I authorize Chattogram Veterinary and Animal Sciences University to loan this thesis to other institutions or individuals for research purposes. I also give permission to reproduce the thesis in whole or in part by photocopying or otherwise, at the request of other institutions or persons for the purpose of scientific research. I express my sincere gratitude and greetings to my research supervisor Professor AMAM Zonaed Siddiki, Department of Pathology and Parasitology, Chattogram Veterinary and Animal Sciences University (CVASU) for his guidance, advice, suggestions and collaboration in conducting the Masters in public health (MPH) program.
With the necessary greetings, I thank the Head of the Department of Pathology and Parasitology and Director of One Health Institute, CVASU Professor Dr. Sharmin Chowdhury of Chattogram Veterinary and Animals Sciences University (CVASU) for the facilities provided to carry out this work. For effective control of various mosquito-borne diseases, the identification of different species of mosquitoes is crucial.
In this study, an attempt was made to apply the DNA barcoding method in addition to classical microscopy as a complementary method to identify mosquito species. Further sequence analyzes revealed successful identification of several mosquito genera, including Aedes and Culex.
Introduction
Different mosquito species are responsible for a lot of nuisance and transmission of deadly pathogens and parasites (Swan and Harding, 2017). Differences between species are visible in different phases of insect life, for example larvae can be identified by their abdominal segments, setae or siphon (Clements, 1992; Swan and Harding, 2017). In the transmission of disease, mosquitoes act as vectors for the spread of pathogenic organisms such as bacteria, viruses and other parasites.
Millions of deaths are caused by these widespread diseases such as West Nile fever, malaria, dengue and yellow fever in adults. Furthermore, morphological identification can be confusing if morphological features are damaged, such as damaged limbs or wings. Although DNA barcoding is promising, it is hampered by expensive equipment and specialized laboratories (Chan et al., 2014).
However, core genes are often more difficult to work with than mitochondrial genes because they are difficult to amplify via PCR. According to experts, there is a remarkable difference between nuclear genes, as they perform better compared to mitochondrial genes (Caterino et al., 2000).
Review of literature
- Vector Importance of Mosquitoes
- Mosquitoes of Bangladesh
- Morphotaxonomy of Mosquitoes
- Aedes aegypti
- Aedes albopictus
- Culex pipiens
- DNA barcoding of mosquitoes
- Parasites and vectors
Ricci et al. (2011) reviewed recent reports on the interdependent association between Wickerhamomyces anomalus (Pichia anomala) and several mosquito vector species as a possible method for implementing mosquito-borne disease control. Murugan et al., (2015) this research highlighted that compounds contained in seaweed are highly effective against larval populations of the filarial vector C. Reeves et al., (2016) all described the method of preservation, storage time and time after feeding. significantly affect the success of PCR amplification.
Khatun et al., (2015) suggested that rural populations in Bangladesh are at risk of emerging mosquito-borne diseases such as Chikungunya. Afroza et al., (2017) reported Aedes aegypti as a common prominent mosquito in Bangladesh which had a predilection for the skin of humans, animals and birds. Scutellum, on the side of the thorax and abdomen are numerous spots that are covered with white-silver colored scales.
The femur of each leg is black and the tip of the knee contains white scales. Longbottom et al., (1901) reported that DNA barcodes allow taxonomists to reconfirm the reference proof specimens. Cywinska et al., (2006) A short fragment of mt DNA from the cytochrome c oxidase 1 (CO1) region was used to provide the first CO1 barcodes for 37 species of Canadian mosquitoes.
Webster et al., (2012) reported that nicotinamide adenine dinucleotide dehydrogenase (NADH), nuclear internal transcribed space, cytochrome b oxidase and 12S rRNA were used as target genes for DNA barcoding. Chiang et al. (2014) demonstrated that DNA barcoding based on the mitochondrial COI gene is comparable to morphological identification for the differentiation of 45 analyzed mosquito species. Shahhosseini et al., (2018) reported the identification of different morphologically indistinguishable culex species and biotypes using multiplex real-time PCR.
Petersen et al., (2013) pointed out that the severity of infection varies significantly and fever develops in almost 25% of those infected. About two-thirds of individuals had significant weakness in the affected limb with paralysis. Linthicum et al., (2016) reviewed the significant threats of Rift Valley fever {(RVF), which is a mosquito-borne viral infection} to public health and agriculture in Africa and the Middle East.
Materials and Methods
- Study area
- Study period
- Collection of samples
- Microscopic examination
- DNA barcoding
- DNA Extraction
- Amplification of COI gene
- Gel Electrophoresis
From mosquito specimens, DNA was extracted using the Favorprep™ Tissue Genomic DNA Extraction Mini Kit according to the manufacturer's instructions. After air-drying, the entire mosquito body or some parts such as legs or wings were added to new eppendorf tubes and 200 μl of binding lysis solution was added to it. After incubation, 200 µl of ethanol concentrate (96–100%) was added to the mixture, then vortexed and Spin centrifuged for 30 seconds.
After centrifugation, we discard the lower part of the mixture (we discard the drops from the inside of the lid). Then the empty spin column was centrifuged at maximum speed at 13000 rpm for 3 minutes to remove the ethanol. Later, the mixture was centrifuged at 13000 rpm for 2 min and the DNA was collected in a new eppendorf tube.
Each individual mosquito or its parts were homogenized and DNA extracted using the procedure described above. Reactions were made in a 25 μl volume with 4 μl of extracted DNA, 2 μl of each primer, 12.5 μl of master mix (2X), and 4.5 μl of double distilled water or nuclease-free water.
Results
Microscopic and Molecular Study
- Microscopic and Molecular study
- Characterization of mosquitoes based on morphological characteristics
When the gender of the mosquito samples was considered, it revealed that the males were the most prominent (n=161). During careful microscopic examination, various characteristic features of different mosquito specimens were noted and considered for their identification. Aedes aegypti has two white lyre-shaped bands on its thorax (back), while Aedes albopictus has a white central band on its thorax.
Identification of adult Aedes albopictus are distinct silvery white scales and shiny black scales on the palpus and tarsi.
Molecular study
- Sequence analysis
- DNA Sequencing and BLAST alignment ................................................ 20-24
The freely available Chromas software was used to analyze sequence data that was confirmed by blast searching. The COI sequence of Aedes aegypti mosquito isolates submitted by others was obtained from NCBI.
There are a number of viruses that are transmitted by mosquitoes, Aedes aegypti and Aedes albopictus such as; Dengue virus, chikungunya, and the like have been reported in Bangladesh (Afroza et al., 2017). During this study, we observed the morphological characteristics of mosquitoes based on their shape, size and pattern such as head, proboscis, maxillary tip, antenna, thorax, wings, legs and abdomen etc. Similarly, Aedes aegypti mosquitoes are visually distinguished by having black and white markings on their bodies and legs.
All these characters were clearly observed and compared with previous reports for their eventual identification as Aedes aegypti. The scutellum is consistently rounded and the posterior margin of the wing is distinctly protruding opposite the termination of vein CuA. Culex quinquefasciatus has a light brown head with the thorax, proboscis, tarsi and wings darker than the rest of the body.
The present study attempted to identify the randomly collected mosquitoes from urban Chattogram by observing their morphological characteristics and analyzed the utility of DNA barcoding approach in vector surveillance by generating a barcode library of mosquitoes found in Bangladesh. With the well-known limitations of morphotaxonomy, the DNA barcoding method could be the most reliable tool for identifying different species. The ability to identify species from any life stage, including eggs, means that DNA barcoding is not only useful in surveillance programs but also biosecurity operations.
DNA barcoding could also be used with next-generation sequencing to identify large numbers of mosquitoes at once (ie, bulk sampling), thereby significantly reducing the processing time involved in species identification and nationwide surveillance. Studies on Aedes aegypti linnaeus occurring in Kolkata, India and its control by soil bacteria. Mapping the spatial distribution of the Japanese encephalitis vector, Culex tritaeniorhynchus Giles, 1901 (Diptera: Culicidae) within Japanese encephalitis risk areas.
Taxonomy of important arthropods infesting cattle in Bangladesh and pathological investigation of tick-borne hemoparasitic diseases of cattle in Chattogram region. Toxicity of seaweed-synthesized silver nanoparticles against the filariasis vector Culex quinquefasciatus and its effect on the predation efficiency of the cyclopid crayfish Mesocyclops longisetus. Maintenance of host DNA integrity in field-preserved blood meals of mosquitoes (Diptera: . Culicidae) for identification by DNA barcoding.
DNA barcodes confirming mosquito species identification and multiplex real-time PCR differentiating Culex pipiens complex and Culex torrentium in Iran. Vector competence of Aedes aegypti (L.) and Culex quinquefasciatus (Say) for Dirofilaria immitis (Leidy). 2009) Malaria prevalence in endemic districts of Bangladesh.
Discussion .................................................................................................... 25- 27
Recommendation and Future Perspective
