Biofiltering capacity of Chambardia rubens (Bivalvia: Unionidae) may modulate expression of stress and growth genes inhibited by the neonicotinoid insecticide acetamiprid in zebrafish.

Neonicotinoid insecticides specifically target insect subtypes of nicotinic acetylcholine receptors. Acetamiprid (ACE: C10H11ClN4), the neonicotinoid insecticide, is used to control crop insect pests worldwide. It is a nitrile, monochloropyridine, and carboxamidine that is highly soluble and accessible to waterways. There, it causes neurotoxic and oxidative perturbance to non-target organisms. The unionid mussel Chamabradia rubens is a common Northern River Nile suspension feeder. The current study aimed to assess ACE filtration from waters by C. rubens, and whether this biological power can reduce ACE effects on fish. Removal of ACE by C. rubens was assessed using LC-MS/MS. Zebrafish Danio rerio adults were exposed to different sublethal doses of ACE in the presence or absence of C. rubens in their aquaria. The results showed that mussels could remove significant ACE amounts from water, where it accumulated mostly in the digestive gland. The presence of C.rubens in zebrafish aquaria having ACE was accompanied by significant upregulation of antioxidant enzyme gene transcripts and total H2O2 scavenging, in contrast to mussel-free ACE-exposed groups. Meanwhile, liver triglycerides rose 5-6-fold in response to ACE in the "Fish-Only" groups, indicating an ACE-induced hepatotoxicity. Also, Insulin-like growth factor 1 (igf1) and fish body mass increased more in "Fish + Mussel" groups than in the "Fish-Only" ones. In aggregate, these findings suggest that the Nile mussel could reduce the oxidative stress and metabolic changes induced in fish by ACE. This can contribute valuable environmental and economic benefits upon the use of this mussel as a biofilter.

Unraveling the Red Sea soft coral Sarcophyton convolutum potentials against oxidative and inflammatory stresses in zebrafish.

The Red Sea is one of the world's hotspots for biodiversity, and for marine natural products (MNPs) as well. These MNPs attract special interest for their capabilities to combat inflammatory and oxidative stress-related diseases, being some of the most serious health problems worldwide nowadays. The current study aimed to identify the bioactive ingredients of the Red Sea soft coral Sarcophyton convolutum, and to assess its protective potentials against oxidative and inflammatory stresses. Coral extract (CE) was analyzed using GC-MS and HPLC. In a protection trial, adult zebrafish were intraperitoneally injected with two doses of crab extract, i.e. 50 and 500 µg/fish in 1 % DMSO as a vehicle, then challenged with 30 µg L-1 of CuSO4 for 48 h. All groups, but the negative control one, were challenged with 30 µg L-1 of CuSO4. Total antioxidant activity, as well as mRNA levels of proinflammatory markers and antioxidant enzyme genes were measured. The results showed richness of S. convolutum extract with various bioactive ingredients, including phenolic compounds, flavonoids, alkanes, fatty acids, sesquiterpenes, and pheromone-like substances. CuSO4 significantly induced the expected signals of inflammatory and oxidative stress, reducing both the antioxidant activity and increasing proinflammatory marker genes. However, CE, especially the low dose, showed significant capability to reduce proinflammatory markers and elevating the total antioxidant activity. Therefore, we concluded that S. convolutum can be a promising source for future efforts of drug discovery and a wide spectrum of pharmaceutical products.

Nile crab Potamonautes niloticus shell extract: Chromatographic and molecular elucidation of potent antioxidant and anti-inflammatory capabilities.

Diseases emerging from oxidative stress and inflammatory imbalance are deeply threatening the modern world. Fisheries by-products are rich in bioactive metabolites. However, they are usually discarded, posing a real environmental burden. Herein we aimed to explore the bioactive compounds, anti-oxidant, and anti-inflammatory capabilities of the shell of the freshwater Nile crab Potamonautes niloticus. Methanolic extract of crab shell was subjected to GC/MS and HPLC analyses of total lipids, flavonoids, and phenolic acids. Also, zebrafish Danio rerio was subjected to inflammatory status using CuSO4, then treated with different doses of shell extract. Total antioxidant capacity and QPCR analyses for gene expression of different antioxidant enzymes, i.e. superoxide dismutase(sod), catalase (cat), and glutathione peroxidase (gpx) and pro-inflammatory cytokines, i.e. tumor necrosis factor alpha (tnf-α), nuclear factor kappa B (nf-κb), interleukin 1-Beta (il-1b) were assessed. The results showed the richness of crab shell extract with ω - 9 (32.78 %), ω - 7 (6.37 %), and ω - 6 (4 %) unsaturated fatty acids. Diverse phenolic acids and flavonoids were found, dominaed by Benzoic acid (11.24 µg mL-1), Syringic acid (11.4 µg mL-1), Ferulic acid (10.55 µg mL-1), Kampferol (9.47 µg mL-1), Quercetin (6.33 µg mL-1), and Naringin (4.16 µg mL-1). Crab extract also increased the total antioxidant capacity and oxidative stress enzymes mRNA levels by 1.3-2.15 folds. It down-regulated pro-inflammatory cytokines mRNA levels by 1.3-2 folds in comparison to positive control (CuSO4-induced) zebrafishes. The net results indicated that Nile crab shell extract is a rich source of anti-oxidant and anti-inflammatory compounds. Therefore, we recommend to continuously explore the bioactive capabilities of exoskeletons of different shellfish species. This can provide additive values for these products and reduce the environmental burden of their irresponsible discarding.

Single nucleotide polymorphism-based methodology for authentication of bovine, caprine, ovine, camel, and donkey meat cuts.

To guarantee food safetyand sustainability, it is necessary to verify meat authenticity. This study focused on the development of single nucleotide polymorphism-based polymerase chain reaction-restriction fragment length polymorphism (SNP-based PCR-RFLP) and forensically informative nucleotide sequence (FINS) methodologies based on PCR amplification of the mitochondrial 12S rRNA gene for discrimination of six red meat species, that is, cattle, buffalo, goat, sheep, camel, and donkey. FINS allowed the unambiguous identification of all species analyzed. In addition, six SNPs, where a restriction site for TasI could be localized using a preliminary in silico analysis, gave a unique RFLP pattern for each species. The results revealed a low level of species substitution (8%) in the tested meat samples. In particular, one buffalo and goat samples have been substituted with cow and sheep, respectively. Finally, the developed techniques herein showed high potentials to be routinely used as reliable and fast tools to avoid meat species substitutions. PRACTICAL APPLICATION: This research deals with genetic techniques to trace meats. This kind of research helps the concerned agencies to build capacity to safeguard consumer sentiments as well as providing better market access and better food price and quality for the consumer.

Serological Survey of Babesia bigemina and Babesia bovis in Cattle and Water Buffaloes from Menoufia Province, Egypt.

PURPOSE: Bovine babesiosis causes morbidity in tropical and subtropical countries worldwide. The present study aimed to determine the seroprevalence of Babesia bigemina and B. bovis in cattle and water buffaloes in Menoufia province, where the second-highest population of bovines in Lower Egypt are raised. MATERIALS AND METHODS: A total of 506 blood samples were collected from cattle (N = 262) and water buffaloes (N = 244) in Menoufia province, Egypt. Seroprevalences of B. bigemina and B. bovis in the samples were determined using recombinant Babesia antigen-specific enzyme-linked immunosorbent assays (ELISA). RESULTS: In cattle, the seroprevalences of B. bigemina and B. bovis were 41.60 and 38.17% (37.40 and 35.88% for IgM and 9.54 and 6.11% for IgG), respectively, whereas those of water buffaloes were 35.66 and 31.97% (27.87 and 21.72% for IgM and 15.16 and 15.16% for IgG), respectively. Statistically significant changes in the seroprevalences of the two infective agents were recorded on the basis of region and season of sample collection. CONCLUSION: In conclusion, babesiosis is frequent and presents a threat of an epidemic among bovines in Menoufia province. In turn, control of bovine babesiosis is required because of its potential to detrimentally affect milk and meat production in Menoufia province.

Multiplex PCR and 12S rRNA gene sequencing for detection of meat adulteration: A case study in the Egyptian markets.

Recently, DNA-based methods have proved to be accurate, fast and sensitive for meat authentication. According to the European Union, the food safety standards require accurate and detailed composition information of the meat products. Therefore, an accurate, fast and cost-effective identification methodology is needed. In this study, multiplex PCR coupled with 12S rDNA sequencing was employed for the detection of meat adulteration in two red meat products (frozen beef liver and cold cut samples, respectively) in Egypt. Multiplex PCR allowed the identification of ruminant, poultry, pork, and donkey residuals in processed red meat products (cold cuts) in a single step PCR reaction. Preliminary uniplex PCR was performed to evaluate primers specificity using DNA extracted from the positive control samples. The primers produced specific fragments for ruminant, poultry, pork, and donkey as follows: 271, 183, 531 and 145 bp, respectively. Multiplex PCR revealed that none of the samples was contaminated by porcine or donkey residuals, but 62.5% of all tested processed beef samples contained poultry contaminants. The sensitivity of this method was 0.01 ng/µL for beef, poultry and donkey and 0.1 ng/µL for pig. Another promising finding is the identification of all frozen beef liver samples as a cattle species (Bos taurus) through PCR-sequencing of a short fragment of 12S rRNA gene. Finally, we recommend the employment of multiplex PCR and PCR-sequencing of 12S rDNA for quality control in routine analysis of processed and frozen meat products.

Molecular tools for assuring human health and environment-friendly frozen shellfish products in the United Arab Emirates markets.

Shellfish consumption in the United Arab Emirates (UAE) exceeds local supply and frozen fish and seafood products are imported to fill the gap. To determine the species in frozen shellfish brands on the UAE markets, 95 frozen samples were subjected to PCR amplification and sequencing of the hypervariable region of the 16S rDNA. This identified 11 different shrimp species and two squid species in the frozen shellfish packs. About 40% of calamari brands contained peanut worm, cattle, and rat 16S rDNA. Also, most shellfish species analyzed had low nucleotide diversity, including two shrimp species (Litopenaeus vannamei and Metapenopsis barbata), which had very limited genetic diversity, low raggedness, and an absence of population expansion. Species misnaming, substitution, overexploitation, origin misreporting, and low genetic diversity were found across frozen UAE shellfish samples analyzed, suggesting inspection and monitoring of frozen seafood sold in UAE markets would be appropriate.

Cucurbita pepo Seed Oil Induces Microsatellite Instability and Tegumental Damage to Schistosoma mansoni Immature and Adult Worms In vitro.

BACKGROUND: Schistosomiasis is a tropical parasitic disease treated exclusively with praziquantel (PZQ). However, PZQ has low efficacy against schistosomula and juveniles. In addition, the emergence of PZQ resistance has prompted the search for new drugs. METHODS: This study investigated the effects of pumpkin (Cucurbita pepo)-seed oil (PSO) on Schistosoma mansoni adults, juveniles, and newly formed schistosomula in vitro by exposing the parasites to increasing concentrations of PSO (20, 40, 60, 80, and 100 µl/mL) with variable incubation periods (24, 48, and 72 hours). Dose-response effects of PSO on mortality rate, worm activity, and tegumental changes were studied. Also, effect on DNA were assessed with microsatellite analysis. RESULTS: All tested stages of S. mansoni were susceptible to PSO, which was more effective than PZQ on juvenile worms and schistosomula. Juveniles and schistosomula S. mansoni were more sensitive to the antischistosomal activity of PSO than adult worms. PSO showed evident changes in the integuments of adults, juveniles, and schistosomula. These changes were more evident with increased concentrations. At the genomic level, PSO induced clear qualitative and quantitative changes in the microsatellite loci R95529 and SMD57 of S. mansoni adults and schistosomula. This microsatellite instability is being reported through the current study for S. mansoni in response to PSO for the first time. CONCLUSION: This study suggested that PSO possesses effective antischistosomal activity against various stages of S. mansoni. Further investigations are needed to figure out the mechanism of action of PSO on this parasite.

Daily rhythms in endocrine factors of the somatotropic axis and their receptors in gilthead sea bream (Sparus aurata) larvae.

Living organisms have adapted to environmental oscillations in light and temperature through evolving biological clocks. Biological rhythms are pervasive at all levels of the endocrine system, including the somatotropic (growth) axis. The objective of the present research was to study the existence of daily rhythms on the somatotropic axis of a marine teleost species, specifically, the gilthead sea bream (Sparus aurata). Larvae of S. aurata at 30 dph (days post hatching), kept under a 9 L:15D (light-dark) photoperiod, were collected every 3 h throughout a 36 h cycle. The expression of the following somatotropic axis genes was analyzed by quantitative PCR: pituitary adenylate cyclase-activating polypeptide 1 (adcyap1), prepro-somatostatin-1 (pss1), growth hormone (gh), growth hormone receptor types 1 and 2 (ghr1 and ghr2, respectively), insulin-like growth factor 1 (igf1) and igf1 receptor a (igf1ra). All genes displayed significant differences among time points and, with the exception of adcyap1, all showed statistically significant daily rhythms. The acrophases of gh, ghr1, ghr2, igf1 and igf1ra were located around the end of the dark phase, between ZT19:44 and ZT0:48 h, whereas the highest expression levels of adcyap1 occurred at ZT18 h. On the other hand, the acrophase of pss1, an inhibitor of Gh secretion, was located at ZT10:16 h, hence it was shifted by several hours with respect to the other genes. The present results provide the first thorough description of somatotropic axis rhythms in gilthead sea bream. Such knowledge provides insights into the role of rhythmic regulation of the Gh/Igf1 axis system in larval growth and metabolism, and it can also improve the implementation of more species-specific feeding regimes.

First detection of Anaplasma ovis in sheep and Anaplasma platys-like variants from cattle in Menoufia governorate, Egypt.

Tick-borne diseases are of global economic importance, especially due to the costs associated with disease treatment and productivity losses in livestock. In this study, 244 livestock animals (cattle N = 92, buffaloes N = 86 and sheep N = 66) from Menoufia, Egypt were tested for Anaplasma, Ehrlichia and Babesia species using PCR. Results revealed detection of A. ovis (9.1%) in sheep while Anaplasma spp. (14.1%), A. marginale (15.2%), B. bigemina (6.5%) and B. bovis (5.4%) in cattle. On the other hand, Anaplasma spp. (1.2%), A. marginale (1.2%) and B. bovis (1.2%), were detected in buffaloes. Significantly higher detection rates were observed in cattle for Anaplasma spp. (P = .020), A. marginale (P = .001) and B. bigemina (P = .022) than in buffaloes. Sequence analysis of Anaplasma spp. isolates from cattle, revealed A. platys-like strains. Phylogenetic analyses of the A. platys-like isolates revealed variation among the strains infecting cattle. The A. marginale buffalo isolate, on the other hand, showed some level of divergence from the cattle isolates. This study reports the first detection of A. ovis in sheep and A. platys-like strains in cattle in Menoufia and Egypt at large. The results of the current study provide valuable information on the epidemiology and genetic characteristics of tick-borne pathogens infecting livestock in Egypt.

Molecular and morphological signatures for extreme environmental adaptability of the invasive mussel Brachidontes pharaonis (Fischer, 1870).

Brachidontes pharaonis (Bivalvia:Mytilidae) is one of the most successful Lessepsian migrants. Its extensive populations' expansion and phenotypic plasticity might reshape the Mediterranean biodiversity. Individuals of B. pharaonis were collected from various sites in the Mediterranean Sea and the Red Sea in Egypt. Species-specific primers for Cytochrome Oxidase Subunit 1 gene were designed. They were applied for analysis of mussel's population genetics and assessment of its aquatic environmental DNA (eDNA) abundance. Morphological, allometric and morphometric characteristics were also described. The newly designed primers could efficiently detect the species presence, abundance, and genetic diversity. The Northern Red Sea and north-westward populations exhibited higher nucleotide diversities than southwards. Phylogeny and principal coordinates' analysis (PCoA) detected three geographical categories for B. pharaonis: one of the Indian Ocean, other of the Middle Red Sea and southwards, and the other extends from the Northern Red Sea to the westernmost part of the Mediterranean. Intraspecific differences in the shell shape, colour, and biometrics were noted. The shells were significantly smaller and lighter in rocky habitats than in sandy ones. The morphometric indices and allometry were significantly different between rocky and sandy environments. In general, B. pharaonis genetic and morphological features appeared to contribute much to the species success in versatile habitats.

DNA and eDNA-based tracking of the North African sharptooth catfish Clarias gariepinus.

The African sharptooth catfish, Clarias gariepinus, contributes much to the River Nile ecosystem by its high omnivorosity, sturdiness, growth rates, and fecundity. It was globally appreciated as a key fluvial aquaculture species. Yet, it is also one of the top world freshwater aliens. Monitoring the genetic diversity of different economically and ecologically important species as well as development of markers that aid their tracing and abundance are fundamental. This is chiefly due to the growing international threats of environmental pollution, reduction, and loss of biodiversity. Herein, the genetic diversity of C. gariepinus along the River Nile in Egypt was assessed through sequencing of the mitochondrial cytochrome oxidase subunit I (COI). Also, a qPCR assay based on C. gariepinus 16srDNA was developed to assess the species abundance through environmental water DNA samples (eDNA). The results showed low genetic diversity of that species in Egypt. Moreover, its populations exhibited high rates of fixation. Testing its eDNA-based marker resulted in an unambiguous quantitative trend in situ, in agreement with reports of local fishermen. These eDNA signals were strong at least 1 Km upstream to the initial sampling areas, even where no C. gariepinus fishing activities are carried out. This possibly indicated a degree of homogenous species-abundance in each of the studied areas. Finally, the results identified a need for better conservation strategies for C. gariepinus, since its low diversity in the Egyptian River Nile may represent a threat against its persistence under the continuously changing environmental conditions. Moreover, using non-invasive sampling methods, e.g. based on aquatic eDNA quantification, can aid much the detection of areas of abundance of C. gariepinus, especially for both the economic importance it contributes and the invasive power it possesses.

Mitochondrial genetic markers for authentication of major Red Sea grouper species (Perciformes: Serranidae) in Egypt: A tool for enhancing fisheries management and species conservation.

Groupers are coral fish species of prime ecological and economic significance. The interactions among them and other coral reefs organisms aid the healthiness and species balance in this fundamental marine niches. Also, groupers are among the top priced fisheries species. The Egyptian habitats of the Red Sea are lacking genetic studies that assess species diversity for the final goal of conservation and fisheries management. Moreover, morphological similarities among these organisms sometimes hinder a proper species identification. Hence, more accurate groupers authentication methods are crucially required. Sixteen grouper species belonging to the genera Epinephelus, Anyperodon, Cephaolopholes, Aethaloperca, Variola, and Plectropomus, present in the Red Sea in Egypt, were investigated for species authentication through mitochondrial DNA variations, applying cytochrome oxidase subunit I (COI) and 12srRNA genes sequencing. GenBank comparisons, phylogenetic analyses and comparisons of pairwise distances were carried out. All these analyses aimed to species authentication and identifying their relations at the international scale. The results exhibited >98% identity with E. fasciatus, A. rogaa, C. oligosticta, E. areolatus, V. louti, P. areolatus, E. malabaricus, C. sexmaculata, E. summana, E. chlorostigma, E. polyphekadion, C. miniataus, A. leucogrammicus, E. tauvina, C. argus, C. hemistiktos. Pairwise distances showed a clear increase upon raising comparison level from among species to among-genera. Combined 12srRNA and COI genes sequencing resulted in an accurate tool for Egyptian Red Sea grouper species unambiguous discrimination. This can provide vital aid to the active efforts for these species conservation and fisheries management in Egypt and the world.

Morphological and molecular clues for recording the first appearance of Artemia franciscana () in Egypt.

Artemia franciscana is a native species to the New World, and became an exotic species to most parts of the world. The Egyptian hypersaline, continental Qaroun Lake (Fayoum Governorate, Middle of Egypt) is subjected to a gradually increasing salinity rates that approximate or exceed these of seawater. Artemia populations there are known to be parthenogenetic. Yet, these populations started to exhibit abnormal morphologies. Therefore, Qaroun Lake samples of Artemia were subjected to several morphological, biometric, and molecular phylogenetic analyses for accurate species identification and phylogeographic origin approximation. These analyses revealed the existence of the alien sexual species of brine shrimp A. franciscana in Qaroun Lake. The characteristics of the subspherical frontal knob with several spines on the top, ovisac lateral triangular lobe on both sides and its projection together with the biometrics confirmed this species morphotype. DNA barcoding and other molecular analyses based on PCR-based amplification and sequencing of the barcode region of the cytochrome oxidase subunit I gene (COI) exhibited that all the collected samples belong to five haplotypes. Egyptian A. franciscana COI sequences phylogeny and pairwise distances analysis exhibited closer proximity to Latin American strains than to the Northern American ones. A. franciscana presence may be ascribed to the migratory birds present in Qaroun Lake protectorate, since no marine aquaculture activity in Qaroun Lake is known. Therefore, and for the best of our knowledge, this is the first record of the invasive A. franciscana in Egypt.

Molecular performance of Prl and Gh/Igf1 axis in the Mediterranean meager, Argyrosomus regius, acclimated to different rearing salinities.

Aquaculture industry in the Mediterranean region exhibits a growing interest for the Mediterranean meager Argyrosomus regius. Some preliminary works showed a good growth performance of the species in nearly isosmotic salinities. However, the patterns of alteration of prolactin (Prl) as well as growth hormone (Gh)/insulin growth factor-1 (Igf1) axis at the molecular level are not yet described in this species. Therefore, we cloned and sequenced partial cDNAs for pituitary prolactin (prl) and growth hormone (gh), hepatic insulin-like growth factor (igf1), and ß-actin (actb). Expression patterns of these transcripts were tested in juveniles of A. regius acclimated to four different environmental salinities: (1) 5 ‰ (hyposmotic); (2) 12 ‰ (isosmotic); (3) 38 ‰ (hyperosmotic; seawater control); and (4) 55 ‰ (extremely hyperosmotic). All investigated transcripts shared high sequence identities with their counterparts in other perciformes. prl mRNA levels showed inverse pattern with increasing salinities. gh mRNA enhanced significantly in both 12 and 55 ‰ salinity groups in comparison with the control group, while igf1 showed its maximum expression levels under the nearly isosmotic environment. The results indicated clear sensitivity of prl, gh and igf1 to changes in environmental salinity, which can possibly control the euryhalinity capacity of this species.

PCR-based assessment of shellfish traceability and sustainability in international Mediterranean seafood markets.

Two mitochondrial markers (cytochrome oxidase COI and 16S rDNA) were employed for species identification of commercial shellfish from two Mediterranean countries. New COI Barcodes were generated for six species: Pleoticus robustus, Metapenaeopsis barbata, Parapenaeus fissuroides, Hymenopenaeus debilis, Metapenaeus affinis and Sepia aculeata. Biodiversity of the seafood species analyzed was greater in Egypt, with nine crustacean and two cephalopod species found compared with only three crustaceans and three cephalopods in Spain. In total, 17.2% and 15.2% products were mislabeled in Egypt and Spain, respectively. Population decline is a problem for some of the substitute species. Others were exotic and/or invasive in exporters' regions. This study offers the first comparable study of shellfish traceability in these Mediterranean markets. The PCR-based method used in this study proved to be reliable, effective and, therefore, could be employed for routine seafood analysis.