Deciphering pyrethroid resistance in Cx. pipiens (L): Implications of cytochrome P450; expression profiling and regulatory microRNA, Fahmy, Nermeen, Egiza Ahmed, Badr Mohamed, Morcos Nadia, Diclaro Joseph, and Abd-ElSamie Emtithal , 2020/04/01, Volume 52, p.101579, (2020) Abstract

Over the past decades, the extensive use of pyrethroids insecticides for vector control has resulted in the development of insecticide resistance. Cytochrome P450 has been recognized to play a critical role in the metabolic detoxification of insecticides. In the current study, Culex pipiens mosquitoes were collected from Giza Governorate in Egypt and tested for insecticide susceptibility against deltamethrin. First detection of Knockdown resistance gene (Kdr) mutations in field collected mosquitoes was performed. Activities of cytochrome oxidase P450 detoxification enzyme that synchronized with the resistance development, was assessed. Expression profiles of cytochrome P450s and their putative corresponding regulating miRNAs, which was previously reported in Cx. pipiens pallens were evaluated in pyrethroid resistant field-collected Cx. pipiens using RT-qPCR and stem-loop RT-qPCR, respectively. Specific stem-loop reverse transcription primers and forward primers were designed for miRNAs profiling. Our results elucidated the pyrethroid resistance development and revealed its relation to the metabolic and target site modification mechanisms with a first report of L1014F-kdr mutation detection. RT-qPCR results have showed an up-regulation in the expression of the studied P450 transcripts. Negative correlations were found between the expression of P450s and their regulatory miRNAs except for CYP9J35, where positive correlation was found with its corresponding miR-13. Interestingly, our data was the first to detect negative correlation between miR-285 and its putative CYP6Cp1 target gene. These findings highlighted the significance of identifying P450 gene along with regulatory miRNAs as a key mechanism implicated in pyrethroid resistance in field Culex vector population. The elucidation of this mechanism would shed light on the development of insecticide resistance and would help in shaping strategies to combat such vectors.

Molecular characterization of viruses found in honeybee (Apis mellifera) colonies infested with Varroa destructor and Nosema cerana in Egypt, El-Samie, Emtithal Abd, Basuny Nasr, and Seyam Heba , 2021/04/01, Volume 57, p.101731, (2021) Abstract

Honey bees (Apis mellifera) have a vital role as pollinators of various crops in the global food supply. Honeybee colonies in Egypt have recently experienced an unexplained rise in annual loss due to a phenomenon known as Colony Collapse Disorder (CCD). In the current study, honey bees were collected from 14 sites from eight governorates in Egypt. The genetic diversity among the collected honey bee populations was investigated using the mitochondrial DNA cytochrome oxidase subunit I (COI). The amplified COI regions were sequenced, analyzed and aligned with other GenBank entries. The nucleotide variability of the CO1 gene was estimated. Multiple viral, varroa mites as well as Nosema ceranae infections were tested in honey bee populations using conventional and RT-qPCR. Based on sequence analysis of the COI, six clearly separated mitotypes were characterized for the first time in these sites in Egypt. Sequence analyses showed that most mitotypes belonged to the A lineage and are very close to the Egyptian native bees, A. m. lamarckii found in the gene databank (NCBI) with 98% similarity. Low genetic diversity between the collected samples was observed. Our results elucidated the detection of Nosema cerana; deformed wing virus (DWV), kakugo virus (KV), black queen/cell virus (BQCV), Israel acute paralysis virus (IAPV), varroa destructor virus-1 (VDV-1) and VDV-1/DWV virus in all regions under investigation in addition to varroa mites. These findings highlighted the importance to maintain proper quarantine measures as well as identify the spectrum of exogenous infectious agents in healthy hives over time which would help in developing more effective control and treatment programs against honey bee viruses and pathogens to facilitate efficient breeding programs and establish a more booming beekeeping industry.

Assessment of anti-cancer potential of Hyalomma dromedarii salivary glands extract: in vitro study, Ibrahim, Wessam, Mohamed Fatma, Samie Emtithal, Moselhy Walaa, and Mohamed Aly , 2021/04/01, Volume 76, p.1215 - 1225, (2021) Abstract

Cancer is still one of the deadly diseases worldwide and the need for naturally-derived effectors is a pursued aim in health research. With the outstanding ability to bypass the host’s various defense mechanisms, tick saliva constitutes an interesting rich source for the discovery of therapeutically-valuable molecules. Therefore, the present work aimed to evaluate the anti-tumor potential of Hyalomma dromedarii salivary glands extract (SGE). Three cell lines were used in this study, namely HCT116 (colorectal cancer), A549 (lung cancer) and HFB4 (normal skin) cells. MTT assay and light microscopy revealed significant dose-dependent inhibition of proliferation with obvious morphological changes in tested cell lines. Colon cells exhibited more sensitivity than lung ones, therefore they were selected for further investigations. Although cytotoxic effects were observed in treated-HFB4 cells, its IC50 value was much higher than that of HCT116 cells. Flow cytometry analysis of treated HCT116 cells showed accumulation of cellular DNA at the G2/M phase and induction of apoptosis. In addition, RT-qPCR indicated down-regulation in the expression of vascular endothelial growth factor (VEGF), fibroblast growth factor-2 (FGF-2), transforming growth factor-β (TGF-β) and interleukin-4 (IL-4) genes. These results demonstrated that H. dromedarii SGE has anti-proliferative, apoptotic and anti-angiogenic potential. Also, they open perspectives for characterization of effective molecules that could be used in developing treatments for colorectal cancer. Further investigations are required to identify the mechanisms, molecules and signaling pathways involved in the inhibitory effect of the SGE.

Partial Characterization of Thrombin Inhibitor(s) Derived from Salivary Glands of the Tick, Hyalomma dromedarii , and Related Anti-Cancer Potential, Ibrahim, Wessam, Mohamed Fatma, Moselhy Walaa, Samie Emtithal, and Mohamed Aly , 2021/01/01, Volume 09, p.1 - 19, (2021) Abstract
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Pyrethroid Resistance Monitoring in Culex pipiens Mosquito Populations from Three Egyptian Governorates., Tageldin1, R., Zayed A., Abd-EI-Samie E., Mahmoud H., and undefined , J. Nucl. Tech. Appl. Sci, Volume 6, Issue 3, (2018)
Detection of tick blood parasites in Egypt using PCR assay I--Babesia bovis and Babesia bigemina., FK, Adham, EM Abd-el-Samie, RM Gabre, and el-Hussein H. , Parasitology Research, Volume 105, Issue 3, p.721-730, (2009) Abstract

Babesia bovis and Babesia bigemina are distributed all over the world; the etiologic agents of the animal babesiosis are considered the most important tick-borne disease. The present research work was the first attempt to determine the prevalence of B. bovis and B. bigemina infection in ticks, in Egypt, by using polymerase chain reaction (PCR). Questing 5,243 hard and soft ticks were collected from different localities throughout the Giza Governorate. Furthermore, DNA from 500 different individual tick species was extracted and PCR was performed. Primers verified from the sequence of Mexico strain of both species were used. Two fragments of 275 and 175 bp of B. bovis and B. bigemina, respectively, were generated. Fragments of the pathogens were recovered with PCR and sequenced. The prevalence of B. bovis and B. bigemina in Boophilus annulatus ticks were 55% and 66%, respectively. Also, presence of 12% dual infection with B. bovis and B. bigemina was observed. Sequence analysis of PCR product of these pathogens shares a high degree of similarity in sequence compared to similar species found in GenBank.

Detection of tick blood parasites in Egypt using PCR assay II- Borrelia burgdorferi sensu lato, K., Fatma, M. Emtithal, M. Refaat, and E. Hala , Journal of the Egyptian Society of Parasitology, Volume 4, Issue 3, p.553-564, (2010) Abstract

The prevalence of Borrelia burgdorferi sensu lato (s.l.), the etiologic agent
of Lyme borrelosis (LB), was determined for the first time in Egypt by using
polymerase chain reaction (PCR). Questing 5243 hard and soft ticks were collected from animal farms throughout Giza Governorate. DNA from 500 individual tick species was extracted and PCR was performed. Primers verified
from the sequence of German strain Pko of Borrelia afzelii were used. Fragments of 642 bp were generated and sequenced. The prevalence of B. burgdorferi sensu lato (s.l.) was 28 % of examined soft and hard ticks. High infection
rate (66%) of B. burgdorferi s.l. was observed in both nymph and adult soft
ticks Ornithodoros savignyi.
Beside, the role of hard ticks as potential vectors of Lyme disease in Egypt,
where the infection rate was between 0.0-50.0%. Sequence analysis of PCR
product of Borrelia burgdorferi sensu lato shares high degree of similarity in
sequence compared to similar species in GenBank.

Developmental Expression of Mitochondrial Gene Cytochrome b in Culex pipiens (L.) (Diptera: Culicidae) , M, Emtithal , Egyptian Journal of Biological Pest Control, Volume 23, p. 95-100, (2013) Abstract

Cytochrome b, coded by mitochondrial DNA, is one of the cytochromes involved in electron transport in the respiratory
chain of mitochondria. A 257 bp fragment of Cytochrome b gene (Cytb) was generated by reverse transcriptionpolymerase chain reaction (RT-PCR) products and sequenced directly. Sequence analysis of PCR product of
Cytb shared a similarity in sequence compared to Culex Cytb found in GenBank. To reveal whether cytochrome b of the
mosquito, Culex pipiens (L.) (Diptera: Culicidae) (CxpCytb) was developmentally regulated; the real-time quantitative
polymerase chain reaction (qPCR), was used to examine Cytb gene expression levels in different developmental stages
of Cx. pipiens. The qPCR showed that CxpCytb was expressed in each developmental stage, with high expression at
eggs, the RNA relative expression level of CxpCytb decreased dramatically in 1st and 2nd instar larvae and increased
again in 4th instar larvae and pupae of mosquitoes, than the lowest point in adult males. Expression of CxpCytb in
teneral adults (2-days-old female) of Cx. pipiens was higher than that found in male adults of the same age. These
results suggest that CxpCytb gene plays an important role in the development of Cx. pipiens and may provide critical
information needed for designing novel control strategies for medically important disease vectors and identifying new
pathways to target for the development of new molecular pesticides

First detection of deformed wing and kakugo viruses in honeybees (Apis mellifera L.) in Egypt by real timepolymerase chain reaction (RT-PCR), M., Emtithal, K. Fatma, E Sawsan, and S Heba , African Journal of Biotechnology, Volume 16, p.738-748, (2017) Abstractd4f20c863599_honey_bee_paper.pdf

The identification of bee viruses is of great importance where there is no information on their natural
occurrence in honeybee populations. Honeybee samples (Apis mellifera L.) obtained from Giza
Governorate, Egypt were evaluated for total RNA from the head, thorax and abdomen, and varroa mites.
Two fragments of 250 and 540 bp of deformed wing (DWV) and kakugo viruses (KV), respectively, were
amplified using conventional real time-polymerase chain reaction (RT-PCR), and were sequenced and
analyzed. All bee body parts resulted in a strongly positive DWV sequence except asymptomatic
honeybee samples that were negative to the DWV infection. The analysis of varroa mite groups (n = 50)
also revealed the presence of DWV which is the first report on this virus in Egypt. Homology search of
DWV-PCR amplified fragments revealed 99 and 98% similarities with DWV isolate Warwick-2009 polyprotein
gene (accession number: GU109335.1) and the Chilensis DWV isolate, respectively (accession
number: JQ413340.1). Also, homology search of KV-PCR amplified fragments using the Genius
program 7.1.2 revealed 97 and 90.0% similarities with the KV isolate_Ox genomic sequence (accession
number: KC786224.1) and the DWV_Ox genomic sequence (accession number: KC786223.1),
respectively. For this study data, a real-time SG RT-Qpcr was found as a fast, simple, sensitive and
useful technique to detect and quantify low viral loads in honeybees and varroa mites.

Molecular Phylogeny and Identification of the Egyptian Wasps (Hymenoptera :Vespidae) Based on COI Mitochondrial Gene Sequences, M.Abd-El-Samie, Emtithal, Elkafrawy Israa, Osama Mai, and Ageez Amr , Egyptian Journal of Biological Pest Control, (2018) Abstract

The Hymenoptera is one of the vital and biggest insect orders comprising the bees, wasps, sawflies, and ants. Wasps
are important to natural and biological pest control because they are predators or parasitoids of pest arthropods. This
study investigated the genetic diversity among the three wasps, Vespa orientalis Linnaeus, Polistes bucharensis Erichson,
and Polistes mongolicus du Buysson, collected from three different governorates in Egypt, using cytochrome oxidase
subunit I (COI) DNA barcoding. PCR was performed to amplify COI fragment. The amplified COI regions (710 bp) were
sequenced and analyzed. All novel nucleotide sequences of COI gene were deposited into the GenBank database. The
genetic distances were estimated using Kimura two-parameter model. In spite of the wide geographical range, minor
genetic diversity was observed between some populations of the three wasp species, revealing unrestricted gene flow
between them. Phylogenetic relationship analysis was performed, using maximum likelihood (ML) method. The results
of the phylogenetic analyses recovered P. bucharensis more closely related to P. dominula and P. gallicus. P. mongolicus
collected from Menofia Governorate formed a distinct branch with 99% support. V. orientalis was sister to the
yellowjacket Dolichovespula adulterine, with 84% support. It can be concluded that DNA barcode is a powerful
tool for rapid and accurate identification of Egyptian wasp species.