Culturable and unculturable potential heterotrophic microbiological threats to the oldest pyramids of the Memphis necropolis, Egypt.

A large percentage of the world's tangible cultural heritage is made from stone; thus, it deteriorates due to physical, chemical, and/or biological factors. The current study explored the microbial community inhabiting two prehistoric sites with high cultural value in the Memphis necropolis of Egypt (Djoser and Lahun Pyramids) using amplicon-based metabarcoding and culture-dependent isolation methods. Samples were examined by epifluorescent microscopy for biological signs before environmental DNA extraction and in vitro cultivation. The metabarcoding analysis identified 644 bacterial species (452 genera) using the 16S rRNA and 204 fungal species (146 genera) using ITS. In comparison with the isolation approach, an additional 28 bacterial species (13 genera) and 34 fungal species (20 genera) were identified. A total of 19 bacterial and 16 fungal species were exclusively culture-dependent, while 92 bacterial and 122 fungal species were culture-independent. The most abundant stone-inhabiting bacteria in the current study were Blastococcus aggregatus, Blastococcus saxobsidens, and Blastococcus sp., among others. The most abundant rock-inhabiting fungi were Knufia karalitana and Pseudotaeniolina globosa, besides abundant unknown Sporormiaceae species. Based on previous reports, microorganisms associated with biodeterioration were detected on color-altered sites at both pyramids. These microorganisms are potentially dangerous as physical and chemical deterioration factors and require proper conservation plans from a microbiological perspective.

aroF and cm2: potential molecular markers for the detection of stone-inhabiting Actinobacteria on cultural heritage sites.

Tangible archeological sites and stone monuments are naturally decayed and deteriorated over time, providing substances that can sustain life, although they provide a complicated ecosystem characterized by low nutrition and desiccation. Stone-inhabiting bacteria (SIB) and especially members of the phylum Actinobacteria dominate such environments, particularly the members of the family Geodermatophilaceae. We used the published data of two confirmed SIB species to mine their genomes for specific molecular markers to rapidly survey the presence of SIB in cultural heritage material prior to further analysis. The search focused on the mycosporine-like amino acids (MAAs) synthesis pathway. MAAs are intracellular compounds biosynthesized by the shikimic acid pathway to synthesize aromatic amino acids and were found related to abiotic resistance features in microorganisms. Based on genome mining, the DAHP II (aroF) and a homolog of the Chorismate mutase gene (cm2) were found mostly in Actinobacteria and few other species. After calibration on five stone-inhabiting Actinobacteria (SIAb) species using conventional PCR, newly designed primers were successfully applied to environmental DNA extracted from two Egyptian pyramidal sites using a qPCR approach. This is the first report of aroF and cm2 as qPCR markers to detect SIAb from cultural heritage material prior to proceeding with further analysis (e.g., metagenomics and meta-barcoding analyses).

A New Extremotolerant Ecotype of the Fungus Pseudotaeniolina globosa Isolated from Djoser Pyramid, Memphis Necropolis, Egypt.

Most of the rock-inhabiting fungi are meristematic and melanized microorganisms often associated with monument biodeterioration. In previous microbial profiling of the Egyptian Djoser pyramid, a Pseudotaeniolina globosa isolate was found. The current study aimed to characterize the P. globosa isolated from the Djoser pyramid compared with an Italian isolate at morphological, physiological, and molecular levels. Experiments were carried out to test temperature, salinity, and pH preferences, as well as stress tolerance to UV radiation and high temperature, in addition to a multi-locus genotyping using ITS, nrSSU or 18S, nrLSU or 28S, BT2, and RPB2 markers. Morphological and molecular data confirmed the con-specificity of the two isolates. However, the Egyptian isolate showed a wider range of growth at different environmental conditions being much more tolerant to a wider range of temperature (4-37 °C) and pH values (3.0-9.0 pH) than the Italian (10-30 °C, 4.0-6.0 pH), and more tolerant to extreme salinity levels (5 M NaCl), compared to the lowest in the Italian isolate (0.2 M NaCl). Besides, the Egyptian isolate was more tolerant to high temperature than the Italian isolate since it was able to survive after exposure to up to 85 °C for 5 min, and was not affected for up to 9 h of UV exposure, while the Italian one could not regrow after the same treatments. The Pseudotaeniolina globosa species was attributed to the family Teratosphaeriaceae of the order Capnodiales, class Dothideomycetes. Our results demonstrated that the Egyptian isolate could be considered an ecotype well adapted to harsh and extreme environments. Its potential bio-deteriorating effect on such an important cultural heritage requires special attention to design and conservation plans and solutions to limit its presence and extension in the studied pyramid and surrounding archaeological sites.

Analysis of Candidate Genes for Growth and Milk Performance Traits in the Egyptian Barki Sheep.

The most common sheep breeds of Egypt are Ossimi, Rahmani, and Barki breeds. The latter one is well adapted to the challenging desert environment, characterized by food shortage and a high temperature fluctuation. Growth performance of Barki sheep has an important economic value in terms of minimizing the shortage of mutton meat in Egypt. Further, milk production is of great importance for feeding newborn lambs. Eight candidate genes, recently associated with production traits in different breeds, were used to study the effect of genotype on lamb growth and ewe milk traits. The examined genes were LEP, IGF1, DGAT1, STAT5A, PRL, CSN1S2, GHR, and GHRHR, of which one representative single nucleotide polymorphism (SNP) located in the coding region was selected for genotyping. Data from 251 Barki sheep were used in this study. Association analysis between SNPs and lamb growth traits identified rs420693815 of the LEP gene to be significantly associated with weaning weight and average daily gain. In ewes, significant effects on milk yield and composition have been estimated for LEP (rs420693815), STAT5A (rs161082816), PRL (rs422713690), and GHRHR (rs414991449). The results indicated that these genes might be considered as interesting candidates for further investigations to improve growth and milk performance in Barki sheep.

Genetic structure of Mugil cephalus L. populations from the northern coast of Egypt.

AIM: The gray mullet, Mugil cephalus, has been farmed in semi-intensive ponds with tilapia and carps in Egypt for years. The current study used the fluorescent amplified fragment length polymorphism (F-AFLP) technique to search for genetic differences between the populations of M. cephalus in the northern region of Egypt and to detect the gene flow between sampled locations and the homogeneity within M. cephalus genetic pool in Egypt. MATERIALS AND METHODS: To fulfill the study objectives 60 (15/location) samples were collected from four northern coast governorates of Egypt (Alexandria "sea," Kafr El-Sheikh "farm," Damietta "farm" and Port Said "sea"). Three replicates of bulked DNA (5 samples/replicate) for each location were successfully amplified using the standard AFLP protocol using fluorescent primers. DNA polymorphism, genetic diversity, and population structure were assessed while positive outlier loci were successfully detected among the sampled locations. Based on the geographical distribution of sampling sites, the gene flow, the genetic differentiation, and correlations to sampling locations were estimated. RESULTS: A total of 1890 polymorphic bands were scored for all locations, where 765, 1054, 673, and 751 polymorphic bands were scored between samples from Alexandria, Kafr El-Sheikh, Damietta and Port Said, respectively. The effective number of alleles (ne) for all bulked samples combined together was 1.42. The expected heterozygosity under Hardy-Weinberg assumption (He) for all bulked samples combined together was 0.28. Bulked samples from Damietta yielded the lowest ne (1.35) and the lowest He (0.23) when inbreeding coefficient (FIS) = 1. Bulked samples from Kafr El-Sheikh scored the highest ne (1.55) and the highest He (0.37). Bulked samples from Alexandria scored 1.40 for ne and 0.26 for He, while bulked samples from Port Said scored 1.39 for ne and 0.26 for He. The observed bulked samples formed three sub-population groups, where none is limited to a certain sampling location. A high differentiation among locations was detected, however, is not fully isolating the locations. Gene flow was 0.58. Positive outliers loci (117) were detected among the four sampled locations while weak significant correlation (r=0.15, p=0.03) was found for the distance between them. CONCLUSION: Even though this species is cultivated in Egypt, the wild population is still present and by the current study a flow of its genes is still exchanged through the northern coast of Egypt. Which contribute to the cultivated populations leading to heterogeneity in its genetic pool and consequently affects the production consistency of M. cephalus in Egypt.