rRNA Gene Loci Research Articles

Regional microbial content of fermented traditional and industrial East Mediterranean sausages from the islands of Cyprus and Mytilini

Objective: To characterize the microbial biodiversity of fermented sausages from the East Mediterranean islands of Cyprus and Mytilini, and to identify differences between the microbial diversity of traditionally and industrially produced Cypriot sausages. Method: The microbial diversity of thirty sausages from Cyprus and Mytilini (traditionally and industrially produced) was analyzed using high throughput sequencing (HTS) (amplicon sequencing) of 16S rRNA gene and ITS loci fragments. By applying microbial signature detection and machine learning algorithms, we identified key microbes that distinguish traditionally produced sausages from those produced industrially. Focusing on selected microbial taxa and using interaction network analysis, we identified associations among the sausages’ microbiota that may affect the shaping of the sausages’ microbial consortia. Results: Cypriot and Mytilini sausages indicated increased relative representation of Lactobacillus and Leuconostoc . Cypriot sausages were distinguished by the presence of the fungi Debaryomyces hansenii and Candida spp. , whereas Mytilini sausages by the bacteria Lactococcus and Streptococcus . Traditionally produced sausages from the Pitsilia region of Cyprus were differentiated by the presence of the species Lactobacillus helveticus , whereas industrially produced sausages were differentiated by the species Leuconostoc mesenteroides . Focusing mainly on Lactobacillus and Leuconostoc , negative associations with pathogenic bacteria, such as Salmonella , and spoilage fungi, such as Fusarium poae , were revealed. Conclusion: The present metataxonomic analysis provided insights into the microbial communities that characterize Cypriot and Mytilini sausages. The findings provide an indication that the microbial diversity might be applied as an additional marker of Cypriot sausages’ authenticity.

Establishment of closed 35S ribosomal RNA gene chromatin in stationary Saccharomyces cerevisiae cells.

As a first step in eukaryotic ribosome biogenesis RNA polymerase (Pol) I synthesizes a large ribosomal RNA (rRNA) precursor from multicopy rRNA gene loci. This process is essential for cellular growth and regulated in response to the cell's physiological state. rRNA gene transcription is downregulated upon growth to stationary phase in the yeast Saccharomyces cerevisiae. This reduction correlates with characteristic changes in rRNA gene chromatin structure from a transcriptionally active 'open' state to a non-transcribed 'closed' state. The conserved lysine deacetylase Rpd3 was shown to be required for this chromatin transition. We found that Rpd3 is needed for tight repression of Pol I transcription upon growth to stationary phase as a prerequisite for the establishment of the closed chromatin state. We provide evidence that Rpd3 regulates Pol I transcription by adjusting cellular levels of the Pol I preinitiation complex component core factor (CF). Importantly, our study identifies CF as the complex limiting the number of open rRNA genes in exponentially growing and stationary cells.

Phylogenetic analysis of a novel Hepatozoon species (Hepatozoon sp. SK3) and an additional yet unknown Hepatozoon species (Hepatozoon sp. BV2) besides H. erhardovae in small rodents from Central Europe.

Hepatozoon spp. are tick-borne apicomplexan parasites of terrestrial vertebrates that occur worldwide. Tissue samples from small rodents and their parasitizing fleas were sampled for molecular detection and phylogenetic analysis of Hepatozoon-specific 18S rRNA gene region. After alignment and tree inference the Hepatozoon-sequences retrieved from a yellow-necked mouse (Apodemus flavicollis) placed into a strongly supported single clade demonstrating the presence of a novel species, designated Hepatozoon sp. SK3. The mode of transmission of Hepatozoon sp. SK3 is yet unknown. It is important to note that this isolate may be identical with the previously morphologically described Hepatozoon sylvatici infecting Apodemus spp.; however, no sequences are available for comparison. Furthermore, the previously reported variants Hepatozoon sp. BV1/SK1 and BV2/SK2 were detected in bank voles (Clethrionomys glareolus). It has been suggested that these variants should be identified as Hepatozoon erhardovae leading to the assumption that BV1 and BV2 are paralogous 18S rRNA gene loci of this species. Evidence has also been presented that fleas are vectors of H. erhardovae. In this study, we show with high significance that only the Hepatozoon sp. BV1 variant, but not BV2, infects the studied flea species Ctenophthalmus agyrtes, Ctenophthalmus assimilis, and Megabothris turbidus (p < 0.001). This finding suggests that Hepatozoon sp. BV2 represents an additional species besides H. erhardovae (= Hepatozoon sp. BV1), for which alternative arthropod vectors or non-vectorial modes of transmission remain to be identified. Future studies using alternative molecular markers or genome sequencing are required to demonstrate that BV1/SK1 and BV2/SK2 are different Hepatozoon species.

Cryptosporidium spp. in large-scale sheep farms in China: prevalence and genetic diversity

Cryptosporidium spp. are significant zoonotic intestinal parasites that induce diarrhea and even death across most vertebrates, including humans. Previous studies showed that sheep are important hosts for Cryptosporidium and that its distribution in sheep is influenced by geography, feeding patterns, age, and season. Environmental factors also influence the transmission of Cryptosporidium. Molecular studies of Cryptosporidium in sheep have been conducted in only a few regions of China, and studies into the effect of sheep-housing environments on Cryptosporidium transmission are even rarer. To detect the prevalence of Cryptosporidium in large-scale sheep-housing farms, a total of 1241 fecal samples were collected from sheep, 727 environmental samples were taken from sheep housing, and 30 water samples were collected in six regions of China. To ascertain the existence of the parasite and identify the species of Cryptosporidium spp., we conducted nested PCR amplification of DNA extracted from all samples using the small-subunit (SSU) rRNA gene as a target. For a more in-depth analysis of Cryptosporidium spp. subtypes, C. xiaoi-and C. ubiquitum-positive samples underwent separate nested PCR amplification targeting the 60 kDa glycoprotein (gp60) gene. The amplification of the Cryptosporidium spp. SSU rRNA gene locus from the whole genomic DNA of all samples yielded a positive rate of 1.2% (20/1241) in fecal samples, 0.1% (1/727) in environmental samples, and no positive samples were found in water samples. The prevalence of Cryptosporidium spp. infection in large-scale housed sheep was 1.7%, which was higher than that in free-ranging sheep (0.0%). The highest prevalence of infection was found in weaning lambs (6.8%). Among the different seasons, the peaks were found in the fall and winter. The most prevalent species were C. xiaoi and C. ubiquitum, with the former accounting for the majority of infections. The distribution of C. xiaoi subtypes was diverse, with XXIIIc (n = 1), XXIIId (n = 2), XXIIIe (n = 2), and XXIIIl (n = 4) identified. In contrast, only one subtype, XIIa (n = 9), was found in C. ubiquitum. In this study, C. xiaoi and C. ubiquitum were found to be the predominant species, and Cryptosporidium was found to be present in the environment. These findings provide an important foundation for the comprehensive prevention and management of Cryptosporidium in intensively reared sheep. Furthermore, by elucidating the prevalence of Cryptosporidium in sheep and its potential role in environmental transmission, this study deepens our understanding of the intricate interactions between animal health, environmental contamination, and public health dynamics.

Prevalence and assemblage identified of Giardia duodenalis in zoo and farmed Asiatic black bears (Ursus thibetanus) from the Heilongjiang and Fujian Provinces of China.

Captive and free-living wildlife serve as significant hosts for Giardia duodenalis. Asiatic black bears, valued for their economic and medicinal importance, are extensively farmed in China and also prevalent in zoos. However, studies on G. duodenalis in these animals in China are limited. Here, 218 feces samples of Asiatic black bears were collected: 36 from a zoo in Heilongjiang Province, and 182 from a farm in Fujian Province. Nested PCR of the SSU rRNA gene, followed by sequencing, was employed to determine the frequency and assemblage distribution of G. duodenalis. Positive samples underwent further analysis through multilocus genotyping (MLG) by amplifying the genes for glutamate dehydrogenase (gdh), β-giardin (bg), and triosephosphate isomerase (tpi). Of the 218 samples, G. duodenalis was detected in 22 cases at the SSU rRNA gene locus, including three from Heilongjiang and 19 from Fujian. Three assemblages were identified: A (n=1), B (n=16), and E (n=2) in Fujian; and B (n=3) in Heilongjiang. Out of the 22 positive samples, 20, 19, and 9 were effectively amplified and sequenced across the tpi, gdh, and bg loci, respectively. Seven samples were genotyped successfully at all three loci, identifying MLG-B1 (n=1), MLG-B2 (n=1), and MLG-B3 (n=1), MLG-B4 (n=1), MLG-B5 (n=2), and MLG-B6 (n=1) as the six assemblage B MLGs. This study marks the first documentation of G. duodenalis in Asiatic black bears in captivity in Fujian and Heilongjiang. The identification of zoonotic assemblages A and B, along with E, underscores potential public health concerns.

Sequence and epigenetic landscapes of active and silent nucleolus organizer regions in Arabidopsis

Arabidopsis thaliana has two ribosomal RNA (rRNA) gene loci, nucleolus organizer regions NOR2 and NOR4 , whose complete sequences are missing in current genome assemblies. Ultralong DNA sequences assembled using an unconventional approach yielded ~5.5- and 3.9-Mbp sequences for NOR2 and NOR4 in the reference strain, Col-0. The distinct rRNA gene subtype compositions of the NORs enabled the positional mapping of their active and inactive regions, using RNA sequencing to identify subtype-specific transcripts and DNA sequencing to identify subtypes associated with flow-sorted nucleoli. Comparisons of wild-type and silencing-defective plants revealed that most rRNA gene activity occurs in the central region of NOR4 , whereas most, but not all, genes of NOR2 are epigenetically silenced. Intervals of low CG and CHG methylation overlap regions where gene activity and gene subtype homogenization are high. Collectively, the data reveal the genetic and epigenetic landscapes underlying nucleolar dominance (differential NOR activity) and implicate transcription as a driver of rRNA gene concerted evolution.

Morphological and molecular characterization of Isospora elliotae n. sp. (Apicomplexa: Eimeriidae) from the Australian magpie Gymnorhina tibicen (Latham, 1801) (Passeriformes: Artamidae) in Western Australia.

A new coccidian species, Isospora elliotae n. sp., from the Australian magpie Gymnorhina tibicen (Latham, 1801) in Western Australia, is described and characterized morphologically and molecularly. Microscopic analysis of a faecal sample identified subspheroidal oocysts (n = 20), 20-22 × 18-20 (20.7 × 18.7); length/width (L/W) ratio 1.05-1.14 (1.10). Wall bi-layered, 1.0-1.3 (1.2) thick, outer layer smooth, c. 2/3 of total thickness. Micropyle and oocyst residuum absent, but usually two polar granules are present. Sporocysts (n = 28) ovoidal, 12-13 × 9-11 (12.6 × 9.7); L/W ratio 1.22-1.35 (1.30). Stieda body present, flattened to half-moon-shaped, c. 0.5 deep × 2.0 wide; sub-Stieda indistinct or barely discernible, c. 1.0 deep × 2.5 wide; para-Stieda body absent; sporocyst residuum present, composed of granules dispersed among the sporozoites. Sporozoites vermiform, with anterior and posterior refractile bodies and nucleus. Segments of three gene loci (18S rRNA, 28S rRNA and COI) were sequenced and I. elliotae n. sp. exhibited 99.8% genetic similarity to Isospora sp. MAH-2013a (KF648870) followed by 99.7% genetic similarity to Isospora neochmiae (Yang, Brice & Ryan, 2016) (KT224380) at the 18S rRNA gene locus. It shared 97.0% genetic similarity with an unnamed Isospora sp. (AY283852) at the 28S rRNA gene locus and it also shared the highest genetic similarity of 99.8% with the unnamed Isospora sp. from an American crow (OL999120) at the COI gene locus. Based on morphological and molecular data, this isolate is a new species named as I. elliotae n. sp.

Morphological and molecular characteristics of a single oocyst for the identification of Eimeria species in domestic rabbits (Oryctolagus cuniculus f. domesticus)

Coccidiosis caused by Eimeria is one of the most common diseases in domestic rabbits (Oryctolagus cuniculus f. domesticus), with 11 Eimeria species in domestic rabbits recognized internationally. To identify Eimeria species more accurately, a method based on the molecular characteristics of a single oocyst with multiple gene loci was established by combining morphological and molecular biology. The results showed that the total infection rate of Eimeria in domestic rabbits was 44.2 % (152/344). Ten Eimeria species were identified in domestic rabbits based on morphological characteristics, namely Eimeria vejdovskyi (39.5 %, 136/344), E. magna (18.0 %, 62/344), E. perforans (17.4 %, 60/344), E. intestinalis (12.5 %, 43/344), E. media (11.9 %, 41/344), E. coecicola (4.4 %, 15/344), E. irresidua (3.8 %, 13/344), E. exigua (2.6 %, 9/344), E. stiedai (2.3 %, 8/344), and E. piriformis (1.5 %, 5/344). The molecular biological identification of Eimeria in domestic rabbits was conducted through single oocyst selection and nested polymerase chain reaction amplification with multiple gene loci. We obtained the sequences of the 18S rRNA, ITS-1 and COI gene loci of E. magna, E. perforans, E. vejdovskyi, E. media, E. intestinalis, and E. coecicola. The results showed that the molecular biology and morphological identification results of single oocysts were consistent and could be used for the molecular identification of Eimeria at the single oocyst level. This study provides an efficient tool for identification of Eimeria in domestic rabbits and the population genetic study of Eimeria in domestic rabbits.

Telomere-to-Telomere genome assemblies of human-infecting Encephalitozoon species

BackgroundMicrosporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete.MethodsIn this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting Encephalitozoon spp. —E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602— using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation.ResultsThe Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes.ConclusionAltogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.

Prevalence, Genetic Homogeneity, and Antibiotic Resistance of Pathogenic Yersinia enterocolitica Strains Isolated from Slaughtered Pigs in Bulgaria.

Yersiniosis is the third most commonly reported foodborne zoonosis in the European Union. Here, we evaluated the prevalence of pathogenic Yersinia enterocolitica among healthy pigs (as a major reservoir) in a slaughterhouse in Bulgaria. A total of 790 tonsils and feces from 601 pigs were examined. Isolation and pathogenicity characterization was carried out by the ISO 10273:2003 protocol and Polymerase Chain Reaction (PCR), detecting the 16S rRNA gene, attachment and invasion locus (ail), Yersinia heat-stable enterotoxin (ystA), and Yersinia adhesion (yadA) genes. Genetic diversity was assessed by pulsed-field gel electrophoresis (PFGE), and antimicrobial resistance by the standard disk diffusion method. Of all the pigs tested, 6.7% were positive for Y. enterocolitica. All isolates belonged to Y. enterocolitica bioserotype 4/O:3. ail, and ystA genes were detected in all positive strains (n = 43), while the plasmid Yersinia virulence plasmid (pYV) was detected in 41. High homogeneity was observed among the strains, with all strains susceptible to ceftriaxone, amikacin and ciprofloxacin, and resistant to ampicillin. In conclusion, a low prevalence of Y. enterocolitica 4/O:3 was found in healthy pigs slaughtered in Bulgaria, not underestimating possible contamination of pork as a potential risk to consumer health.

Detection and Molecular Characterization of Giardia and Cryptosporidium spp. Circulating in Wild Small Mammals from Portugal.

Cryptosporidium spp. and Giardia spp. are important diarrhea-causing protozoan parasites worldwide that exhibit broad host ranges. Wild small mammals can harbor host-adapted and potentially zoonotic species of both parasites. The aim of this study was to investigate Cryptosporidium spp. and Giardia spp. in wild rodents and shrews in Portugal, focusing on the protist's occurrence and genetic diversity. Molecular screening by PCR at the small subunit (SSU) rRNA gene locus of 290 fecal samples from wood mice (Apodemus sylvaticus), southwestern water voles (Arvicola sapidus), Cabrera's voles (Microtus cabrerae), Lusitanian pine voles (Microtus lusitanicus), Algerian mice (Mus spretus) and greater white-toothed shrews (Crocidura russula) in Northeast Portugal revealed the low occurrence of Cryptosporidium spp. (1%) and high occurrence of Giardia spp. (32.8%). The analysis revealed that "species" was the only significant factor associated with the increasing probability of Giardia spp. infection, with the highest prevalence reported in southwestern water voles and Lusitanian pine voles. Cryptosporidium and Giardia species determination at the SSU rRNA gene locus revealed C. muris and G. microti as the only circulating species, respectively. Subtyping of the glutamate dehydrogenase (gdh) and beta-giardin (bg) genes provided evidence of the high genetic diversity within the G. microti clade. This study suggests that rodent-adapted G. microti occurs to a large extent in cricetid hosts and supports the limited role of wild rodents and shrews as natural sources of human infections in Northeast Portugal regarding the investigated parasites. Moreover, this is the first record of G. microti in southwestern water voles, Lusitanian pine voles, Algerian mice, wood mice and Cabrera's voles and C. muris in Cabrera's voles. Finally, this study improves the database of sequences relevant for the sequence typing of G. microti strains and provides new insights about the epidemiology of Giardia spp. and Cryptosporidium spp. in wild rodents and shrews, two parasite genera of high importance for public and animal health.

Fermented table olives from Cyprus: Microbiota profile of three varieties from different regions through metabarcoding sequencing.

The knowledge about the microbial diversity of different olives varieties from diverse regions in the Mediterranean basin is limited. This work aimed to determine the microbial diversity of three different fermented olive varieties, collected from different regions in Cyprus, via Next Generation Sequencing (NGS) analysis. Olives were spontaneously fermented for 120 days, microbial DNA was extracted from the final products, and subjected to 16S rRNA gene and ITS1 loci metabarcoding analysis for the determination of bacterial and fungal communities, respectively. Results revealed that the bacterial profile of the studied varieties was similar, while no noteworthy differences were observed in olives from different regions. The bacterial profile was dominated by the co-existence of Lactobacillus and Streptococcus, while the genera Lactococcus and Salinivibrio and the family Leuconostocaceae were also present in increased relative abundances. Regarding fungal communities, the analysis indicated discrimination among the different varieties, especially in Kalamata ones. The most abundant fungi were mainly the genera Aspergillus, Botryosphaeria, Meyerozyma, and Zygosaccharomyces for Cypriot olives, the genera Botryosphaeria, Saccharomyces, Geosmithia, and Wickeromyces for Kalamata variety, while the dominant fungi in the Picual variety were mainly members of the genera Candida, Penicillium, Saccharomyces, Hanseniospora and Botryosphaeria. Potential microbial biomarkers that distinguish the three varieties are also proposed. Moreover, interaction networks analysis identified interactions among the key taxa of the communities. Overall, the present work provides useful information and sheds light on an understudied field, such as the comparison of microbiota profiles of different varieties from several regions in Cyprus. The study enriches our knowledge and highlights the similarities and the main differences between those aspects, booming in parallel the need for further works on this frontier, in the attempt to determine potentially olives' microbial terroir in Cyprus. Our work should be used as a benchmark for future works in this direction.

The Chromosome Number and rDNA Loci Evolution in Onobrychis (Fabaceae).

The evolution of chromosome number and ribosomal DNA (rDNA) loci number and localisation were studied in Onobrychis Mill. Diploid and tetraploid species, as well as two basic chromosome numbers, x = 7 and x = 8, were observed among analysed taxa. The chromosomal distribution of rDNA loci was presented here for the first time using fluorescence in situ hybridisation (FISH) with 5S and 35S rDNA probes. Onobrychis species showed a high polymorphism in the number and localisation of rDNA loci among diploids, whereas the rDNA loci pattern was very similar in polyploids. Phylogenetic relationships among the species, inferred from nrITS sequences, were used as a framework to reconstruct the patterns of basic chromosome number and rDNA loci evolution. Analysis of the evolution of the basic chromosome numbers allowed the inference of x = 8 as the ancestral number and the descending dysploidy and polyploidisation as the major mechanisms of the chromosome number evolution. Analyses of chromosomal patterns of rRNA gene loci in a phylogenetic context resulted in the reconstruction of one locus of 5S rDNA and one locus of 35S rDNA in the interstitial chromosomal position as the ancestral state in this genus.

Eimeria spp. (Eimeriidae) in the migratory whooper swan (Cygnus cygnus) Linnaeus, 1758 (Anatidae) from Sanmenxia Swan Lake National Urban Wetland Park in the middle reaches of the Yellow River in China.

This study i dentifies four Eimeria spp. recorded from fecal samples of migratory whooper swans (Cygnus cygnus) in Sanmenxia Swan Lake National Urban Wetland Park in Sanmenxia city in the middle reaches of the Yellow River, China. Eimeria hermani, Eimeria nocens, Eimeria stigmosa, and Eimeria magnalabia were compatible in all characteristic features with their respective original descriptions. In addition to the preliminary morphological identification, this study provides a preliminary genotypic identification of these four Eimeria spp. via sequencing of the 18S rRNA, 28S rRNA, and COI gene loci that are suitable for the genotypic differentiation of these coccidia. This is the first report of molecular data for the four Eimeria spp. in migratory whooper swans. Finally, this study discusses the environmental risks of these coccidia for migratory whooper swans in Sanmenxia Swan Lake National Urban Wetland Park.

The Prevalence and Genetic Diversity of Babesia divergens in Ixodes ricinus Nymphs Collected from Farm- and Woodland Sites in Ireland.

The parasite, Babesia divergens causes redwater fever in cattle and a rare, albeit life-threatening disease in humans. In Ireland, B. divergens has always been considered an important pathogen as the high incidence of redwater fever precluded areas of the country from cattle farming. Moreover a relatively large proportion of human cases were reported here. Red deer (Cervus elaphus), which often harbour babesias that are genetically very similar (if not identical) to B. divergens, are quite widespread. In this study 1369 nymphal Ixodes ricinus ticks collected from various habitats were screened for the presence of B. divergens using TaqMan followed by conventional nested PCR. Fragments of the 18S rRNA gene locus (560 bp) were compared against published Irish B. divergens isolates from cattle, humans and red deer. Overall just 1% of I. ricinus nymphs were infected with B. divergens, with similar infection rates in ticks collected from farm- and woodland. Most (90%) 18S rRNA gene fragments derived from woodland ticks were 100% identical to published sequences from cattle and humans. One differed by a single nucleotide polymorphism (SNP) as did two isolates from ticks collected in bogland. Two isolates derived from nymphs collected in farmland differed by 2 and 4 SNPs respectively.

Characterization and diversity of native Azotobacter spp. isolated from semi-arid agroecosystems of Eastern Kenya.

Declining food production in African agroecosystems is attributable to changes in weather patterns, soil infertility and limited farming inputs. The exploitation of plant growth-promoting soil microbes could remedy these problems. Such microbes include Azotobacter; free-living, nitrogen-fixing bacteria, which confer stress tolerance, avail phytohormones and aid in soil bioremediation. Here, we aimed to isolate, characterize and determine the biodiversity of native Azotobacter isolates from soils in semi-arid Eastern Kenya. Isolation was conducted on nitrogen-free Ashby's agar and the morphological, biochemical and molecular attributes evaluated. The isolates were sequenced using DNA amplicons of 27F and 1492R primers of the 16S rRNA gene loci. The Basic Local Alignment Search Tool (BLASTn) analysis of their sequences revealed the presence of three main Azotobacter species viz., Azotobacter vinelandii, Azotobacter salinestris and Azotobacter tropicalis. Kitui County recorded the highest number of recovered Azotobacter isolates (45.4%) and lowest diversity index (0.8761). Tharaka Nithi County showed the lowest occurrence (26.36%) with a diversity index of (1.057). The diversity was influenced by the soil pH, texture and total organic content. This study reports for the first time a wide diversity of Azotobacter species from a semi-arid agroecosystem in Kenya with potential for utilization as low-cost, free-living nitrogen-fixing bioinoculant.

Genetic diversity within the 18S rRNA and actin locus of Cryptosporidium scrofarum (Apicomplexa: Cryptosporidiidae) infecting domestic pigs (Sus scrofa domesticus) of India

The genetic diversity was studied of Cryptosporidium scrofarum (syn Cryptosporidium pig genotype II) of domestic pigs (Sus scrofa domesticus) from Punjab, India. Nested PCR amplification targeting the 18S rRNA and actin gene loci from Cryptosporidium positive samples was carried out, and the amplicons were sequenced. Phylogenetic comparison of a partial 18S rRNA gene revealed that they were genetically most similar to C. scrofarum isolated from other parts of the world. However, comparison of sequences representing a fragment of the genomic actin locus identified a new genotype conserved within the isolates sampled from India but distinct from other published sequences, suggesting the presence of a different Indian genotype.

Molecular cytogenetical and biochemical studies on some Lupinus species

BackgroundLupins are cultivated as human consumption grains and forage legumes. The chromosomes of lupins are too small to be karyotyped by conventional techniques, because they reveal a general lack of distinctive cytological features. In the current study, Fluorescence in situ Hybridization (FISH) was used to locate 5S and 45S ribosomal gene sites on the chromosomes of Lupinus albus ssp albus, Lupinus albus ssp graecus, Lupnus termis (all with 2n = 50), and Lupinus polyphyllus lindl var. polyphyllus (2n = 48), FISH together with seed storage protein electrophoretic patterns were used to find out the relationship among these species.ResultsThe double-target FISH on the chromosomes of the studied species with rDNA probes revealed that the two types of rRNA genes are located on different chromosomes. The detected loci of rRNA genes partially reflected the taxonomical similarity among the two Lupinus albus subspecies and L. termis. Lupinus polyphyllus lindl var. polyphyllus was exception by having unique large chromosome mostly is covered by one signal of 45S rDNA, whereas its homologous chromosome seems to be normal-sized and have the other 45S rDNA locus. The similarity matrix among the Lupinus species as computed according to Jaccardʼs Coefficient from the SDS-PAGE, showed that L. albus ssp. Albus and L. albus ssp. Graecus are the most similar species (~ 97%), and then comes L. termis, and L. polyphyllus lindl var. polyphylus has been placed in separate clade and still the most related species to it among the studied species is L. termis (~ 70%).ConclusionIt could be postulated from FISH and seed storage protein electrophoretic patterns that the relationships among the studied species is as follows, Lupinus albus ssp albus, is the most related species to Lupinus albus ssp graecus then comes Lupnus termis and Lupinus polyphyllus lindl var. polyphyllus at a distal position.

Morphological and molecular characterization of a new species of Isospora Schneider, 1881 (Apicomplexa: Eimeriidae) from the western wattlebird Anthochaera lunulata Gould in Western Australia

A new coccidian species, Isospora lunulatae n. sp., from the western wattlebird Anthochaera lunulata Gould in Western Australia is described and characterised molecularly. Microscopic analysis of a faecal sample identified subspheroidal oöcysts measuring 27–34 × 26–31 (30.6 × 29.4) μm (n = 20), with a length/width (L/W) ratio of 1.0–1.1 (1.0). Oöcysts have a bi-layered wall, 0.9–1.2 (1.0) μm thick; the outer layer is smooth, representing c.2/3 of total thickness. Micropyle and oöcyst residuum are both absent, but a polar granule is present. Sporocysts are ovoidal, 17–19 × 10–12 (18.3 × 10.7) μm, with a L/W ratio of 1.6–1.8 (1.7) and occupying about 21% of the area (each one) within the oöcyst. Stieda body is flattened to rounded, measuring on average 0.9 × 1.8 μm; sub-Stieda body is rounded to rectangular, measuring on average 1.5 × 2.6 μm; para-Stieda body is absent. Sporocyst residuum has an irregular shape consisting of numerous granules and appears membrane-bound. Sporozoites are vermiform 12.8 × 3.0 μm on average, with prominent striations at the more pointed end and two refractile bodies below striations. Segments of three gene loci (18S rRNA, 28S rRNA and cox1) were sequenced and I. lunulatae n. sp. exhibited 99.6% genetic similarity to Isospora phylidonyrisae Yang, Brice, Berto & Ryan, 2021 at the 18S rRNA gene locus, 99.8% genetic similarity to Isospora anthochaerae Yang, Brice & Ryan, 2014 and shared a 98.1% genetic similarity with Isospora manorinae Yang, Brice, Jian & Ryan, 2016 at the cox1 gene locus. Morphological and molecular data support the distinct species status of the new species.

Chromosomal analysis of eight species of dragonflies (Anisoptera) and damselflies (Zygoptera) using conventional cytogenetics and fluorescence in situ hybridization: Insights into the karyotype evolution of the ancient insect order Odonata

All Odonata species studied to date using fluorescence in situ hybridization (FISH) belong to the dragonfly (Anisoptera) families Corduliidae and Libellulidae. It was shown that 18S rRNA gene loci locate on one of the largest pairs of autosomes in every species, whereas the “insect” telomere motif (TTAGG)n is absent in all but one species. For better understanding the chromosomal organization and evolution of Odonata, we used C-banding and FISH to study the karyotypes and map TTAGG sequences and major rRNA loci on chromosomes of three more dragonfly species from the families Corduliidae, Libellulidae, and Aeshnidae. Moreover, we obtained the first FISH-data on the suborder Zygoptera (damselflies) by analyzing five species of the families Coenagrionidae and Calopterygidae. We showed that all studied dragonfly species had 2n = 24A + X. The same karyotype was observed in the damselfly family Coenagrionidae, whereas in species of the Calopterygidae, the karyotype 2n = 26A + X was found. Both dragonfly and damselfly species had a pair of m-chromosomes; constitutive heterochromatin tended to be concentrated in the terminal regions of their chromosomes. The use of (TTAGG)n and 18S rRNA gene probes in dual-color FISH did not generate (TTAGG)n fluorescent signals in any species; major rRNA clusters were revealed on one of the largest pairs of autosomes in all Anisoptera species but on m-chromosomes in all Zygoptera species. Our results suggest that the former 18S location pattern was ancestral in the Odonata and the latter pattern had an ancient origin and could arise in a common ancestor of the damselfly superfamilies Calopterygoidea and Coenagrionoidea.