Symposium on Dengue - 2015
http://repository.kln.ac.lk/handle/123456789/10378
2024-03-29T06:32:02ZBiology and diversity of Copepods in Sri Lanka and their potential use as predator of Aedes larvae
http://repository.kln.ac.lk/handle/123456789/10901
Biology and diversity of Copepods in Sri Lanka and their potential use as predator of Aedes larvae
Anukulan, J.W.; Ratnayake, R.M.A.S.; Perera, R.S.M.; Hapukotuwa, R.B.; Iqbal, M.C.M.
BACKGROUND: Dengue is a major viral disease in tropical and subtropical countries. Although chemical control of the vector is efficient it causes much damage to the natural environment including destroy of other useful insects. There are many natural predators that prey on mosquito larvae such as copepods, dragonfly, damselfly and larvivorous fish. Among them the use of fish has been practiced extensively to control mosquito larvae. Another potential predator of mosquito larvae is a small aquatic organism called copepod, which is extensively used for biological control in South East Asian countries. We are conducting a preliminary survey on the copepods in Sri Lanka and their ability to prey on larvae of dengue vector Aedes spp. METHODS: Water collected from reservoirs in Kandy (species A), Batticaloa (species B) and Kurunegala (species C) were sieved with a net with 50μm mesh size. The trapped copepods were collected into sampling bottles by washing the net thoroughly. The copepods were observed under a stereo microscope to determine their size. The predation studies were conducted with two ratios: 25 first instar Aedes larvae and 5adult copepods(5:1) and 50 first instar Aedes larvae and 5 adult copepods (10:1). The 5:1 ratio was replicated four times while applying the 10:1 ratio thrice. RESULTS: The body length of the Batticaloa (species B) copepods were larger than the Kandy and Kurunegala copepods. The total mortality of larvae (TM) from the study of 5:1 was species A TM=45, species B, TM=70 and species C, TM=49. The average mortality (AM) was, speciesA, AM=11.25±5.6, species B, AM=17.5±3.1 and species C, AM=12.25±2.5.Mosquito larva predation rate per day by a copepod was, species A 2.25±1.12, species B 3.45±0.62 and species C 2.45±0.5.In the second study with a ratio of 10:1, the total mortality was, species A, TM= 86, species B, TM= 123 and species C, TM= 108. Average mortality was, species A, AM= 28.67±14.17, species B, AM=41±6.24 and species C, AM=39.33±14.53. Mosquito larval predation rate per day by a copepod was, species A, 2.87±1.42, species B, 4.1±0.62 and species C, 3.93±1.45. CONCLUSION: Species B from the Batticaloa, which were with larger in body size showed a better predation ability of Aedes when compared with species A (Kandy) and C (Kurunegala).
Postgraduate Students Presentations
2015-01-01T00:00:00ZAwareness on dengue and solid waste management among selected school students in Gampaha District of Sri Lanka
http://repository.kln.ac.lk/handle/123456789/10900
Awareness on dengue and solid waste management among selected school students in Gampaha District of Sri Lanka
Radhika, N.M.L.; Gunathilaka, P.A.D.H.N.; Abeyewickreme, W.; Kasturiratne, K.T.A.A.
BACKGROUND: Community mobilization is often respected as a powerful tool in ensuring the effective controlling of dengue via promoting effective and sustainable dengue vector control strategies. The current study attempts to investigate the level of awareness on dengue and solid waste management, among selected school students in Gampaha District which is the second highest dengue reported district in Sri Lanka. METHODOLOGY: Awareness programmes on dengue (transmission, vector ecology and preventive measures) and solid waste management were conducted in 10 schools belong to Kelaniya Medical Officer of Health (MOH) area selecting a minimum group of 200 students from each school within the age of 13 - 15 years. Tools such audio-visual presentations, leaflets and videos were used for the awareness programmes. A questionnaire was given to each student prior to the awareness and after the awareness programs as pre and post evaluations respectively in order to assess the general awareness on dengue and solid waste management before and after the awareness programme. One-Way ANOVA, paired t-test and multiple regression techniques were utilized in evaluation of the degree of awareness and key factors affecting the level of awareness, prior and after the conducting the awareness programmes. RESULTS: The degree of initial awareness on dengue and solid waste management of the target school community was recognized to be significantly characterized by the grade of the students and the Education Zonal Category [EZU] (into which their school belongs to) in accordance with One-Way ANOVA (p < 0.05). A significant increment in post awareness of the target school community was recognized due to the conducting of awareness programmes in accordance with the statistics of paired t-test (p < 0.05). Furthermore, based on the multiple linear regression modelling, the post awareness was significantly influenced by the grade of the students and the initial awareness (p < 0.05), regardless of the EZU (p > 0.05). CONCLUSIONS: The awareness of school students on dengue and solid waste management is significantly affected by the grade of the students and EZCs. Awareness programmes are an effective media of raising the awareness of school communities on dengue and solid waste management, while motivating them to actively contribute for the controlling of dengue, via establishing a strong inter sectorial network among schools and other relevant institutions within the Gampaha District.
Postgraduate Students Presentations
2015-01-01T00:00:00ZEngineering miR-shRNA based molecule to interfere replication of dengue virus in transgenic Aedes aegypti mosquitoes: Bioinformatics approach
http://repository.kln.ac.lk/handle/123456789/10899
Engineering miR-shRNA based molecule to interfere replication of dengue virus in transgenic Aedes aegypti mosquitoes: Bioinformatics approach
Ramyasoma, H.P.B.K.D.; Dassanayake, R.S.; Gunawardene, Y.I.N.S.; Kajan, M.; Abeyewickreme, W.
BACKGROUND: The genus Flavivirus of the family Flaviviridae includes several vector-borne viruses to which the four serotypes of dengue viruses (DENV-1,-2,-3 and 4) belong to and DENV viruses have a messenger like positive polarity, single-stranded RNA genome approximately 11kb in length which encodes three structural proteins (C-prM-E) and seven Non-Structural proteins (NS1-NS2A-NS2B-NS3-NS4A-NS4B-NS5). RNA interference (RNAi) and its properties as a tool has heralded a new era in functional genomics and short double stranded RNAs mediated by RNAi has become a powerful tool for post transcriptional gene silencing. Therefore, this study took the advantage the latter biological phenomenon and designed a multiple miR-shRNA (multi-mir-shRNA) molecule using bioinformatic approach to be effective to block the replication of all dengue serotypes of Sri Lanka. METHODS: Genome sequences of DENV strains belonged to serotypes 1 and 3 isolated from Sri Lanka deposited in GenBank were analyzed for potential sequences for the best siRNA target sites and identified two such sites from DENV 1 and DENV 3 from non structural protein coding sequence of NS5 and structural protein coding sequence of prM consensus regions, respectively. Two more siRNA targets reported from previous study chosen from upstream and downstream of non coding region effective for silencing all DENV serotypes were also used in designing mir-shRNA sequences. The stem region of miR1175 pre-miRNA sequence (miRBase ref: MI0013470) of Aedes aegypi was then replaced with each selected siRNA targets to generate the DENV effective miR-shRNAs and generated miR-shRNAs connected together by placing restriction endonuclease sites between each other to obtain multi-mir-shRNA containing four loop and stem structures. The transgenic gene cassette containing Ae. aegypti carboxypeptidase A promoter, multi-miR-shRNAs and poly adenylation signal of Simian Virus 40 Major Capsid VP1 was then constructed. Expression of this effecter molecule can be achieved by the induction of Ae. aegypti carboxypeptidase A (AeCPA) promoter following blood meal which then ensures activation of RNAi at the time of virus enter into the midgut of mosquito. RESULTS: Folding patterns of the transcript of the designed multi-mir-shRNA cluster were analyzed using online bioinfomatic tool, mfold and the secondary structure of this transcript shown to have optimum endogenous miRNA cleavge/processing with the lowest -ΔG indicating the ability of this design to exert RNAi in mosquito Ae. aegypti. CONCLUSION: Designing multi-miR-shRNA in bioinformatic means an effective way to construct the effector molecule that could exert the maximum RNAi against DENV. However, the effect of design will have to be demonstrated first by transforming to Ae. aegypti mosquitoes and then by evaluating the inhibition of DENV replication in mosquito.
Postgraduate Students Presentations
2015-01-01T00:00:00ZMaternal transformation of Wolbachia isolated from infected mosquito hosts to Aedes aegypti using micro-injection based procedure: an approach towards integrated dengue vector control
http://repository.kln.ac.lk/handle/123456789/10898
Maternal transformation of Wolbachia isolated from infected mosquito hosts to Aedes aegypti using micro-injection based procedure: an approach towards integrated dengue vector control
Wijegunawardana, N.D.A.D.; Gunawardene, Y.I.N.S.; Chandrasena, T.G.A.N.; Dassanayake, R.S.; Ruanareerate, T.; Kittayapong, P.; Abeyewickreme, W.
BACKGROUND: The maternal transformation of Wolbachia (Wb) to Aedes mosquitoes is an approach that has been widely used in Aedes mosquito population control strategies in the integrated dengue vector control efforts. This study details usage of such approach in the maternal transformation of Aedes aegypti using the Wb isolated from infected mosquito hosts using micro-injection based procedure for the first time in Sri Lanka. METHODS: Twenty five ovaries of each adult female Aedes albopictus and Culex quinquifaciatus mosquitoes (14-18 days old) were dissected and Wb was isolated and suspended in 20 μl of 1X Phosphate Buffered Saline (PBS). The isolated samples were kept on ice to maximize the bacteria viability. The exudate was then used in the trans-infection of Ae. aegypti mosquitoes. Infection of Wb into the mosquito Ae. aegypti was carried out by maternal/adult microinjection. To create stable trans-infected lines, the regions near the 3rd leg emerged from the thorax of Ae. aegypti were microinjected to incorporate Wb into the developing germ line and then to facilitate the transmission of Wb to offspring. The volume used for injection based on the swelling of the abdomen, which was monitored throughout the experiment while maintaining the injection pressure, time and number of injecting times. Virgin female Aedes mosquitoes survived after microinjection was fed with 1 ml of 10% sugar solution containing 10,000 units Penicillin/Streptomycin per 100 ml. Survival rate was monitored at 1, 3 and 24 hour/s following microinjection procedure. Survived female mosquitoes resulted from injected line (generation 0 [G0]) was placed in vials with three uninfected male mosquitoes to establish iso-female lines. After egg laying 3 times, G0 females were sacrificed and total DNA was extracted, and PCR was carried out (wsp primers 81F and 691R) using DNA extracted from Ae. aegypti to detect the presence of Wb DNA and its density in the latter mosquito using dilution series of extracted DNA. To select for a stable infection, only offspring from females that came positive for Wb by PCR screening was used as parental stocks. For the samples positive by wsp PCR, strain identification was carried out by performing PCR with 4 different primer sets; wPip, wAlbA, wAlbB and wsp specific for Wb. Females that tested negative for Wb were discarded along with their progeny. The resulting triple infected line will be monitored periodically by PCR to confirm infection status. RESULTS: Wolbachia-triple infected line of Ae. aegypti mosquito was created by using isolated wAlbA+wAlbB+wPip Wolbachia strains. Infection status of newly developed line was monitored by PCR up to G2 and will be continued to G8. Experimental work will be started from G5 to evaluate the use of newly develop Ae. aegypti mosquito line for Wb-based approach under laboratory condition. CONCLUSION: Direct maternal micro-injection technique was used to create Wb infected mosquito line of Ae aegypti, which could be applied in Wb-based dengue vector control activities in Sri Lanka once the line is established.
Postgraduate Students Presentations
2015-01-01T00:00:00Z