New Approach For Vector Control Biology Essay

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Dengue (DENV) is an important arboviral disease consisting of four serotypes (Dengue 1, 2, 3, and 4) that are endemic in more than 100 countries in tropical and sub-tropical regions worldwide. Currently almost two fifths of the world's population is threatened with DENV and an estimated 50 million infections occur worldwide each year. Dengue is acquired by female Aedes aegypti mosquitoes when they obtain a blood meal from infected host. Over an incubation period of four to seven days, the virus travels from a mosquito's midgut to its salivary gland where the virus can then be injected into another person during subsequent blood feeding (WHO. 2009). Currently there are no effective vaccines available for treatment of dengue; prevention relies mainly on vector control as the only solution.

A new approach for vector control is through the introduction of the bacterial Wolbachia pipientis into Ae. aegypti populations. The bacterial Wolbachia pipientis is intracellular maternally inherited and is predicted to naturally infect more than 60% of all insect species worldwide (Hilgenboecker, Hammerstein et al. 2008). Wolbachia pipientis can invade host populations by positively influencing host fitness (Dobson, Rattanadechakul et al. 2004; Weeks, Turelli et al. 2007; Brownlie, Cass et al. 2009). Infection in Ae. aegypti with Wolbachia has proven to be effective in limiting dengue virus replication within the mosquitoes (Moreira, Iturbe-Ormaetxe et al. 2009; Bian, Xu et al. 2010). The mosquitoes infected with Wolbachia are being developed for field release to replace wild mosquito populations that are uninfected with the bacterium. If the Wolbachia infected mosquitoes spread into wild populations, then they will effectively be "vaccinated" against dengue.

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The midgut of Ae. aegypti mosquito contains diverse range of microorganisms' especially rod-shaped gram-negative bacteria in the midgut along the blood digestion process (Gusmao, Santos et al. 2010). These microorganisms residing along the midgut may have important roles for insect nutrition, reproduction, development, behaviour and resistance to pathogen colonization and immune capability (Dillon and Dillon 2004). Studying the roles of these microorganisms is essential in understanding the effect of these microorganisms to mosquito viability and physiology. In this study, we aim to develop and assay to investigate any differences in microorganisms between Ae. aegypti infected with the wMel strain of Wolbachia that blocks dengue (unpublished data) in comparison to a control Wolbachia cured line, wMel.tet. We have developed and employed a high-resolution melt analysis (HRMA) based on bacterial 16s rRNA to differentiate these high species specific microorganisms which reside in the midgut of female Ae. aegypti mosquito (Yang, Ramachandran et al. 2009).

Materials and Methods

For this experiment, two laboratory lines of Ae. aegypti were used, the wMel strain of Wolbachia, previously generated by transinfection with wMel strain of Wolbachia and wMel.tet, Wolbachia cured control line. The mosquitoes were maintained at 25oC, 75%-85% relative humidity, with 12:12 hours light: dark photoperiod. Ten mosquito female pupae were collected and reared inside a 15 by 15 cm cage. The females' mosquitoes were fed with 10% sucrose solution until it is ready for extraction.

After the mosquitoes reach the age of five days old, they were dissected under the dissecting microscope (ZEISS - SFEMI 2000), on a glass slide containing sterile PBS. The midgut was carefully removed from the mosquito abdomen and transferred into a 1.5mL tube containing 200µl of PBS. Mosquito DNA were extracted using DNeasy spin columns (QIAGEN, Australia); following the manufacturers' spin column protocol: Purification of total DNA from animal tissues.

Primers used in this experiment were designed to amplify specific regions of the bacterial 16s rRNA sequence. Genetic 16s sequences of 10 commonly found midgut bacterial in Ae. aegypti (Gusmao, Santos et al. 2010) were extracted from Genbank (http://www.ncbi.nlm.nih.gov/genbank/). The bacterial were further grouped into 4 distinct family of bacterial (See Appendix A for taxonomy tree) and their sequences were aligned using ClustalW (http://www.ch.embnet.org/software/ClustalW.html). Aligned 16s sequences were reviewed for possible conserved regions for primers designing and three separate sets of primers were designed for this experiment (See Appendix B for exact primers sequences).

The primers were tested to be working by running the primers with generic RPS17 DNA under polymerase chain reaction (PCR) followed by 1% gel electrophoresis. The PCR reaction conditions were as follows: 1.0µl of RPS17 DNA, 2.0µl of 10 x Buffer, 0.5µl of 1mMdNTPs, 0.5µl of 20µM 16s primers, 0.15µl Taq DNA polymerase and water up to 20µl. Cycling parameters for PCR reactions include initial denaturation step at 94oC for 3 minutes, followed by 34 cycles of a denaturation step at 94oC for 30 seconds(s), primer annealing step at 55oC for 30s, an extension step at 72oC for 60s and a final elongation at 72oC for 10 minutes. A volume of 13µl from each PCR reaction was separated on 1% agarose gels, stained with ethidium bromide and visualised under ultraviolet illumination.

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Each sample was subjected to high- resolution melting analysis on the LightCycler 480 instrument (Roche). Prior to setting up the experiment, mosquito midgut DNA concentration was measured by spectrophotometer (Nanodrop ND - 1000 spectrophotometer) to ensure consistency in the genomic DNA used. Samples with higher than 3-4ng/µl were diluted with buffer AE used during the extraction process to the optimum concentration (5 - 30ng per 20µl reaction). Magnesium concentration used in this experiment is 2mM instead of 1.5mM after a gel electrophoresis was done with 1.5mM concentration and showed poor sample amplification. Each PCR analysis was performed in a 20µl total volume comprised of 10µl of 2 x concentration master mixes, 1µl of 20x concentration of each primer, 1.6µl of 25mM MgCl2, 2.4µl of PCR-grade water and 5µl of sample DNA. The PCR master mix contains FastStart Taq DNA polymerase, reaction buffer, dNTP mix (with dUTP) and high resolution melting dye containing SYBR Green I (Roche). Each PCR analysis contained one primer pair.

The PCR reaction was performed using LightCycler 480 Real-Time PCR system (Roche). Cycling conditions were as follows: Pre-incubation at 95oC for 10minutes, followed by 45 cycles of a denaturation step at 95oC for 10s, primer annealing step at 65oC for 15s, an extension step at 72oC for 10s. High resolution melting was done at 95oC for 1 minute, 40oC for 1 minute, 50oC for 1s and 95oC with data acquisition performed 25 times per 1oC increase in temperature. Lastly ending with cooling at 40oC for 10s. HRMA for each PCR sample were performed in duplicate and analyzed using LightCycler 480 gene scanning software. All results were analyzed using LightCycler 480 gene scanning software with analysis method: Tm calling for all samples. All melting peaks data includes Tm, area, width and height were acquired from all samples and recorded in a spreadsheet. Statistical analysis using t test was done for all melting peak results.

Results

In this study, we aim to identify any microorganism's differences between Wolbachia infected mosquitoes (wMel) and Wolbachia cured line (wMel.tet). Melting peaks of Wolbachia infected mosquitoes under primer 1 condition shows mostly single peak with a Tm ranging from 85 to 88 (FIG 1A). In comparison, melting peaks of Wolbachia cured mosquitoes under primer 1 shows mostly single peak with a Tm ranging from 85 to 88 (FIG 1B). Melting peaks of Wolbachia infected mosquitoes under primer 2 conditions shows mostly triple peaks with a Tm ranging from 86 to 88 (FIG 1C). In contrast, melting peaks of Wolbachia cured mosquitoes under primer 2 condition shows multiple variable peaks with primary peak Tm ranging from 86 to 87 (FIG 1D). Lastly for Wolbachia infected mosquitoes under primer 3 conditions, melting peaks were mostly single peak with a Tm range of 85 to 86 (FIG 1E). In comparison, melting peaks of Wolbachia cured mosquitoes under primers 3 condition shows mostly single peaks with a Tm range of 85 to 86 (FIG 1F) (Table 1). In addition, melting peak analysis of Wolbachia infected line revealed no distinct expression of Wolbachia bacterial peak in analysis.

All peaks recoded in this experiment were label with a peak number using Tm as a gauge for each category. In peak 1, the Tm ranges from 85 to 87.99, for peak 2, the Tm ranges from 83 to 84.99, for peak 3, the Tm ranges from 80 to 82.99 and lastly for peak 4 the Tm are any peaks below 80. Scores were recorded for each peak and the results are shown in Table 1.

 

Primary Peak

Secondary Peaks

 

Peak 1

(85 - 87.99oC)

Peak 2

(83 - 84.99oC)

Peak 3

(80 - 82.99oC)

Peak 4

(<80oC)

Primer 1

wMel

100%

20%

20%

20%

wMel.tet

100%

10%

20%

10%

Primer 2

 

 

 

 

wMel

100%

70%

60%

10%

wMel.tet

90%

50%

10%

50%

Primer 3

 

 

 

 

wMel

100%

0%

10%

0%

wMel.tet

100%

0%

0%

10%

Table 1: Analysis of the percentage of individual peaks amplified by HRMA

Statistical analysis was done by comparing Tm values with each individual peak between Wolbachia infected and Wolbachia cured lines. This was only done only on peak one as there were not enough samples for statistical analysis from peak two to four. Both Wolbachia infected and Wolbachia cured lines under primer set one (n = 10, t = 1.20, df = 18, P = 0.244) and primer set two (n =9, t = -0.744, df = 17, P = 0.467) conditions respectively revealed no significant differences between them. However for samples run under primer set three (n = 10, t = -2.63, df = 18, P = 0.0171), significant differences can be seen and shows that there were more diversity between samples run under primer set three (FIG 2).

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FIG 2: Tm values differences between Wolbachia infected (wMel) and Wolbachia cured lines (wMel.tet). Bars represent means + sem of individual primer set. *P = 0.0171 by t test. N = 9 to10 samples per primer set.

Discussion

Microorganisms' diversity found within the midgut of female Ae. aegypti are essential in understanding how diversity of midgut microorganisms could play a part in mosquito viability and physiology. In this experiment, melting peak analysis generated by HRMA serves as an indication for different species diversity identified between both mosquitoes' lines. Results obtained from melting peak analysis revealed no significant differentiation between Wolbachia infected and Wolbachia cured line. In addition, there was no distinct revelation of Wolbachia melting peak seen in all Wolbachia infected line which should have been.

Melting peaks analysis between Wolbachia infected and Wolbachia cured lines should revealed a clear distinction of Wolbachia bacterial peak in Wolbachia infected line as compared to Wolbachia cured line. However this was not apparent which is different from what we have hypothesised. The detection of Wolbachia serves as an internal positive control for the methods. The failure to detect these differences can be explained by the similarity in genetic sequences of the primers products between Wolbachia and the four major families of bacterial found in midgut of Ae. aegypti. It also does not bode well for our ability to detect the presence of other major subgroups of bacteria in the gut which are unknown to us.

When Wolbachia pipientis 16s genetic sequences were aligned with the four major families of bacteria found in midgut of Ae. aegypti, it was found that the average genetic distance between Wolbachia pipientis and the four major families of bacterial for primer 2 and 3 was 5.9% and 8.2% respectively. Genetic distance was not calculated for primer 1 due to the large deletion in Wolbachia gene within the primer 1 sequences. Better differentiation should be seen for samples under primer set 3 and this is reflected by the statistical significant difference in results seen for samples run under primer 3. However there is still no clear differentiation seen for primer set 3 and this could be because the genetic distance is not diverse enough to show a clear differentiation of the different peaks despite being significantly different. Therefore it is believed most of the peak 1 seen in all primers combination for Wolbachia infected lines contains Wolbachia pipientis and other genetically similar bacterial found in the midgut but they could not be differentiated.

Future approaches

In any future experiments, both culture dependent and culture independent methods can be used to identify the bacterial diversity found within midgut of female Ae. aegypti. For culture dependent methods, midgut bacterial found in female Ae. aegypti can be culture on LB agar plate, isolated and perform PCR to correlate with peak of interest found in HRMA.

For culture independent methods, we may employ a Phylochip method, which would involve DNA-DNA hybridisation of mid-gut extracted DNA to an oligonucleotide array containing probes to all known genera of bacteria. This method is very sensitive and can even detect bacteria that are rare in the gut. Alternatively, we may employ pyro-sequencing which is less able to detect rare species but has the added benefit of providing a relative quantification of difference bacteria present and can detect bacteria that are unknown or not previously characterised.

(Word count: 1954)