Morphological and molecular characterization of Eimeria magna infecting local rabbit (Oryctolagus cuniculus) in Alkarg City, Saudi Arabia.

Coccidiosis is one of the most prevalent diseases found in local rabbits (Oryctolagus cuniculus), which is caused by the Eimeria. The study aimed to more reliably identify Eimeria species (Eimeria magna) infecting Local Rabbits in Alkarg City, Saudi Arabia, based the method on the molecular properties and morphological and molecular biological techniques. Sub-spheroidal oocysts measuring 21-27 × 12-16 (24 × 14.4) µm (20 n) and with a length/width (L/W) ratio of 0.9-1.1 (1.0) were identified by microscopic analysis of a fecal sample. Oocysts feature a bi-layered wall that is 1.0-1.2 (1.1) µm thick. About two-thirds of the wall's thickness is made up of a smooth outer layer. A polar granule is present, but neither a micropyle nor an oocyst residuum is present. The ovoidal sporozoites measure 15-18 × 8-11 (16.5 × 9.5) µm, have an L/W ratio of 1.6-1.8 (1.7), and take up around 21% of the oocyst's total surface. The mean size of the sub-Stieda body is 1.4 × 2.3 µm, while the average size of the Stieda body is 0.9 × 1.8 µm. The para-Stieda body is lacking. Sporocyst residuum appears membrane-bound and has an uneven form made up of several granules. With two refractile bodies below the striations and pronounced striations at the more pointed end, sporozoites are vermiform, measuring an average of 11.6 × 4.0 µm. The results of the sequencing for the 18S rDNA gene confirmed the species of Eimeria parasites found in the host (rabbits). The current parasite species is closely related to the previously described and deposited E. magna and deeply embedded in the genus Eimeria (family Eimeriidae). According to the findings, single oocyst molecular identification of Eimeria may be accomplished through consistent use of the morphological and molecular results. It is possible to draw the conclusion that the current research supplies relevant facts that help assess the potential infection and future control measures against rabbit coccidiosis to reduce the financial losses that can be incurred by the rabbit industry in Saudi Arabia.

Ultrastructural and molecular characterization of Glugea sp. (microsporidia), a parasite of the Red Sea fish Carangoides bajad (Carangidae).

Glugea sp. found infecting the liver of the teleost fish Carangoides bajad from the Red Sea, Egypt, is described based on light microscopy and ultrastructural characteristics combined with phylogenetic analyses. This microsporidium forms whitish xenomas up to ~4 mm in size. Xenomas display numerous parasitophorous vacuoles totally filled by mature spores, no other life cycle stages were observed. Mature spores ellipsoidal and measuring 6.3 × 4.0 µm in size. The polaroplast appears composed of two distinct regions: an electron-dense vesicular region and a densely packed lamellar region. The polar tubule forms approximately 24-27 coils arranged in three layers encircling the posterior vacuole. The small subunit (SSU) rRNA gene and its ITS region were sequenced and showed the highest similarity of 99.4% to other Glugea spp. Bayesian inference and maximum likelihood analyses place the novel isolate within the Glugea clade, more specifically within a subclade that predominantly grouped species described from fish inhabiting the Arabian Gulf or Red Sea. The results validate the parasite's classification in the Glugea genus. Nevertheless, until more detailed ultrastructural and molecular data are obtained, the identification of the current Glugea species is hampered by the absence of some developmental stages and the high degree of genetic similarity.

An investigation of the acaricidal activity of benzyl alcohol on Rhipicephalus annulatus and Rhipicephalus sanguineus and its synergistic or antagonistic interaction with commonly used acaricides.

The most economically significant ectoparasites in the tropics and subtropics are ixodid ticks, especially Rhipicephalus annulatus and Rhipicephalus sanguineus. Years of extensive use of the readily available acaricides have resulted in widespread resistance development in these ticks, as well as negative environmental consequences. Benzyl alcohol (BA) has been frequently used to treat pediculosis and scabies, and it may be an effective alternative to commonly used acaricides. The main aim of the present study was to evaluate the acaricide activity of BA and its combination with the regularly used chemical acaricides against R. annulatus and R. sanguineus. Different concentrations of BA alone and in combination with deltamethrin, cypermethrin and chlorpyrifos were tested in vitro against adult and larvae of both tick species. The results showed that BA is toxic to R. annulatus and R. sanguineus larvae, with 100% larval mortality at concentrations of ≥50 mL/L, and LC50 and LC90 attained the concentrations of 19.8 and 33.8 mL/L for R. annulatus and 18.8 and 31.8 mL/L for R. sanguineus, respectively. Furthermore, BA in combination with deltamethrin, cypermethrin and chlorpyrifos exhibited synergistic factors of 2.48, 1.26 and 1.68 against R. annulatus larvae and 1.64, 11.1 and 1.14 against R. sanguineus larvae for deltamethrin + BA, cypermethrin + BA and chlorpyrifos + BA, respectively. BA induced 100% mortality in adult R. annulatus at concentrations of ≥250 mL/L with LC50 and LC90 reached the concentrations of 111 and 154 mL/L, respectively. Additionally, BA had ovicidal activity causing complete inhibition of larval hatching at 100 mL/L. The combination of BA with deltamethrin and cypermethrin increased acetylcholinesterase inhibition, whereas the combination of BA with chlorpyrifos decreased glutathione (GSH) activity and malondialdehyde levels. In the field application, the combination of BA 50 mL/L and deltamethrin (DBA) resulted in a significant reduction in the percentage of ticks by 30.9% 28 days post-treatment when compared with groups treated with deltamethrin alone. In conclusion, BA causes mortality in laboratory and field studies alone and in combination with cypermethrin or deltamethrin. BA can be used for control of ticks of different life stages, that is, eggs and larvae, through application to the ground.

Potential role of the methanolic extract for Olea europaea (stem and leaves) on sporulation of Eimeria papillata oocysts: In vitro study.

Many plants are efficient anticoccidial agents owing to their content of active chemicals. Drug-resistant Eimeria species have emerged as a result of excessive drug use. The current work aimed to investigate the oocysticidal activity (Eimeria papillata) of Olea europaea stem extract (OESE) and leaf extract (OELE) in vitro. The results of gas chromatography-mass spectrometry analysis for OELE and OESE showed the presence of 12 and 9 phytochemical compounds, respectively. Also, chemical examination revealed that the plant extracts are rich in phenols, flavonoids and tannins. Additionally, the best radical scavenging activity of OESE and OELE was at a concentration of 100 µg/ml, reaching 92.04 ± 0.02 and 92.4 ± 0.2%, respectively. The in vitro study revealed that concentrations of 200 mg/ml from OESE and OELE caused significant inhibition (100%) of process sporulation for E. papillata oocysts, in contrast to the other commercial products, which displayed varying degrees of suppression sporulation. Our findings showed that OESE and OELE have anticoccidial activity, which motivates further the conduction of in vivo studies in the search for a less expensive and more efficient cure.

Repurposing the drug, amprolium as a novel molluscicide against the land snail (Eobania vermiculata).

Amprolium (AMP) is an organic compound used as a poultry anticoccidiostat. The aim of this work is to repurpose AMP to control the land snail, Eobania vermiculata in the laboratory and in the field. When snails treated with ½ LC50 of AMP, the levels of alkaline phosphatase (ALP), total lipids (TL), urea, creatinine, malondialdehyde (MDA), catalase (CAT), and nitric oxide (NO) were significantly increased, whereas the levels of acetylcholinesterase (AChE), total protein (TP), and glutathione (GSH) decreased. It also induced histopathological and ultrastructural changes in the digestive gland, hermaphrodite gland, kidney, mucus gland, and cerebral ganglion. Furthermore, scanning electron micrographs revealed various damages in the tegumental structures of the mantle-foot region of E. vermiculata snails. The field application demonstrated that the AMP spray caused reduced percentages in snail population of 75 and 84% after 7 and 14 days of treatment. In conclusion, because AMP disrupts the biology and physiology of the land snail, E. vermiculata, it can be used as an effective molluscicide.

Repellency, Fumigant Toxicity, Antifeedent and Residual Activities of Coridothymus capitatus and Its Main Component Carvacrol against Red Flour Beetle.

Tribolium castaneum is a challenging pest of stored products, causing significant economic losses. The present study explored the efficacy of Coridothymus capitatus essential oil and its primary constituent, carvacrol, as eco-friendly alternatives for managing this pest. To evaluate their insecticidal potential, repellency, fumigant toxicity, and antifeedant properties, progeny inhibition assays were performed. Carvacrol exhibited superior repellency compared to the essential oil, achieving a 92% repellency rate at 2 mg/cm2. Both compounds demonstrated significant fumigant toxicity against T. castaneum, with LC50 values of 168.47 and 106.5 µL/L for the essential oil and carvacrol, respectively, after 24 h. Carvacrol also outperformed the essential oil in antifeedant activity, inducing an 80.7% feeding deterrence at 1.17 mg/g. Moreover, both treatments effectively suppressed the development of the pest's progeny. These results collectively underscore the potent insecticidal properties of C. capitatus essential oil and carvacrol, particularly carvacrol, as promising candidates for the sustainable management of T. castaneum in stored product protection.

Identification of Eimeria acervulina (Apicomplexa: Eimeriidae) infecting the broiler chicken Gallus gallus domesticus through morphology and molecular analyses.

Coccidiosis is an intestinal protozoan disease that affects the poultry industry worldwide. The severity of this disease varies depending on the identity of the infectious agents. Therefore, this study was carried out to identify the Eimeria species that affect broiler chickens, Gallus gallus domesticus, through morphological and molecular phylogenetic analyses. Twenty-five faecal samples were collected from the broiler chickens in a commercial poultry farm in Riyadh (Saudi Arabia). Using the floatation technique, faeces were examined microscopically for the Eimeria occurrence. Identification of Eimeria species was performed based on morphological criteria and molecular tools (DNA amplification for the partial small subunit ribosomal RNA (18S rRNA), internal transcribed spacer (ITS)-1, and mitochondrial cytochrome c oxidase I (COI) genes. In this study, 32% (8 out of 25) of collected samples were found to be positive for coccidiosis. After sporulation in potassium dichromate (K2Cr2O7), the sporulated oocysts were observed as ovoid and measured 18.37-23.19 µm (19.87) long and 15.07-18.67 µm (16.46) wide, with the anterior location of a polar granule and absence of micropyle. These Eimeria oocysts were assumed to size and shape characteristics of Eimeria acervulina. Molecular analysis was conducted on the sequences of the polymerase chain reaction products from the three genes studied (18S rRNA, ITS-1, and COI). At the three genes, results showed that the resultant sequences clustered with E. acervulina from different regions confirming morphological description. This study highlighted the importance of molecular techniques to detect avian Eimeria species more than the traditional morphology-based tool to optimise the appropriate anticoccidial strategies for long-term control in the studied area.

Identification of sheep eimerian parasites, Eimeria crandallis and Eimeria faurei, employing microscopic and molecular tools.

Coccidiosis, caused by apicomplexan Eimeria species, is a protozoan disease that affects various species of wild and domestic animals. However, data available on Eimeria diversity in ruminants in Saudi Arabia is meagre. Therefore, this study was designed to investigate some eimerian parasites infecting sheep (Sawakni and Harrie breeds) using microscopy and molecular methods for the first time in Saudi Arabia. Twenty-four fecal samples were collected from sheep farms. Based on the floatation technique, eimerian oocysts were observed in 8 of the 24 (33.33%) fecal samples. The coccidian-positive samples were subjected to fecal culture in a shallow layer of 2.5% potassium dichromate (K2 Cr2 O7 ). Detected eimerian oocysts were described micromorphometrically as the basis for traditional oocyst identification. Morphologically, the sporulated oocysts were similar to those of sheep eimerian parasies; Eimeria faurei and Eimeria crandallis. PCR products from the two eimerian species detected from Sawakni and Harrie breeds were sequenced and were found to be distinct from each other with mutations at five positions. One of them clustered with E. crandallis with 99.8%-100% identity with sequences available in GenBank. E. crandallis was obtained from two Sawakni sheep and two Harrie sheep. The other sequences grouped with E. faurei with 99.8% identity with the only sequences available in GenBank. E. crandallis was detected from both Sawakni and Harrie breeds whereas E. faurei was detected only from Sawakni sheep. The findings of this study have implications for the importance of morphometric identification with advanced molecular tools to confirm the identities of sheep Eimeria species and to address the taxonomic study of this eimeriid parasite at the species level.

Morphological and mitochondrial approaches of Hatschekia sargi (Copepoda: Hatschekiidae) as a parasite of Epinephelus chlorostigma.

Little information is available until now about the copepods infecting different fish species. Therefore, this study aimed to provide light on siphonostomatoids infecting Epinephelus chlorostigma. Twenty fish specimens were taken from the Red Sea coast (Jeddah, Saudi Arabia), and ectoparasitic copepods were investigated. Light microscopy and molecular tools were used to examine copepods isolated from fish. Parasitological indexes were calculated and showed that 60% of the examined fish were infected with a mean intensity of 12 parasite/fish. Morphological examination revealed that this copepod species is characterized by all unique features of the genus Hatschekia with special reference to Hatschekia sargi. The taxonomic position of the recovered species in the Hatschekiidae family within Siphonostomatoida was confirmed using phylogenetic analysis based on partial mitochondrial cytochrome c oxidase subunit I (mt COI) gene sequences. The mt COI gene query revealed that the recovered Hatschekia species is closely related to Hatschekia maculatus (gb| JQ664005.1). This study discovers a new host for Hatschekia species isolated from Saudi Arabia and conducts the first genomic investigation of the mt COI gene.

Silencing of heat shock protein 90 (hsp90): Effect on development and infectivity of Ichthyophthirius multifiliis.

BACKGROUND: Recently, an increasing number of ichthyophthiriasis outbreaks has been reported, leading to high economic losses in fisheries and aquaculture. Although several strategies, including chemotherapeutics and immunoprophylaxis, have been implemented to control the parasite, no effective method is available. Hence, it is crucial to discover novel drug targets and vaccine candidates against Ichthyophthirius multifiliis. For this reason, understanding the parasite stage biology, host-pathogen interactions, molecular factors, regulation of major aspects during the invasion, and signaling pathways of the parasite can promote further prospects for disease management. Unfortunately, functional studies have been hampered in this ciliate due to the lack of robust methods for efficient nucleic acid delivery and genetic manipulation. In the current study, we used antisense technology to investigate the effects of targeted gene knockdown on the development and infectivity of I. multifiliis. Antisense oligonucleotides (ASOs) and their gold nanoconjugates were used to silence the heat shock protein 90 (hsp90) of I. multifiliis. Parasite stages were monitored for motility and development. In addition, the ability of the treated parasites to infect fish and cause disease was evaluated. RESULTS: We demonstrated that ASOs were rapidly internalized by I. multifiliis and distributed diffusely throughout the cytosol. Knocking down of I. multifiliis hsp90 dramatically limited the growth and development of the parasite. In vivo exposure of common carp (Cyprinus carpio) showed reduced infectivity of ASO-treated theronts compared with the control group. No mortalities were recorded in the fish groups exposed to theronts pre-treated with ASOs compared with the 100% mortality observed in the non-treated control fish. CONCLUSION: This study presents a gene regulation approach for investigating gene function in I. multifiliis in vitro. In addition, we provide genetic evidence for the crucial role of hsp90 in the growth and development of the parasite, suggesting hsp90 as a novel therapeutic target for successful disease management. Further, this study introduces a useful tool and provides a significant contribution to the assessing and understanding of gene function in I. multifiliis.

Persea americana extract protects intestinal tissue from Eimeria papillata-induced murine Infection.

Coccidiosis is the most prevalent disease-causing widespread economic loss among farm and domestic animals. Currently, several drugs are available for the control of this disease but resistance has been confirmed for all of them. There is an urgent need, therefore, for the identification of new sources as alternative treatments to control coccidiosis. The present work aimed to study the effect of the Persea americana extract (PAE) as an anti-coccidial, anti-oxidant, and anti-apoptotic modulator during murine intestinal Eimeria papillata infection. A total of 25 male mice were divided into five groups, as follows: Group1: Non-infected-non-treated (negative control), Group2: Non-infected-treated group with PAE (500 mg/kg b.w). Group3: Infected-non-treated (positive control), Group4: Infected-treated group with PAE (500 mg/kg b.w.), and Group5: Infected-treated group with Amprolium (120 mg/kg b.w.). Groups (3-5) were orally inoculated with 1 × 103 sporulated E. papillata oocysts. After 60 min of infection, groups (4 and 5) were treated for 5 consecutive days with the recommended doses of PAE and amprolium. The fact that PAE has an anti-coccidial efficacy against intestinal E. papillata infection in mice has been clarified by the reduction of fecal oocyst output on the 5th day post-infection by about 85.41%. Moreover, there is a significant reduction in the size of each parasite stage in the jejunal tissues of the infected-treated group with PAE. PAE counteracted the E. papillata-induced loss of glutathione peroxidase (GPx), superoxide dismutase (SOD), and total antioxidant capacity (TCA). E. papillata infection also induced an increase in the apoptotic cells expressed by caspase-3 which modulated after PAE treatment. Moreover, the mRNA expression of the goblet cell response gene, mucin (MUC2), was upregulated from 0.50 to 1.20-fold after treatment with PAE. Based on our results, PAE is a promising medicinal plant with anti-coccidial, anti-oxidant, and anti-apoptotic activities and could be used as a food additive.

D-limonene nanoemulsion: lousicidal activity, stability, and effect on the cuticle of Columbicola columbae.

The current study was conducted to investigate the efficacy and stability of D-limonene (DL) and its nanoemulsion (DLN) against pigeon feather lice (Columbicola columbae) and their mode of action. DL pure form and DLN were prepared and characterized freshly and after storage for 50 days. In vitro bioassay on live lice was conducted with different concentrations of DL, DLN, and deltamethrin (DM). The results revealed significant mortality rates in the DL-, DLN-, DM-treated groups when compared with the control (p < 0.05). The scanning electron micrographs of lice treated with DL and DLN revealed collapsed bodies with destruction in the cuticle of the mouthparts and damaged antennae. The 50 days stored DLN showed stability in their effectiveness when compared with the freshly prepared formulation. DL and DLN caused significant inhibition (p ≤ 0.05) in acetylcholinesterase activity (AchE). Malondialdehyde level (MDA) was significantly increased while glutathione was significantly decreased in DL- and DLN-treated lice. In conclusion, DL and DLN have significant lousicidal activities. DLN showed better stability than DL after storage for 50 days. In addition, the mode of action of DL may associate with its effect on the cuticle of the lice body, inhibition of AchE, and increasing oxidative stress in the treated lice.

In Vitro: The Effects of the Anticoccidial Activities of Calotropis procera Leaf Extracts on Eimeria stiedae Oocysts Isolated from Rabbits.

Hepatic coccidiosis is an infectious and mortal disease that causes global economic losses in rabbits. The research aimed to assess the efficacy of Calotropis procure leaf extracts on the inhibition of Eimeria stiedae oocysts and to determine the optimal dosage for suppressing the parasite's infective phase. In this experiment, oocyst samples per milliliter were tested, and 6-well plates (2 mL) of 2.5% potassium dichromate solution containing 102 non-sporulated oocysts on Calotropis procera leaf extracts were exposed after 24, 48, 72, and 96 h, and the treatments were as follows: a nontreated control, 25%, 50%, 100%, and 150% of C. procera for oocyst activities. In addition, amprolium was utilized as a reference drug. The Calotropis procera was analyzed by GC-Mass, and results showed that the botanical extract contained 9 chemical components that were able to inhibit the oocysts of E. stiedae at 100% and 150% concentrations by about 78% and 93%, respectively. In general, an increase in the incubation period and a greater dose resulted in a decrease in the inhibition rate. The results showed that C. procera has an effective ability, inhibitory potential, and protective effect on the coccidian oocyst sporulation of E. stiedae. It can be used in the disinfection and sterilization of poultry and rabbit houses to get rid of Eimeria oocysts.

Administration of Ethanolic Extract of Spinacia oleracea Rich in Omega-3 Improves Oxidative Stress and Goblet Cells in Broiler Chickens Infected with Eimeria tenella.

This study investigated the anticoccidial activity of spinach (Spinacia oleracea) whole-plant extract against Eimeria tenella, both in vitro and in vivo. For this purpose, one hundred 8-day-old broiler chicks of both sexes were divided into four groups (n = 25 in each group). Chicks in the first group served as the negative control (non-treated-non-infected). Chicks in the second group were challenged at 18 days old with 5 × 104E. tenella sporulated oocysts. The third group was challenged with 5 × 104 sporulated E. tenella oocysts at 18 days old after receiving spinach extract at a dose of 50 mg/kg at 8 days old. The fourth group received 0.2 mg/kg diclazuril (Coxiril® 0.2%) in their diet two days before being orally infected with 5 × 104 sporulated E. tenella oocysts and this continued till day 10 post-infection (PI). The growth performance, clinical symptoms, oocyst shedding, histological findings, and biochemical parameters were used to evaluate the efficacy on day 8 PI when the infection was at its peak. A gas chromatography examination revealed that omega-3 fatty acids were the main constituents of the spinach extract, followed by oleic acid, palmitic acid, and phytol, with amounts of 23.37%, 17.53%, 11.26%, and 7.97%, respectively. The in vitro investigation revealed that the spinach extract at concentrations of 10% and 5% inhibited the oocyst sporulation by 52.1% and 45.1%, respectively. The 5% concentration was selected for the in vivo trial based on the results of the in vitro study. The infected-untreated group showed high levels of OPG; lower body weight; a greater number of parasite stages; few goblet cells; decreased SOD, CAT, and GPX levels; and increased MDA and NO levels. The spinach-treated group, on the other hand, showed a significant decrease in oocyst output per gram of feces (OPG), increased body weight, decreased parasitic stages, and a nearly normal number of goblet cells. Additionally, it reduced malondialdehyde (MDA) and nitric oxide (NO), while increasing superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX). In conclusion, spinach produced significant antioxidant effects, increased body weight, reduced the number of oocysts and parasite stages in the caecum, and restored the number of goblet cells relative to those of an uninfected control. Furthermore, spinach extract inhibits the sporulation percentage of E. tenella oocysts. The ethanolic extract of S. oleracea (whole plant) contained high concentrations of fatty acids, palmitic acid, Phytol, betulin, and ursolic aldehyde, all of which are known to regulate the antioxidant pathway and modulate inflammatory processes and may be the main reason for its anticoccidial activity.

Toxicity and Repellency Efficacy of Benzyl Alcohol and Benzyl Benzoate as Eco-Friendly Choices to Control the Red Flour Beetle Tribolium castaneum (Herbst. 1797).

Tribolium castaneum is a damaging pest of stored grains, causing significant losses and secreting lethal quinones, which render the grains unfit for human consumption. Chemical insecticides are the most commonly used approach for control; however, they create insecticide resistance and affect the health of humans, animals, and the environment. As a result, it is critical to find an environmentally friendly pest-management strategy. In this study, two naturally occurring chemicals, benzyl alcohol (BA) and benzoyl benzoate (BB), were investigated for insecticidal activity against T. castaneum using different assays (impregnated-paper, contact toxicity, fumigant, and repellency assays). The results showed that BA had a significant insecticidal effect, with the LC50 achieved at a lower concentration in the direct-contact toxicity test (1.77%) than in the impregnated-paper assay (2.63%). BB showed significant effects in the direct-contact toxicity test, with an LC50 of 3.114%, and a lower toxicity in the impregnated-paper assay, with an LC50 of 11.75%. Furthermore, BA exhibited significant fumigant toxicity against T. castaneum, with an LC50 of 6.72 µL/L, whereas BB exhibited modest fumigant toxicity, with an LC50 of 464 µL/L. Additionally, at different concentrations (0.18, 0.09, 0.045, and 0.0225 µL/cm2), BA and BB both showed a notable and potent repelling effect. BA and BB significantly inhibited acetylcholinesterase, reduced glutathione (GSH), and increased malondialdehyde (MDA) in treated T. castaneum. This is the first report of BA insecticidal activity against the red flour beetle. Also, the outcomes of various assays demonstrated that the application of BA induces a potent bio-insecticidal effect. BA may be a promising eco-friendly alternative to control T. castaneum due to its safety and authorization by the EFSA (European Food Safety Authority).

Molecular identification of the rodent-borne pathogen Rodentolepis nana using the genetic markers of ITS-1, 18 S, and 28 S rDNA.

BACKGROUND: Rodentolepis nana (syn. Hymenolepis nana), the most common cyclophyllid tapeworm infecting rodents, is a well-studied gastrointestinal parasite in mice and belongs to the family Hymenolepididae. METHODS: The present study focuses on the molecular analysis for the nuclear genes (ITS-1, 18 S, and 28 S rDNA) used for the accurate recognition of the recovered Rodentolepis species. RESULTS: The annotated partial ITS-1, 18 S, and 28 S rDNA gene regions were deposited in GenBank (gbǀ MW310394.1, gbǀ MW327585.1, and gbǀ MW324479.1, respectively) and further used in the maximum likelihood method (ML) to clarify their genetic relationships at the species level. The interrogation sequence of R. nana was aligned and belonged to the family Hymenolepididae, in the same group as all Hymenolepis species, which were distinct from Cyclophyllidea cestodes, especially species belonging to Anoplocephalidae and Taeniidae. Sequence data support the paraphyly of Hymenolepis species. CONCLUSIONS: The phylogeny supports the availability of the ITS-1, 18 S, and 28 S rDNA genes as reliable genetic markers for evolutionary relationships.

Global taxonomic and phylogenetic assembly of AM fungi.

Arbuscular mycorrhizal (AM) fungi are a ubiquitous group of plant symbionts, yet processes underlying their global assembly - in particular the roles of dispersal limitation and historical drivers - remain poorly understood. Because earlier studies have reported niche conservatism in AM fungi, we hypothesized that variation in taxonomic community composition (i.e., unweighted by taxon relatedness) should resemble variation in phylogenetic community composition (i.e., weighted by taxon relatedness) which reflects ancestral adaptations to historical habitat gradients. Because of the presumed strong dispersal ability of AM fungi, we also anticipated that the large-scale structure of AM fungal communities would track environmental conditions without regional discontinuity. We used recently published AM fungal sequence data (small-subunit ribosomal RNA gene) from soil samples collected worldwide to reconstruct global patterns in taxonomic and phylogenetic community variation. The taxonomic structure of AM fungal communities was primarily driven by habitat conditions, with limited regional differentiation, and there were two well-supported clusters of communities - occurring in cold and warm conditions. Phylogenetic structure was driven by the same factors, though all relationships were markedly weaker. This suggests that niche conservatism with respect to habitat associations is weakly expressed in AM fungal communities. We conclude that the composition of AM fungal communities tracks major climatic and edaphic gradients, with the effects of dispersal limitation and historic factors considerably less apparent than those of climate and soil.

Synergistic larvicidal and repellent effects of essential oils of three Origanum species on Rhipicephalus annulatus tick.

Ticks are of great economic importance worldwide, both because they represent major obstacles to livestock productivity and because of their ability to transmit diseases to humans and animals. Although synthetic acaricides are the most common method for tick control, their overuse has led to the development of resistance as well as unacceptable residual levels in animal products and in the environment in general. There is therefore an urgent need to identify alternative treatments. Among such alternative approaches for tick control is plant essential oil (EO) therapy. In the present study, we investigated the synergistic effect of EOs of three oregano species-Origanum onites, O. majorana and O. minutiflorum-against Rhipicephalus annulatus larvae. Gas chromatography-mass spectrometry profiles of the three EOs revealed that carvacrol was their major component, with a concentration of 86.2% in O. majorana, 79.1% in O. minutiflorum and 77.4% in O. onites. The results of larvicidal assays revealed that the doses that lead to the death of 50% of the ticks (LC50) were 22.99, 25.08 and 27.06 µL/mL for O. majorana, O. minutiflorum and O. onites EOs, respectively, whereas the doses that lead to the death of 99% (LC99) were 41.26, 43.62 and 48.96 µL/mL. In addition, the LC50 and LC99 of the three oils combined was lower (viz., 4.01 and 6.97 µL/mL) than that of each oil alone. The tested EOs were also able to repel larvae of R. annulatus to varying degrees, with O. onites oil exhibiting the greatest repellent effect, as shown by the lowest RC50 dose, followed by O. minutiflorum and O. majorana. Interestingly, this means that the oil that was least effective in killing the larvae was the most effective in repelling them. The calculated synergistic factor of any combination was higher than 1 which means that combinations have a synergistic effect. In conclusion, the combination of all three oils showed higher toxic and repellent activities than either oil separately or combinations of any two oils, suggesting synergistic effects with low doses. Further studies including field trials and the establishment of the mode of action and side effects are urgently needed to expand on these findings, and other tick stages such as adults should also be tested.

Role of antioxidant activity of essential oils in their acaricidal activities against Rhipicephalus annulatus.

Essential oils of Origanum majorana and Satureja thymbra as well as carvacrol are natural products that are known to have potent antioxidant activities. The current study was designed to investigate the role of the antioxidant properties of these natural products in their acaricidal activities against Rhipicephalus annulatus larvae. The synergistic and/or antagonistic effects of the addition of vitamins E and C and hydrogen peroxide (H2O2) to these natural products were also evaluated. Larval packet tests were used to evaluate the acaricidal activities against the larvae of R. annulatus. The antioxidant effectiveness of these products was determined by a DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay. The addition of vitamin E at 100 mg/mL to O. majorana and S. thymbra decreased the concentrations required to achieve the death of half of the larvae (LC50) to 0.44 and 0.47%, respectively. The combination of O. majorana and S. thymbra attained the LC50 at 1.54% which was decreased to 0.69% after addition of vitamin E. Also, the addition of vitamin E to carvacrol reduced the LC50 to 0.27%. The total antioxidant activity of these natural products increased significantly in presence of vitamin E. The addition of H2O2 inhibited the acaricidal activity of all tested materials, especially at low concentrations. All treatments induced an increase in lipid peroxidation, whereas carvacrol-treated larvae revealed the lowest values for the superoxide dismutase. Glutathione peroxidase and catalase activity decreased in larvae treated with S. thymbra combined with vitamin E. In conclusion, the addition of vitamins E and C increased the acaricidal activities of the tested compounds, whereas the addition of H2O2 decreased these activities. The antioxidant activities of essential oils and their active components may play an important role in mediating their acaricidal activities.

Long-Term Exposure to the Water of Wadi El-Rayan Lakes Induced Testicular Damage and Endocrine Disruption in Mugil cephalus.

This study aimed to investigate the testicular function of Mugil cephalus that inhabit Wadi El-Rayan lakes. Testes of fish inhabiting the upper lake (site 2) and the lower lake (site 3) of Wadi El-Rayan showed significant decreases in gonadosomatic index, high accumulation levels of six metals, and eight organochlorine pesticide residues. Compared to reference fish, high percentages of histological alterations as testicular degeneration, germ cell reduction, testicular inflammation, vacuolization, and loss of tubular arrangement were observed in sites 2 and 3. Moreover, endocrine disruption signs were recorded based on the percentage of ovotestis appearance and the ovotestis severity index values. The maximum defective testicular antioxidant mechanisms were recorded in site 3 as indicated by sharp decreases in catalase, superoxide dismutase, glutathione reduced levels, and high thiobarbituric acid reactive substances. Finally, long-term exposure to Wadi El-Rayan water may impair the reproductive health of fish via testicular oxidative damage and endocrine disruption.