein pattern. Differentiation and identification of human pathogens as well as plant/animal pathogens is of increasing importance in medical care (e.g. infection, sepsis, and antibiotics resistance), biotechnology, food sciences and detection of biological warfare agents. A distinction between microorganisms on the species and strain level was made by comparing peptide/protein profiles to patterns already stored in databases. These profiles and patterns were obtained from the surface of vegetative forms of microorganisms or even their spores by MALDI MS. Thus, an unknown sample can be compared against a database of known pathogens or microorganisms of interest. To benefit from newly available, metal-based disposable microscope-slide format MALDI targets that promise a clean and even surface at a fraction of the cost from full metal targets or MTP (microtiter plate) format targets, IC/ISMS analysis was performed on these and the data evaluated. Various types of bacteria as well as fungal spores were identified unambiguously on this disposable new type of metal nano-coated targets. The method even allowed differentiation between strains of the same species. The results were compared with those gained from using full metal standard targets and found to be equal or even better in several aspects, making the use of disposable MALDI targets a viable option for use in IC/ISMS, especially e.g. for large sample throughput and highly pathogenic species.

Coupling Spore Traps and Quantitative PCR Assays for Detection of the Downy Mildew Pathogens of Spinach (Peronospora effusa) and Beet (P. schachtii).

Downy mildew of spinach (Spinacia oleracea), caused by Peronospora effusa, is a production constraint on production worldwide, including in California, where the majority of U.S. spinach is grown. The aim of this study was to develop a real-time quantitative polymerase chain reaction (qPCR) assay for detection of airborne inoculum of P. effusa in California. Among oomycete ribosomal DNA (rDNA) sequences examined for assay development, the highest nucleotide sequence identity was observed between rDNA sequences of P. effusa and P. schachtii, the cause of downy mildew on sugar beet and Swiss chard in the leaf beet group (Beta vulgaris subsp. vulgaris). Single-nucleotide polymorphisms were detected between P. effusa and P. schachtii in the 18S rDNA regions for design of P. effusa- and P. schachtii-specific TaqMan probes and reverse primers. An allele-specific probe and primer amplification method was applied to determine the frequency of both P. effusa and P. schachtii rDNA target sequences in pooled DNA samples, enabling quantification of rDNA of P. effusa from impaction spore trap samples collected from spinach production fields. The rDNA copy numbers of P. effusa were, on average, ≈3,300-fold higher from trap samples collected near an infected field compared with those levels recorded at a site without a nearby spinach field. In combination with disease-conducive weather forecasting, application of the assays may be helpful to time fungicide applications for disease management.

Development of loop-mediated isothermal amplification method for detection of Kudoa septempunctata (Myxozoa: Multivalvulida) in olive flounder (Paralichthys olivaceus).

A loop-mediated isothermal amplification (LAMP) assay was developed and validated for early, rapid, and sensitive detection of Kudoa septempunctata, a myxosporean parasite found in olive flounder (Paralichthys olivaceus). Recently, several outbreaks associated with ingestion of raw olive flounder muscles harboring mature K. septempunctata spores have been reported, and it is becoming obvious that fresh K. septempunctata spores can cause problems in humans when ingested. Thus, it is necessary to develop reliable detection method of K. septempunctata, to prevent outbreaks and ensure food safety. The LAMP assay has advantages over other molecular detection methods for detecting K. septempunctata in olive flounder muscle, in terms of simplicity, rapidity, and sensitivity. The reaction condition was optimized as 63 °C, 45 min, with three sets of specific primers. The results can be simply confirmed with the naked eye after adding SYBR Green I or by conventional electrophoresis followed by ethidium bromide staining. This LAMP assay did not show any cross-reaction with other kudoid myxosporeans (Kudoa lateolabracis, Kudoa thyrsites) can be found in olive flounder muscles and was validated by testing Kudoa septempunctata spore-spiked samples and field samples. The results showed that the LAMP assay is ten times more sensitive than the conventional polymerase chain reaction in this study and can be applied for early detection for monitoring and epidemiological studies of K. septempunctata in olive flounder aquaculture farms.

Improved recovery of Bacillus spores from nonporous surfaces with cotton swabs over foam, nylon, or polyester, and the role of hydrophilicity of cotton in governing the recovery efficiency.

Evaluating different swabbing materials for spore recovery efficiency (RE) from steel surfaces, we recorded the maximum RE (71%) of 10(7) Bacillus subtilis spores with Tulips cotton buds, followed by Johnson's cotton buds and standard Hi-Media cotton, polyester, nylon, and foam (23%) swabs. Among cotton swabs, instant water-absorbing capacity or the hydrophilicity index appeared to be the major indicator of RE, as determined by testing three more brands. Tulips swabs worked efficiently across diverse nonporous surfaces and on different Bacillus spp., registering 65 to 77% RE.