Peptidoglycan-Targeted [18F]3,3,3-Trifluoro-d-alanine Tracer for Imaging Bacterial Infection.

Imaging is increasingly used to detect and monitor bacterial infection. Both anatomic (X-rays, computed tomography, ultrasound, and MRI) and nuclear medicine ([111In]-WBC SPECT, [18F]FDG PET) techniques are used in clinical practice but lack specificity for the causative microorganisms themselves. To meet this challenge, many groups have developed imaging methods that target pathogen-specific metabolism, including PET tracers integrated into the bacterial cell wall. We have previously reported the d-amino acid derived PET radiotracers d-methyl-[11C]-methionine, d-[3-11C]-alanine, and d-[3-11C]-alanine-d-alanine, which showed robust bacterial accumulation in vitro and in vivo. Given the clinical importance of radionuclide half-life, in the current study, we developed [18F]3,3,3-trifluoro-d-alanine (d-[18F]-CF3-ala), a fluorine-18 labeled tracer. We tested the hypothesis that d-[18F]-CF3-ala would be incorporated into bacterial peptidoglycan given its structural similarity to d-alanine itself. NMR analysis showed that the fluorine-19 parent amino acid d-[19F]-CF3-ala was stable in human and mouse serum. d-[19F]-CF3-ala was also a poor substrate for d-amino acid oxidase, the enzyme largely responsible for mammalian d-amino acid metabolism and a likely contributor to background signals using d-amino acid derived PET tracers. In addition, d-[19F]-CF3-ala showed robust incorporation into Escherichia coli peptidoglycan, as detected by HPLC/mass spectrometry. Based on these promising results, we developed a radiosynthesis of d-[18F]-CF3-ala via displacement of a bromo-precursor with [18F]fluoride followed by chiral stationary phase HPLC. Unexpectedly, the accumulation of d-[18F]-CF3-ala by bacteria in vitro was highest for Gram-negative pathogens in particular E. coli. In a murine model of acute bacterial infection, d-[18F]-CF3-ala could distinguish live from heat-killed E. coli, with low background signals. These results indicate the viability of [18F]-modified d-amino acids for infection imaging and indicate that improved specificity for bacterial metabolism can improve tracer performance.

Synthesis and characterization of piperic acid conjugates with homochiral and heterochiral dipeptides containing phenylalanine and their application in skin cancer.

The current work demonstrates the synthesis and characterization of piperic acid conjugates with homochiral/heterochiral dipeptides containing phenylalanine as anti-skin cancer agents. The conjugates PA-DPhe-LPhe-OH, FC-1; PA-LPhe-DPhe-OH, FC-2; PA-DPhe-DPhe-OH, FC-3; and PA-DPhe-DPhe-OH, FC-4 were synthesized, characterized and assessed for cytotoxicity against melanoma cell lines of human and murine origin. Among all, PA-DPhe-DPhe-OH (FC-3) conjugate was identified as a potential cytotoxic lead against melanoma cells by delineating the anti-proliferative and anti-migratory potential together with its anti-inflammatory potential against pro-inflammatory interleukins (IL-1ß, IL-6, and IL-8). Evidences from western blotting, fractionation, and immunocytochemistry experiments suggest that Stat-3 is a critical signaling molecule involved in the FC-3 mechanism of action. The results denote that FC-3 profoundly ablates Stat-3 expression, phosphorylation, and nuclear translocation. Stat-3 mRNA analysis revealed that FC-3 did not alter the transcription of Stat-3. However, in cells where proteasome mediated degradation was inhibited, FC-3 failed to check the Stat-3 expression implying that FC-3 augments the proteasomal degradation of Stat-3. Of note, FC-3 failed to reverse the IL-6 mediated hyperactivation of Stat-3 in A375 cells. Critically, in Stat-3 deficient cancer cells, the anti-clonogenic and anti-migratory potential of FC-3 was significantly subdued. Further, the in vivo efficacy of FC-3 was validated in the two-step (DMBA/TPA) chemically induced mouse skin cancer model. The FC-3-treated cohorts of mice unveiled a significant decrease in the cumulative number of tumors besides attenuation of tumor growth with respect to the vehicle-treated mice. Lastly, in corroboration with our in vitro findings, serum collected from mice groups at various intervals during the treatment regimen demonstrated decrement in IL-1ß and IL-6 levels in FC-3 treated groups compared to the vehicle-treated group.

Lethal and sublethal effects of flubendiamide and spirotetramat against the leaf worm, Spodoptera litura(Fabricius) under laboratory conditions.

The leaf worm, Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae) is a notorious insect pest of many economically important cultivated crops like cotton, maize, tobocco and vegetables causing severe economic losses from 50-100%. In most crops, damage arises due to voracious feeding by the larvae and leads to the skeletonizing of leaves. Toxicological studies were performed to estimate lethal and sublethal levels of flubendiamide and spirotetramat against S. litura. Effects of these estimated values were assessed on different biological traits of S. litura including life duration, survival and next generation potential. Both flubendiamide and spirotetramat showed toxic responses against second instar larvae of S. litura under laboratory conditions. Larval duration and survival rate of S. litura to were significantly different. Exposure to test insecticides resulted in negative effect on the demography of S. litura as longer life cycle and decreased fecundity. Changes in net reproductive rate and intrinsic rate of increase also helped to decide the fate of these insecticides. Low reproductive potential and low hatching percentage due to exposure to test insecticides can help to manage next generation of target pest. These two new chemistry insecticides can be recommended for their effective and long-term utilization against this important leaf feeder which may help its management and decrease economic losses faced by the growers. Their impact on larval duration and low survival rate at lethal levels guides about their potential in pest control.

Synthesis, Characterization, and Antimicrobial Activity of Ultra-Short Cationic ß-Peptides.

The development of new antibiotics is urgently required because of the rapidly growing resistance against conventional antibiotics. The antimicrobial peptides show potential as small antibiotic molecules. The stability of peptides is a primary concern for the use of peptides as drugs. Introducing ß-amino acids into peptide sequences can be useful in preventing biological degradation by proteolytic enzymes. Herein, we describe the synthesis, characterization, and antimicrobial activity of ultra-short cationic ß-peptides, LA-ß3,3-Pip-ß2,2-Ac6c-PEA, P1; LA-ß3,3-Pip(G)-ß2,2-Ac6c-PEA, P2; LAU-ß3,3-Pip-ß2,2-Ac6c-PEA, P3, and LAU-ß3,3-Pip(G)-ß2,2-Ac6c-PEA, P4. Peptides P1-P4 were evaluated against Gram-negative, Gram-positive, MRSA, and multi-drug resistant E. coli (MDR-E. coli). P3 exhibited the most potent antimicrobial activity against E. coli, S. epidermidis, S. aureus, K. pneumoniae, S. mutans, and E. faecalis, with MIC values 0.5, 2, 0.5, 1, 2, and 1 µg/mL, respectively. P3 exhibited time- and concentration-dependent bactericidal activities against E. coli, S. aureus, and E. faecalis with a killing rate of 1.6 logs/h. The treatment of E. coli with peptide P3 showed membrane disruption. In addition, P3 exhibited the inhibition of biofilm produced by E. coli, synergism with antibiotics (ciprofloxacin, streptomycin, and ampicillin), 100% cell viability against AML12, RAW 264.7, and HEK-293 cell lines at 1, and 10 µg/mL concentrations.

Synthesis, characterization, and wound healing properties of α/γ hybrid peptides.

The present work describes the synthesis, characterization, and wound healing properties of α/γ hybrid peptides: Boc-Phe-γ4 -Phe-Val-OMe (S1), Boc-D Phe-γ4 -Phe-Val-OMe (S2), Boc-Ala-γ4 -Phe-Val-OMe (S3), Boc-D Ala-γ4 -Phe-Val-OMe (S4), Boc-Leu-γ4 -Phe-Val-OMe (S5), and Boc-D Leu-γ4 -Phe-Val-OMe (S6). Peptides S1-S6 were screened against human keratinocytes (HaCaT) and RAW 264.7 cells. Among all, S1- and S2-treated cells exhibited high cell viability; S1 and S2 induced keratinocyte migration and inhibited the production of the cytokines IL-6 and TNF-α. In vivo results demonstrated that the hybrid peptides S1 and S2 accelerate wound healing in Wistar rats with 83% and 88% at 50 µg/ml, and 74% and 76% at 25 µg/ml, respectively.

Heterochiral dipeptide d-phenylalanyl- l-phenylalanine (H-D Phe-L Phe-OH) as a potential inducer of metastatic suppressor NM23H1 in p53 wild-type and mutant cells.

In recent years, significant progress has been made to the use-case of small peptides because of their diversified edifice and hence their versatile application scope in cancer therapy. Here we identify the heterochiral dipeptide H-D Phe-L Phe-OH (F1) as a potent inducer of the metastatic suppressor NM23H1. We divulge the effect of F1 on the major EMT/metastasis-associated genes and the implications on the invasion and migration ability of cancer cells. The anti-invasive potential of F1 was directly correlated with NM23H1 expression. Mechanistically, F1 treatment elevated p53 levels as validated by localization and transcriptional studies. In the NM23H1 knockdown condition, F1 failed to induce any p53 expression/nuclear localization, indicating that the upregulation in p53 expression by F1 is NM23H1 dependent. We also demonstrate how the antimetastatic potential of F1 is primarily mediated through NM23H1 irrespective of the p53 status of the cell. However, both NM23H1 and a functional p53 protein in conjunction govern the apoptotic and cytostatic potential of F1. Coimmunoprecipitation studies unraveled the augmentation of the p53 and NM23H1 interaction in p53 wild-type cells. However, in p53 mutated cells, no such enrichment was evidenced. We employed mouse isogenic cell lines (4T-1 and 4T-1 p53) to determine the in vivo efficacy of F1 (spontaneous and experimental models). Decreased tumor volume in the cohort injected with 4T-1 p53 cells demonstrated that while the antimetastatic potential of F1 was reliant on NM23H1, p53 activation was required for ablation of primary tumor burden. Our findings unravel that F1 treatment induces significant abrogation of the migration, invasion and metastatic potential of both p53 wild-type and p53 deficient cancers mediated through NM23H1.

Synthesis, conformation and cytotoxic activity of short hybrid peptides containing conformationally constrained 1-(aminomethyl)cyclohexanecarboxylic acid and gabapentin.

The present work describes the synthesis,conformation and cytotoxic activities of short ß/γ hybrid peptides, Boc-ß2,2-Ac6c-Gpn-NHMe, BG1; Boc-(ß2,2-Ac6c-Gpn)2-OMe, BG2; Boc-(ß2,2-Ac6c-Gpn)3-OMe, BG3; H-ß2,2-Ac6c-Gpn-NHMe, BG4; H-(ß2,2-Ac6c-Gpn)2-OMe, BG5; H-(ß2,2-Ac6c-Gpn)3-OMe, BG6, Boc-ß2,2-Ac6c-Gpn-OMe, BG7 and H-ß2,2-Ac6c-Gpn-OMe, BG8. Mixed C6/C7 conformations were observed for ß/γ hybrid peptides. Further, BG1-BG8 were screened against MCF-7 (Breast cancer), A549 (Lung Cancer), PC-3 (Prostate cancer), HCT-116 (Colon cancer), and MDA-MB-231 (Breast cancer) cell lines. Among all, BG6 exhibited potent cytotoxicity against all cancer cell lines with IC50 ranging from 1.6 µM to 6.3 µM with relatively low cytotoxicity against normal epithelial breast cell line fR-2 and human embryonic kidney cell line HEK-293. Minimal hemolytic activity was observed for BG6 against human erythrocytes. Peptide BG6 displayed anti-migratory and anti-invasive potentials showing strong interactions with intrinsic apoptotic markers Bcl-2, Bax, and cleaved-PARP, as well as the induction of the mitochondria maladjustment mediated apoptosis.

Synergistic antimicrobial and antibiofilm activities of piperic acid and 4-ethylpiperic acid amides in combination with ciprofloxacin.

In the present work, piperic acid and 4-ethylpiperic acid (EPA) amides with amino acids (C1-C8) were bio-evaluated for their antimicrobial activity and biofilm inhibition against Gram-positive and Gram-negative bacterial strains. Among all, EPA-ß3,3-Pip(Bzl)-OMe, C2 displayed the potent antimicrobial activity with MIC of 6.25 µg ml-1 against Gram-negative bacteria Escherichia coli. In combination studies, the FIC indices suggested that C1 and C2 have a synergistic effect with ciprofloxacin against E. coli and Bacillus subtilis, whereas C5 exhibited a synergistic effect with ciprofloxacin against all the tested bacteria. The inhibitory effect of amides C1, C2, and C5 on the biofilm formation of test strains was significantly potentiated by co-administration with ciprofloxacin. Furthermore, the effective concentrations of C2 in combination reduced drastically compared to alone for biofilm inhibition. At these concentrations, C2 showed negligible hemolytic and cytotoxic activities.

Taxonomy and distribution of agriculturally important plusiinae (Lepidoptera: Noctuidae) from southern Punjab, Pakistan.

Taxonomic identification and classifications of insect pest genera Chrysodeixis and Ctenoplusia of the subfamily Plusiinae is very compulsory due to their phytophagous nature and potential to damage the cash as well as cereal crops. Taxonomy plays a key role in proper not only in identification and classification of the pest but also in designing a successful managing strategy. In current study, specimens of Chrysodeixis and Ctenoplusia genera were collected from different geographical areas of south Punjab, Pakistan and their diagnostic features were examined following taxanomic keys. The data of temperature, relative humidity and rainfall were also recorded during the study period. Genitalia was extracted by dissecting of the abdomen and inspected under Stereo microscope. The results revealed two new species, Chryodeixis maxus and Ctenoplusia oleraceaus, from south Punjab region in addition to previously reported species: Chrysodeixis furihatai. Suitable management of the voracious insect pest at appropriate time may help in sustaining the agriculture in Pakistan.

Functional Analysis of Autophagy-Related Gene ATG12 in Potato Dry Rot Fungus Fusarium oxysporum.

Autophagy is an intracellular process in all eukaryotes which is responsible for the degradation of cytoplasmic constituents, recycling of organelles, and recycling of proteins. It is an important cellular process responsible for the effective virulence of several pathogenic plant fungal strains, having critical impacts on important crop plants including potatoes. However, the detailed physiological mechanisms of autophagy involved in the infection biology of soil-borne pathogens in the potato crop needs to be investigated further. In this study, the autophagy-related gene, FoATG12, in potato dry rot fungus Fusarium oxysporum was investigated by means of target gene replacement and overexpression. The deletion mutant ∆FoATG12 showed reduction in conidial formation and exhibited impaired aerial hyphae. The FoATG12 affected the expression of genes involved in pathogenicity and vegetative growth, as well as on morphology features of the colony under stressors. It was found that the disease symptoms were delayed upon being inoculated by the deletion mutant of FoATG12 compared to the wild-type (WT) and overexpression (OE), while the deletion mutant showed the disease symptoms on tomato plants. The results confirmed the significant role of the autophagy-related ATG12 gene in the production of aerial hyphae and the effective virulence of F. oxysporum in the potato crop. The current findings provid an enhanced gene-level understanding of the autophagy-related virulence of F. oxysporum, which could be helpful in pathogen control research and could have vital impacts on the potato crop.

Tetrahydropiperic acid (THPA) conjugated cationic hybrid dipeptides as antimicrobial agents.

The present work describes the synthesis of hybrid dipeptides H-Lys-Gpn-PEA, C1; H-Lys-ß3,3AC6C-PEA, C2, and THPA conjugated dipeptides, THPA-Lys-Gpn-PEA, C3, and THPA-Lys-ß3,3AC6C-PEA, C4. All the peptides were evaluated against both Gram-negative and Gram-positive bacterial strains. Among all, peptide C4 exhibited the most potent activity with MIC 1.56 µM against P. aeruginosa (MTCC 424) and S. aureus (MTCC 737). Further, time-kill kinetics, fluorescence assays, and scanning electron microscopy (SEM) studies were performed in order to understand the mechanism of action and efficacy of peptide C4, The fluorescence assays and SEM images demonstrated the bacterial killing through membrane disruption. The peptide C4 exhibited very low hemolytic activity with negligible cytotoxicity against normal human breast cell line FR2.

CO1-Based DNA barcoding for assessing diversity of Pteropus giganteus from the state of Azad Jammu Kashmir, Pakistan.

The flying fox (Pteropus giganteus) also familiar with the name of the greater Indian fruit Bat belongs to the order Chiroptera and family Pteropodidae. Current research emphasis on the DNA barcoding of P. giganteus in Azad Jammu Kashmir. Bat sequences were amplified and PCR products were sequenced and examined by bioinformatics software. Congeneric and conspecific, nucleotide composition and K2P nucleotide deviation, haplotype diversity and the number of haplotypes were estimated. The analysis showed that all of the five studied samples of P. giganteus had low G contents (G 19.8%) than C (27.8%), A (25.1%) and T (27.3%) contents. The calculated haplotype diversity was 0.60% and the mean intraspecific K2P distance was 0.001% having a high number of transitional substitutions. The study suggested that P. giganteus (R=0.00) do not deviate from the neutral evolution. It was determined from the conclusion that this mtDNA gene is a better marker for identification of Bat species than nuclear genes due to its distinctive characteristics and may serve as a landmark for the identification of interconnected species at the molecular level and in the determination of population genetics.

Charaterization of mosquito larval habitats in Qatar.

Mosquito borne diseases have remained a grave threat to human health and are posing a significant burden on health authorities around the globe. The understanding and insight of mosquito breeding habitats features is crucial for their effective management. Comprehensive larval surveys were carried out at 14 sites in Qatar. A total of 443 aquatic habitats were examined, among these 130 were found positive with Culex pipiens, Cx. quinquefasciatus, Cx. mattinglyi, Ochlerotatus dorsalis, Oc. caspius and Anopheles stephensi. The majority of positive breeding habitats were recorded in urban areas (67.6%), followed by livestock (13.8%), and least were in agriculture (10.7%). An. stephensi larvae were positively correlated with Cx. pipien, Cx. quinquefasciatus, and negatively with water salinity. Large and shaded habitats were the most preferred by An. stephensi. In addition, Cx. pipiens was positively associated with the turbidity and pH, and was negatively associated with vegetation and habitat size. A negative association of Cx. quinquefasciatus with dissolved oxygen, water temperature, and salinity, while positive with habitat surface area was observed. Oc. dorsalis was negatively correlated with pH, water temperature, depth, and habitat surface area, whereas salinity water was more preferable site for females to lay their eggs. These results demonstrate that environmental factors play a significant role in preference of both anopheline and culicine for oviposition, while their effective management must be developed as the most viable tool to minimize mosquito borne diseases.

Investigation of α/γ hybrid peptide self-assembled structures with antimicrobial and antibiofilm properties.

The present work describes the synthesis and characterization of α/γ hybrid peptides, Boc-Phe-γ4 -Phe-Val-OMe, P1; Boc-Ala-γ4 -Phe-Val-OMe, P2; and Boc-Leu-γ4 -Phe-Val-OMe, P3 together with the formation of self-assembled structures formed by these hybrid peptides in dimethyl sulfoxide (DMSO)/water (1:1). The self-assembled structures were characterized by infrared (IR) spectroscopy, circular dichroism (CD), and scanning electron microscopy (SEM). Further, α/γ hybrid peptide self-assembled structures were evaluated for antibacterial properties. Among all, the self-assembled peptide P1 exhibited the antimicrobial activity against Escherichia coli and Klebsiella pneumoniae, while self-assembled peptide P3 inhibited the biofilms of Salmonella typhimurium and Pseudomonas aeruginosa. In this study, we have shown the significance of self-assembled structures formed from completely hydrophobic α/γ hybrid peptides in exploring the antibacterial properties together with biofilm inhibition.

Role of Autophagy-Related Gene atg22 in Developmental Process and Virulence of Fusarium oxysporum.

Autophagy is a universal catabolic process preserved in eukaryotes from yeast to plants and mammals. The main purpose of autophagy is to degrade cytoplasmic materials within the lysosome/vacuole lumen and generate an internal nutrient pool that is recycled back to the cytosol during nutrient stress. Here, Fusarium oxysporum was utilized as a model organism, and we found that autophagy assumes an imperative job in affecting the morphology, development, improvement and pathogenicity of F. oxysporum. The search of autophagy pathway components from the F. oxysporum genome database recognized putative orthologs of 16 core autophagy-related (ATG) genes of yeast, which additionally incorporate the ubiquitin-like protein atg22. Present study elucidates the unreported role of Foatg22 in formation of autophagosomes. The deletion mutant of Foatg22 did not demonstrate positive monodansylcadaverine (MDC) staining, which exposed that Foatg22 is required for autophagy in F. oxysporum. Moreover, the ∆Foatg22 strains exhibited a decrease in hyphal development and conidiation, and reduction in pathogenicity on potato tubers and leaves of potato plant. The hyphae of ∆Foatg22 mutants were less dense when contrasted with wild-type (WT) and overexpression (OE) mutants. Our perceptions demonstrated that Foatg22 might be a key regulator for the control of dry rot disease in tuber and root crops during postharvest stage.

Effects of temephos resistance on life history traits of Aedes albopictus (Skuse) (Diptera: Culicidae), a vector of arboviruses

Abstract The effects of temephos resistance on the fitness cost of the wild populations of Aedes albopictus was evaluated. The larvae of two wild populations were exposed to the diagnostic dose of 0.02 mg and 0.012 mg/L. The larvae which survived after the 24 h exposure to diagnostic dose were considered as resistant and F1 generations were chosen for the comparisons of fitness parameters with the control strain, which includes larval developmental time, adult longevity, fecundity, wing length and hatchability. We found that temephos resistance had negative effects on larval developmental time which was longer for Gelugor strain among the populations with the median range of 10 days and a shorter longevity was observed with the median range of 13 days for males and 16 days for females. Whereas, an effective reduction of 29.8 and 38.6% was observed in fertility and fecundity of Gelugor strain as compared to control strain. In contrast, no clear differences were found in biological parameters of Balik Palau and USM strain, except fecundity and fertility with a reduction of 13.4 and 15.5%, respectively. Whereas, no significant differences were seen in the wing size between the populations with the mean length (mm) of 2.40 for Gelugor, 2.44 for Balik Palau and 2.46 for USM control (p > 0.05). Present results indicated that the temephos resistance is associated with the developmental and reproduction potential of resistant population of A. albopictus and the fitness has been compromised.

Adulticidal Susceptibility Evaluation of Aedes albopictus Using New Diagnostic Doses in Penang Island, Malaysia.

Insecticide-based vector control approaches are facing challenges due to the development of resistance in vector mosquitoes. Therefore, a proper resistance surveillance program using baseline lethal concentrations is crucial for resistance management strategies. Currently, the World Health Organization's (WHO) diagnostic doses established for Aedes aegypti and Anopheles species are being used to study the resistance status of Aedes albopictus. In this study, we established the diagnostic doses for permethrin, deltamethrin, and malathion using a known susceptible reference strain. Five field-collected populations were screened against these doses, following the WHO protocol. This study established the diagnostic dose of malathion at 2.4%, permethrin at 0.95%, and deltamethrin at 0.28%, which differ from the WHO doses for Aedes aegypti and Anopheles spp. Among the insecticides tested on the 5 wild populations, only deltamethrin showed high effectiveness. Different susceptibility and resistance patterns were observed with permethrin, malathion, and dichloro-diphenyl-trichloroethane (DDT) at 4%. This study may assist the health authorities to improve future chemical-based vector control operations in dengue-endemic areas.

Contribution of public places in proliferation of dengue vectors in Penang Island, Malaysia

Objective: To determine abundance, distribution and diversity of potential breeding container habitats of the dengue vectors in public places including schools, restaurants, mosques and parks in southwest areas of Penang Island, Malaysia. Methods: Premises at restaurants, schools, parks and mosques were surveyed simulta-neously and inspected visually for container habitats and production of immature mosquitoes from March 2015 to March 2016. Abundance (mean ± SE) of breeding containers between sites was compared using One-way ANOVA. Independent sample t-test was used to compare total number of Aedes albopictus (Ae. albopictus) and Aedes aegypti (Ae. aegypti) surveyed. Results: The surveyed locations yielded a total of 3741 breeding containers and 19537 immature mosquitoes from four areas. Concurrent artificial and natural containers pro-duced 78.4%immature Ae. albopictus and 6.3%Ae. aegypti mosquitoes in wet season, with 14.2% Ae. albopictus and 1.1% Ae. aegypti mosquitoes in dry season. Artificial containers accounted for 98.1% of the total containers recorded, with restaurants being the most productive locations (8012) and schools being the least productive (2234). Conclusions: It was concluded that public places are good sources of potential container habitats of Aedes mosquitoes in Penang Island, Malaysia and Ae. albopictus has exclu-sively replaced the home-grown Ae. aegypti even in urban areas. Therefore, treatment of artificial containers in such locations is critical in Aedes mosquito control campaigns during dengue outbreaks.

CO1-Based DNA barcoding for assessing diversity of Pteropus giganteus from the state of Azad Jammu Kashmir, Pakistan

Abstract The flying fox (Pteropus giganteus) also familiar with the name of the greater Indian fruit Bat belongs to the order Chiroptera and family Pteropodidae. Current research emphasis on the DNA barcoding of P. giganteus in Azad Jammu Kashmir. Bat sequences were amplified and PCR products were sequenced and examined by bioinformatics software. Congeneric and conspecific, nucleotide composition and K2P nucleotide deviation, haplotype diversity and the number of haplotypes were estimated. The analysis showed that all of the five studied samples of P. giganteus had low G contents (G 19.8%) than C (27.8%), A (25.1%) and T (27.3%) contents. The calculated haplotype diversity was 0.60% and the mean intraspecific K2P distance was 0.001% having a high number of transitional substitutions. The study suggested that P. giganteus (R=0.00) do not deviate from the neutral evolution. It was determined from the conclusion that this mtDNA gene is a better marker for identification of Bat species than nuclear genes due to its distinctive characteristics and may serve as a landmark for the identification of interconnected species at the molecular level and in the determination of population genetics.

Resumo A raposa-voadora (Pteropus giganteus), também conhecida como morcego indiano, pertence à ordem dos Chiroptera e à família Pteropodidae. A presente pesquisa dá ênfase ao código de barras de DNA de P. giganteus em Azad Jammu e Caxemira. Sequências genéticas dos morcegos foram amplificadas, e os produtos de PCR foram sequenciados e examinados por software de bioinformática. De espécies congenérica e coespecífica, foram estimados composição nucleotídica e desvio de nucleotídeos K2P, diversidade de haplótipos e número de haplótipos. A análise mostrou que todas as cinco amostras estudadas de P. giganteus apresentaram baixos teores de G (19,8%) em comparação com C (27,8%), A (25,1%) e T (27,3%). A diversidade de haplótipos calculada foi de 0,60%, e a distância média intraespecífica de K2P foi de 0,001%, com um elevado número de substituições transicionais. O estudo sugeriu que P. giganteus (R = 0,00) não se desviou da evolução neutra. É possível concluir que o gene mtDNA é um marcador favorável para identificação de espécies de morcegos do que genes nucleares por causa de suas características distintivas e pode servir como um marco para a identificação de espécies interconectadas em nível molecular e para a determinação genética de populações.

Revised Discriminating Lethal Doses For Resistance Monitoring Program on Aedes albopictus Against Temephos and Malathion in Penang Island, Malaysia.

Dengue vector control still heavily relies on the use of chemical insecticides, and the widespread use of insecticides has led to resistance in mosquitoes. The diagnostic dose is a key part of resistance monitoring. The present study corroborates the discriminating lethal doses of temephos and malathion based on dose-response of known susceptible strain of Aedes albopictus following the World Health Organization (WHO) diagnostic test procedure. Late 3rd and early 4th instars were tested with a range of larvicides to determine the lethal concentrations (LC50 and LC99) values. A slightly higher diagnostic dose of 0.020 mg/liter as compared with the WHO-established value of 0.012 mg/liter was observed for temephos. Meanwhile, a malathion diagnostic dose of 0.200 mg/liter is also reported here since there are no such reported values by WHO. Doubling the LC99 values of susceptible strains, 3 of the 5 wild-collected populations showed resistance to temephos and 2 showed incipient resistance; all 5 populations showed incipient resistance to malathion. The revised and established lethal diagnostic dose findings from the current work are crucial to elaborate on the variation in susceptibility of Ae. albopictus in future resistance monitoring programs in Malaysia.