HMGB1 prefers to interact with structural RNAs and regulates rRNA methylation modification and translation in HeLa cells.

BACKGROUND: High-mobility group B1 (HMGB1) is both a DNA binding nuclear factor modulating transcription and a crucial cytokine that mediates the response to both infectious and noninfectious inflammation such as autoimmunity, cancer, trauma, and ischemia reperfusion injury. HMGB1 has been proposed to control ribosome biogenesis, similar as the other members of a class of HMGB proteins. RESULTS: Here, we report that HMGB1 selectively promotes transcription of genes involved in the regulation of transcription, osteoclast differentiation and apoptotic process. Improved RNA immunoprecipitation by UV cross-linking and deep sequencing (iRIP-seq) experiment revealed that HMGB1 selectively bound to mRNAs functioning not only in signal transduction and gene expression, but also in axon guidance, focal adhesion, and extracellular matrix organization. Importantly, HMGB1-bound reads were strongly enriched in specific structured RNAs, including the domain II of 28S rRNA, H/ACA box snoRNAs including snoRNA63 and scaRNAs. RTL-P experiment showed that overexpression of HMGB1 led to a decreased methylation modification of 28S rRNA at position Am2388, Cm2409, and Gm2411. We further showed that HMGB1 overexpression increased ribosome RNA expression levels and enhanced protein synthesis. CONCLUSION: Taken together, our results support a model in which HMGB1 binds to multiple RNA species in human cancer cells, which could at least partially contribute to HMGB1-modulated rRNA modification, protein synthesis function of ribosomes, and differential gene expression including rRNA genes. These findings provide additional mechanistic clues to HMGB1 functions in cancers and cell differentiation.

Morphological and molecular characterization of plagiorchiid trematodes (Plagiorchis: Plagiorchiidae, Digenea) from bats with redescription of Plagiorchis mordovii Shaldybin, 1958.

Five Plagiorchis spp. parasitize bats in European Russia: Plagiorchis elegans, Plagiorchis koreanus, Plagiorchis mordovii, Plagiorchis muelleri, and Plagiorchis vespertilionis. Their identification is difficult due to a high morphological similarity. The morphological variability of these species is poorly studied. The taxonomic position of P. mordovii remains debatable. The purpose of our study was to analyse Plagiorchis spp. from European bats using a combination of morphological and molecular-phylogenetic approaches and to establish the taxonomic position of the problematic species P. mordovii.Plagiorchis spp. were shown to be variable morphologically and morphometrically both from various host species and from different specimens of the same species. We presented a new taxonomic key for identification of the Plagiorchis spp. from European bats, provided a complete description of Plagiorchis mordovii, and confirmed the validity and the generic affiliation of this species.

A new ring nematode, Xenocriconemella costaricense sp. nov., (Nematoda: Criconematidae) from Costa Rica.

During nematode surveys of natural vegetation in forests of La Cima de Copey de Dota, San José, San José province, Costa Rica, a Xenocriconemella species closely resembling X. macrodora and related species was found. Integrative taxonomical approaches demonstrated that it is a new species described herein as X. costaricense sp. nov. The new species is parthenogenetic (only females have been detected) and characterised by a short body (276-404 µm); lip region with two annuli, not offset, not separated from body contour; first lip annulus partially covering the second lip annulus. Stylet thin, very long (113-133 µm) and flexible, occupying 30.5-47.8% of body length. Excretory pore located from one or two annuli anterior to one or two annuli posterior to level of stylet knobs, at 42 (37-45) µm from anterior end. Female genital tract monodelphic, prodelphic, outstretched, and occupying 35-45% of body length, with vagina slightly ventrally curved (14-18 µm long). Anus located 6-11 annuli from the tail terminus. Tail conoid and bluntly rounded terminus, the last 2-3 annuli oriented dorsally. Results of molecular characterisation and phylogenetic analyses of D2-D3 expansion segments of 28S rRNA, ITS, and partial 18S rRNA, as well as cytochrome oxidase c subunit 1 gene sequences further characterised the new species and clearly separated it from X. macrodora and other related species (X. iberica, X. paraiberica, and X. pradense).

A new species of the genus Hexamermis Steiner, 1924 (Nematoda: Mermithidae) from northern Iran: a nematode with an unusual uterine morphology.

Hexamermis zirabi sp. n., recovered from a natural habitat of Mazandaran province, north of Iran, is described based on morphological and molecular data. The new species is characterized by its six cephalic papillae; cuticle with distinct cross fibers; conoid or sharply tapered head; mouth terminal; six hypodermal cords; J-shaped vagina oriented to the anterior end of body; uterus with Z-organs or sclerotized bodies; tail similar in both sexes and bluntly rounded; spicules paired, separate, slightly curved, shorter than body width at cloaca, with rounded tip; and male genital papillae arranged in five rows. In addition to the morphological study, molecular phylogenetic analyses using a partial large subunit (28S D2-D3) were also performed, and the new species formed a highly supported (1.00% Bayesian posterior probability (BPP)) clade with Hexamermis popilliae.

Morphological and genetic identification of the gill monogenean parasite (Diclidophora merlangi) that infects Twobar Seabream Fish (Acanthopagrus bifasciatus) in the Arabian Gulf, Saudi Arabia.

Ectoparasites, particularly monogeneans, negatively affect fish health and growth. This study identified monogenean parasites in the twobar seabream, Acanthopagrus bifasciatus (Sparidae), inhabited the Arabian Gulf (Saudi Arabia). Following that, forty A. bifasciatus fish samples were visually examined for monogeneans. Parasite species were collected from the gills and then analyzed morphometrically, morphologically, and molecularly using the partial regions of the large subunit of ribosomal RNA (28S rRNA) and mitochondrial cytochrome C oxidase subunit I (COI) genes. Fish species were also identified using a DNA barcoding approach based on the COI gene. The monogenean species of Diclidophora merlangi (Diclidophoridae) were found in 45% of the fish species studied. The generic features of the Diclidophora genus distinguish this species. This species discriminated itself from congeners by having a muscular bulb with 17 grooved and recurved hooks, 218±10 (184-267) post-ovarian testes, and four pairs of pedunculated clamps of relative sizes. Partial 28S rRNA sequencing from monogeneans revealed that they grouped with members of the genus Diclidophora, forming a monophyletic group that supported the morphological descriptions. Molecular identification revealed that D. merlangi has a unique barcode made up of a COI sequence. The host identity was established as A. bifasciatus based on the COI gene sequences. Furthermore, a molecular phylogenetic study was performed to determine the phylogenetic affinity of parasite species and fish hosts. This study on Diclidophora species is considered the first record of this genus in the examined area.

Metformin Pre-Treatment as a Means of Mitigating Disuse-Induced Rat Soleus Muscle Wasting.

Currently, no ideal treatment exists to combat skeletal muscle disuse-induced atrophy and loss of strength. Because the activity of AMP-activated protein kinase (AMPK) in rat soleus muscle is suppressed at the early stages of disuse, we hypothesized that pre-treatment of rats with metformin (an AMPK activator) would exert beneficial effects on skeletal muscle during disuse. Muscle disuse was performed via hindlimb suspension (HS). Wistar rats were divided into four groups: (1) control (C), (2) control + metformin for 10 days (C+Met), (3) HS for 7 days (HS), (4) metformin treatment for 7 days before HS and during the first 3 days of 1-week HS (HS+Met). Anabolic and catabolic markers were assessed using WB and RT-PCR. Treatment with metformin partly prevented an HS-induced decrease in rat soleus weight and size of slow-twitch fibers. Metformin prevented HS-related slow-to-fast fiber transformation. Absolute soleus muscle force in the HS+Met group was increased vs. the HS group. GSK-3ß (Ser9) phosphorylation was significantly increased in the HS+Met group vs. the HS group. Metformin pre-treatment partly prevented HS-induced decrease in 18S+28S rRNA content and attenuated upregulation of calpain-1 and ubiquitin. Thus, pre-treatment of rats with metformin can ameliorate disuse-induced reductions in soleus muscle weight, the diameter of slow-type fibers, and absolute muscle strength.

Exploration of Multi-Gene DNA Barcode Markers to Reveal the Broad Genetic Diversity of Field Ticks (Acari: Ixodidae) in a Tropical Environment of Hainan Island, China.

Ticks are hematophagous arthropods and obligate ectoparasites of humans and other animals. This study focused on the molecular discrimination of ticks in the tropical environment of Hainan according to multi-gene DNA barcode markers with the expectation of accurately distinguishing species. A total of 420 ticks, including 49 adult ticks, 203 nymphal ticks, and 168 larval ticks, were collected in the field, and the 49 adult ticks were identified as Rhipicephalus turanicus, Dermacentor marginatus, and Haemaphysalis longicornis. The mitochondrial 16S rRNA, ribosomal 28S rRNA D2, and ribosomal internal transcribed spacer 2 (ITS2) regions were used as DNA barcode markers to discriminate species. According to basic local alignment search tool analysis against the GenBank database, 16S rRNA positively identified ticks in the Rhipicephalus, Dermacentor, and Haemaphysalis genera; the 28S rRNA D2 region identified ticks in the Rhipicephalus and Dermacentor genera; and ITS2 identified ticks as D. marginatus. Pairwise sequence comparisons based on these three regions were visualized with a Sequence Demarcation Tool matrix. Substitution saturation tests using data analysis and molecular biology and evolution revealed little substitution saturation (Iss < Iss.c, p < 0.05) in the 16S rRNA region for the Haemaphysalis genus; 28S rRNA D2 region for the Rhipicephalus, Dermacentor, and Haemaphysalis genera; and ITS2 region for the Rhipicephalus and Dermacentor genera. Distinctive sequences for which it is difficult to obtain good matches with the sequences available in GenBank exist in the ticks of Hainan. Future studies should obtain complementary sequences to refine and update the database for the molecular characterization of ticks.

Morphological and molecular characterization of Metaxonchium magnum sp. n. from Iran (Nematoda, Dorylaimida, Belondiridae), with new insights into the phylogeny of the genus.

A new species of the genus Metaxonchium is described from a natural habitat in Iran. Metaxonchium magnum sp. n. is characterized by its 3.62-4.65 mm long body, lip region cap-like and offset by constriction and 13-16 µm wide, odontostyle fusiform and 14-17 µm long, neck 1016-1359 µm long, both parts of the pharynx separated by a short isthmus-like narrowing, pharyngeal expansion occupying 74.2 (73-77)% of total neck length in females and 70.4 (66-72)% in males, female genital system mono-opistho-ovarian, didelphic, anterior genital branch a large uterine sac with a small terminal mass occupying 7-14% of body length, posterior uterus long and tripartite with a Z-like differentiation, V = 50-52, caudal region short and rounded (24-41 µm, c = 99-161, c' = 0.5-0.7), spicules 90-105 µm long and 10-13 spaced ventromedian supplements with hiatus. Analysis of D2-D3 28S rDNA sequences of the new species suggests that Metaxonchium might not be a monophyletic taxon, a matter that should be confirmed after future research.

Morphological and molecular evidence for the recognition of Allocreadium bursensis n. sp. (Trematoda: Allocreadiidae) from Angora loach Oxynoemacheilus angorae from Turkey.

This is the first study reporting parasites from the freshwater cyprinid Oxynoemacheilus angorae (Steindachner 1897) caught in Nilüfer Stream, Bursa, in the Northwest Anatolian Region of Turkey. Allocreadium bursensis n. sp. was described from the intesine of O.angorae based on morphological and genetic characteristics. Allocreadium bursensis n. sp. was differentiated from other Allocreadium spp. in having a combination of external (ventral and oral suckers ratio; body length and width and its ratio to forebody) and internal (cirrus pouch position; uterus extension in hindbody; egg size; disposition of anterior border of vitellarium; esophagus length) features. Phylogenetic hypotheses based on maximum parsimony, maximum likelihood, Bayesian inferrence, and neighbor joining analyses of sequence data strongly supported the hypothesis that A. bursensis is nested within the clade of Allocreadium species hosted by cypriniform fish, and it is more closely related to the Far Eastern species A. pseudoisoporum (Primorsky region, Russia) than to the African A. apokryfi. According to genetic p-distances, the taxonomic status of trematodes collected in Turkey was established as independent relative to nine of the valid Allocreadium spp.: 1.8-5.8% in 28S gene and 18.8-22.6% in cox1 gene. The present study increases the number of Allocreadium species and their definitive hosts recorded in Turkey and raises the number of Palearctic representatives of Allocreadium spp. to 26.

First reports of Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae (Tylenchomorpha) from Greece and further records of four other nematode species.

Nematode samplings in various areas and crops of Greece were carried out and the recovered nematode species were characterized using morphological and molecular data. Seven species of plant-parasitic nematodes were recovered, three of which are reported for the first time in Greece, including Hemicycliophora poranga, Helicotylenchus dihystera and Tylenchorhynchus zeae. Four other recovered species had already been reported in Greece, including Bitylenchus hispaniensis, Helicotylenchus microlobus, Nanidorus minor and Scutellonema brachyurus. D2-D3 segments of 28S rRNA gene for all of these nematode species are provided.

Morphological and molecular identification of trematode cercariae related with humans and animal health in freshwater snails from a lake and a dam in Myanmar.

Freshwater snails play an essential role in the transmission of trematode parasitic flatworms that can infect wild and domestic animals, as well as humans. This study aimed to investigate the rate of cercarial infections in freshwater snails collected from two study areas, Inlay Lake and Yezin Dam, in Myanmar. A total of 4,740 snail samples were collected from Inlay Lake (n = 3,837) and Yezin Dam (n = 903), and infection rate by cercarial emergence was examined. Cercarial DNA samples were analysed by PCR. Based on morphological characteristics, eleven snail species and eight cercarial types were identified. Snails of Melanoides tuberculata in the family Thiaridae were found as the most abundant, followed by Indoplanorbis exustus of the family Planorbidae, in both study areas. The infection rate by cercarial emergence in snails in Inlay Lake and Yezin Dam was 5.8% (224/3,837) and 48.6% (439/903), respectively. Echinostome cercariae showed the highest infection rate in both study areas. Phylogenetic analysis of cercarial internal transcribed spacer 2 (ITS2) sequences revealed that at least seven cercaria types belonged to five digenean trematode families, two of which were zoonotic trematodes in the families of Opisthorchiidae/Heterophyidae and Schistosomatidae. Furthermore, cercarial 28S ribosomal RNA gene analysis showed that the furcocercous cercariae in Yezin Dam were identified as Schistosoma spindale, a causative agent of ruminant schistosomiasis. This is the first report on zoonotic trematode cercariae in snails in Myanmar. The findings indicate that various snail species act as intermediate host for trematode species that infect aquatic animals, mammals and humans in the country.

Oligonucleotide Insecticides for Green Agriculture: Regulatory Role of Contact DNA in Plant-Insect Interactions.

Insects vastly outnumber us in terms of species and total biomass, and are among the most efficient and voracious consumers of plants on the planet. As a result, to preserve crops, one of the primary tasks in agriculture has always been the need to control and reduce the number of insect pests. The current use of chemical insecticides leads to the accumulation of xenobiotics in ecosystems and a decreased number of species in those ecosystems, including insects. Sustainable development of human society is impossible without useful insects, so the control of insect pests must be effective and selective at the same time. In this article, we show for the first time a natural way to regulate the number of insect pests based on the use of extracellular double-stranded DNA secreted by the plant Pittosporum tobira. Using a principle similar to one found in nature, we show that the topical application of artificially synthesized short antisense oligonucleotide insecticides (olinscides, DNA insecticides) is an effective and selective way to control the insect Coccus hesperidum. Using contact oligonucleotide insecticide Coccus-11 at a concentration of 100 ng/µL on C. hesperidum larvae resulted in a mortality of 95.59 ± 1.63% within 12 days. Green oligonucleotide insecticides, created by nature and later discovered by humans, demonstrate a new method to control insect pests that is beneficial and safe for macromolecular insect pest management.

The Role of Glycogen Synthase Kinase-3 in the Regulation of Ribosome Biogenesis in Rat Soleus Muscle under Disuse Conditions.

It is well-established that prolonged exposure to real or simulated microgravity/disuse conditions results in a significant reduction in the rate of muscle protein synthesis (PS) and loss of muscle mass. Muscle protein synthesis is largely dependent upon translational capacity (ribosome content), the regulation of which is poorly explored under conditions of mechanical unloading. Glycogen synthase kinase-3 (GSK-3) (a negative regulator of PS) is known to be activated in rat soleus muscle under unloading conditions. We hypothesized that inhibition of GSK-3 activity under disuse conditions (hindlimb suspension, HS) would reduce disuse-induced downregulation of ribosome biogenesis in rat soleus muscle. Wistar rats were randomly divided into four groups: (1) vivarium control (C), (2) vivarium control + daily injections (4 mg/kg) of AR-A014418 (GSK-3 inhibitor) for 7 days, (3) 7-day HS, (4) 7-day HS + daily injections (4 mg/kg) of AR-A014418. GSK-3beta and glycogen synthase 1 (GS-1) phosphorylation levels were measured by Western-blotting. The key markers of ribosome biogenesis were assessed via agarose gel-electrophoresis and RT-PCR. The rate of muscle PS was assessed by puromycin-based SUnSET method. As expected, 7-day HS resulted in a significant decrease in the inhibitory Ser9 GSK-3beta phosphorylation and an increase in GS-1 (Ser641) phosphorylation compared to the C group. Treatment of rats with GSK-3 inhibitor prevented HS-induced increase in GS1 (Ser641) phosphorylation, which was indicative of GSK-3 inhibition. Administration of GSK-3 inhibitor partly attenuated disuse-induced downregulation of c-Myc expression as well as decreases in the levels of 45S pre-rRNA and 18S + 28S rRNAs. These AR-A014418-induced alterations in the markers of ribosome biogenesis were paralleled with partial prevention of a decrease in the rate of muscle PS. Thus, inhibition of GSK-3 during 7-day HS is able to partially attenuate the reductions in translational capacity and the rate of PS in rat soleus muscle.

Do 'cheese factory-specific' mites (Acari: Astigmata) exist in the cheese-ripening cabinet?

To determine whether the mites used in the ripening process of traditional cheeses are genetically unique to cheese factories, we investigated mites from three types of traditional cheeses, that use mites in the ripening process: 'Würchwitzer Milbenkäse' from Germany and 'Mimolette' and 'Artisou' from France. In addition, traditional ripened cheeses were purchased from cheese specialty stores in France (Mimolette) and Japan ('Laguiole' from France) as well as stores in temporary markets in France ('Salers' and 'Cantal vieux') and the mites obtained from those cheeses were analyzed in this study. Partial sequences of the 28S rRNA gene (28S) were determined and used to reconstruct a phylogenetic tree. Tyrolichus casei, the dominant cheese mite species from the ripening cabinets of three traditional cheese producers and two cheese specialty stores in France and Japan, had identical partial 28S sequences. All specimens from Cantal vieux from a store in the temporary market in France had an identical sequence with Acarus siro and Acarus immobilis in the determined region of the 28S sequences. Mite individuals from Salers from a store in the temporary markets in France shared the same haplotype as Acotyledon paradoxa. For the T. casei individuals from five different localities (19 individuals in total), the nuclear loci were obtained using MIG-seq. More than several thousand genomic regions are amplified simultaneously by multiplex PCR, and targeting regions surrounded by inter-simple sequence repeats (ISSRs) in the genome were sequenced using the MiSeq system (Illumina). SNPs extracted from this genome-wide analysis showed that no genetic structure existed in the populations from any region. Among the five samples from the three regions, which were more than 500 km apart and from completely different environments, the mites had no geographic bias, but all mite individuals were genetically nearly identical. Thus, we found no evidence to support the existence of 'cheese factory-specific' T. casei mites, at least in terms of genetic analysis.

Molecular phylogeny of Cosmetocleithrum Kritsky, Thatcher & Boeger, 1986 (Monogenoidea: Dactylogyridae), gill parasites of Neotropical catfishes (Siluriformes).

Cosmetocleithrum Kritsky, Thatcher & Boeger, 1986 (Dactylogyridae) represents one of the most species-rich groups (22 species currently recognized as valid) of all dactylogyrid parasites infecting Neotropical catfishes. Species of Cosmetocleithrum exhibit a remarkable affinity towards catfishes of the Doradidae and the Auchenipteridae. However, phylogenetic relationships between members of this genus have not been yet analysed. This study analysed newly obtained partial sequences of the 28S ribosomal RNA gene of seven species of Cosmetocleithrum, including its type species C. gussevi Kritsky, Thatcher & Boeger, 1986, along with several other dactylogyrids infecting siluriform, gymnotiform, perciform and characiform fishes. Cosmetocleithrum appeared as an evolutionary recent group, composed of two well-defined lineages: lineage 1 includes parasites of doradids - namely, C. bulbocirrus, C. confusum, C. parvum and C. bifurcum - whereas lineage 2 is composed of species from doradids - that is, C. rarum, C. gussevi, C. gigas, C. trachydorasi and C. falsunilatum - together with parasites of auchenipterids - namely, C. laciniatum and C. baculum. The search for synapomorphies to characterize taxonomic groups within Cosmetocleithrum appears challenging, since the morphology of their haptoral elements is quite conservative, and that of the copulatory complex is highly variable between species. The results of the present study support the recent synonymization of Paracosmetocleithrum Acosta, Scholz, Blasco-Costa, Alves & Silva, 2018 with Cosmetocleithrum. Whereas the 28S ribosomal DNA data resolved Cosmetocleithrum as monophyletic, the statistical support for the lineage was low, rendering its phylogenetic position between other Neotropical dactylogyrids yet undefined.

A new needle nematode, Longidorus maginicus n. sp. (Nematoda: Longidoridae) from southern Spain.

During nematode surveys in natural vegetation in Sierra Mágina, Jaén province, southern Spain, a Longidorus species closely resembling Longidorus carpetanensis was found, but application of integrative taxonomic approaches clearly demonstrated that it is a new species described herein as Longidorus maginicus n. sp. The new species is amphimictic, characterized by a moderately long body (4.2-5.2 mm); lip region anteriorly flattened, slightly separated from the rest of body by a depression, 9.0-11.0 µm wide and 3.5-6.0 µm high; amphidial fovea not lobed; relatively short odontostyle (61.0-70.5 µm); guiding ring located 23.5-27.0 µm from anterior end; vulva located at 42.0%-51.3% of body length; female tail 39.0-61.0 µm long, conoid, dorsally convex with rounded terminus (c' = 1.3-2.1), with two or three pairs of caudal pores; and males common (1:2 ratio males:females), with moderately long spicules (39.0-48.5 µm) and 1 + 6-9 ventromedian supplements and three juvenile developmental stages. According to the polytomous key, codes for the new species are (codes in parentheses are exceptions): A2-B1-C2-D2-E1-F2(3)-G2-H5(4)-I2-J1-K6. The results of molecular analysis of D2-D3 28S, internal transcribed spacer region, partial 18S rDNA, and cytochrome oxidase c subunit 1 (coxI) gene sequences further characterized the new species status, and separated it from L. carpetanensis and other related species.

First Report of Xiphinema Ifacolum Luc, 1961 (Dorylaimida: Longidoridae) from Nigeria.

A population of a species of dagger nematode (Xiphinema) belonging to the non-americanum group was recovered from the fields of kola nut (Cola sp.) in southern Nigeria. The morphological and morphometric data obtained from this population were consistent with the characteristics of the species Xiphinema ifacolum. In addition, molecular identification based on D2-D3 expansion segments of 28S rRNA and partial mitochondrial COI gene regions confirmed its identity. According to our knowledge, this is the first report of the species from Nigeria, and the second report from Africa, after the original description from Foulaya, Guinea.

Heteromicrocotyla polyorchis Unnithan, 1961 (Monogenea: Heteromicrocotylidae), a gill parasite of the yellow-spotted trevally, Carangoides fulvoguttatus (Carangidae) from Saudi Arabia: Morphology and phylogeny.

Little information for parasitic infections of Carangoides fulvoguttatus was recorded. The present study was intended to investigate the gill parasite Heteromicrocotyla polyorchis of this fish and to provide a full morphological description and clarify its taxonomic status through phylogenetic analysis of the 28S rRNA gene region. A total of sixty fish specimens have been collected from the studied area (the Red Sea in Jeddah Province, Saudi Arabia) and gills were isolated and examined for identification of parasites. Using light electron microscopy, the recovered monogenean parasite's morphology was exhaustively characterized and described. Microscope examinations found that this parasite species represent Heteromicrocotyla polyorchis, and it could be distinguished from congeners of the same genus by armed genital atrium and cirrus sac, follicular post-ovarian testes, unique shape and number of clamps on both haptor sides, and the dorsally curved tip of the male copulatory organ. Morphological features were combined with molecular analysis of the 28S rRNA gene region. The selected gene for the isolated Heteromicrocotyla species was analyzed using appropriate primers to assist in phylogeny with those in the GenBank database. The present monogenean species was characterized by unique genetic sequences that were analyzed and deposited in the GenBank for the first time under the accession number MW406473. Phylogenetic analyses reported that the maximum identity between the current Heteromicrocotyla species and taxa of Heteromicrocotylidae was between 91.42 and 92.09% and confirmed its taxonomic status in this family with a well-distinct clade. The present study supported the second report of H. polyorchis as carangid fish ectoparasites and investigates the first appearance in C. fulvoguttatus inhabiting Saudi Arabia.

Reverse-transcription PCR increases sensitivity of broad-range fungal detection in bronchoalveolar lavage fluid.

Broad-range PCR targeting 28S D1-D2 ribosomal DNA (rDNA) identifies numerous fungi but has limited sensitivity in clinical specimens. Ribosomal RNA (rRNA) vastly outnumbers rDNA, suggesting reverse transcription (RT)-PCR could improve detection. Among contrived samples, RT-PCR decreased 28S PCR cycle threshold values by 10--12 cycles and lowered the limit of detection > 2000-fold. Among 32 bronchoalveolar lavage specimens, RT-PCR detected 12/15 (80%) fungal PCR- or culture-positive specimens, versus 6/12 (50%) by 28S PCR, 9/12 (75%) by any fungal PCR, and 13/15 (87%) by culture. RT-PCR newly identified fungi in 4/17 (24%) PCR- and culture-negative specimens. RT substantially increased 28S PCR sensitivity overall. LAY SUMMARY: Fungal infection remains difficult to diagnose in the laboratory. Here, we have shown that detecting ribosomal RNA and DNA, rather than only ribosomal DNA, in a broad range fungal assay results in a significant enhancement in the ability to detect and identify fungal pathogens in clinical samples.

Morphological and molecular characterization of Cystoisospora yuensis n. sp. and Cystoisospora rastegaievae (Protozoa: Eimeriidae) in amur hedgehogs, Erinaceus amurensis (Schrenk, 1859).

Twenty-four fecal samples were collected from captive amur hedgehogs (Erinaceus amurensis) in Zhengzhou, China. Based on morphological and molecular analysis, the overall prevalence of Cystoisospora was 62.5% (15/24). These samples contained two types of coccidian oocysts, including C. rastegaievae (50.0%, 12/24) and a new species named C. yuensis n. sp. (12.5%, 3/24). Sporulated oocysts (n = 30) of C. yuensis n. sp. are ovoid, (20.6 ± 1.4) µm × (20.9 ± 0.9) µm, with a shape index (length/width) of 1.0 and a smooth and bi-layered oocyst wall, 1.3 µm thick (outer layer 0.8 µm, inner 0.5 µm). A polar granule is present, but micropyle cap, micropyle, and oocyst residuum are absent. The sporocysts are ovoid-shaped, (9.3 ± 0.6) µm × (8.5 ± 1.1) µm, with a shape index (length/width) of 1.1. Stieda, substieda bodies, and refractile bodies are absent. Residuum is scattered and distributed around the entire sporocysts. At the 18S rRNA locus, C. yuensis n. sp. exhibited the highest identity to C. timoni (99.3%) from a slender-tailed meerkat. It has 98.0% identity at the 28S rRNA locus and 99.3% at the ITS locus. Based on morphological and molecular data, this isolate is a new species of Cystoisospora. Additionally, we have provided data on the prevalence of C. rastegaievae in China and sequences of the 18S rRNS, 28S rRNA, and ITS loci.