Abstract
Objective
To develop an artificial and modified Wolbachia removal technique using tetracycline from naturally Wolbachia infected Aedes albopictus (Ae. albopictus) so as to be able to produce generations of Wolbachia free offsprings.
Methods
In this study, seven different tetracycline treatment methods were conducted to obtain the best removal method. Four methods focused on larvae tetracycline treatment, one method on both larvae and adult tetracycline treatment and the last two methods on adult mosquito sucrose treatment.
Results
All larval tetracycline treatments resulted in either high larvae mortality, sterile F0 adult mosquitoes or unsuccessful Wolbachia removal. Treatment of both larvae and adults resulted in reduced larvae mortality, successful Wolbachia removal but slow mosquito fecundity. As for the adult treatment, 1.0 mg/mL as previously published was not able to completely remove Wolbachia in F1 generation whereas 1.25 mg/mL successfully removed Wolbachia from F1 and F2 mosquitoes in 2 weeks.
Conclusions
This method is different from the previously published methods as it provides an improved Wolbachia removal technique from Ae. albopictus with high egg hatchability, low larvae mortality, increased fecundity and better Wolbachia removal rate.
Keywords: Wolbachia, tetracycline, Aedes albopictus
1. Introduction
Wolbachia pipientis is an intracellular bacteria found in most of the arthropods, nematodes and isopods[1],[2]. They are vertically transmitted rickettsia endosymbiont bacteria[3]. In order to ensure the parasite being successfully transmitted maternally, Wolbachia tend to alter reproduction properties of their host[4]. Common alteration that have been reported are male killing, feminization, parthenogenesis and cytoplasmic incompatibility (CI)[5],[6]. Wolbachia modifies the spermatogenesis causing no viable offspring to be produced when infected male mates with uninfected female or female infected differently from the male[7]. Well understood CI can be used to reduce population of the host.
Aedes albopictus (Ae. albopictus) is an arthropod known to be naturally infected with Wolbachia pipientis bacteria. Most strains of Ae. albopictus screened in Malaysia were superinfected with both two Wolbachia strains (wAlbA and wAlbB). Aedes aegypti and Ae. albopictus are the major vectors for dengue in Malaysia. They are lethal vectors which transmit many deadly pathogens including dengue fever virus, chikungunya fever virus and West Nile Virus[8]. The combination of dengue blocking activity and rapid spread due to CI has led researchers to suggest that Wolbachia can be used to develop public health strategies to reduce dengue incidence in human[9],[10].
In order to study the CI and effect of Wolbachia on Malaysian Ae. albopictus, it is necessary to have both Wolbachia infected and uninfected strains.
Ae. albopictus is a species naturally infected with Wolbachia therefore obtaining a natural strain without Wolbachia would be very rare[11]. Therefore an artificial Wolbachia removal technique is needed.
Previous studies have suggested treatment of larvae with tetracycline antibiotic to remove Wolbachia from Ae. albopictus[3],[11]. However, reduced fecundity and egg viability was observed when the above mentioned method was implemented. Another study proposed treatment of only the adult mosquito with tetracycline antibiotic[11]. This managed to overcome the reduced fecundity and egg viability issue. However, when the method was implemented, the resulting offsprings were found not to be totally free from Wolbachia.
In this study, a modified Wolbachia removal method from Ae. albopictus is reported. It has minimal effect on the mosquito fecundity and egg viability and was able to produce generations of Wolbachia free offsprings.
2. Materials and methods
2.1. Mosquito strain
A strain of Ae. albopictus obtained from Bukit Lagong, Selayang, Kuala Lumpur, Malaysia in August 2013 was used in this study. Mosquitoes were maintained in cages with 10% sucrose with 100 mg B-Complex solution. They were blood fed and eggs were collected weekly. Mosquito infection status was confirmed using polymerase chain reaction (PCR) amplification and sequencing.
2.2. Infection status
A minimum of 30 blood fed mosquitoes were randomly caught for each new generation, blood fed, allowed to lay eggs and then extracted using Dneasy Blood and Tissue Extraction Kit according to the protocol provided by the manufacturer (Qiagen, CA, USA). Extracted DNA were stored at -20 °C until needed. All samples were screened for the presence of Wolbachia using multiplex PCR with Promega (Promega, Madison, WI) reagents for amplification of the wsp gene with diagnostic primers (Genomics BioSci & Tech, China).
The wAlbA strain gene was amplified with the wsp 328F and 691R primer pair whereas wAlbB strain gene was amplified with the wsp 183F and 691R primer pair. PCR was conducted in a 20 µL reaction per individual. This consisted of 10 µL ddH2O, 4 µL 5X Green GoTaq® Flexi Buffer, 1.6 µL magnesium chloride, 0.4 µL dNTPs, 0.6 µL of each primer (183F, 328F and 691R), 0.2 µL of GoTaq® Flexi DNA polymerase and 2 µL template. Samples were denatured for 5 min at 94 °C, followed by 35 cycles of 1 minute at 94 °C, 1 min at 55 °C and 1 min at 72 °C. A negative control was run along with each batch of PCR amplification by substituting 2 µL of sample with 2 µL of ddH2O[12].
A total of 8 µL of each sample was run in 1% agarose gel to detect the presence of amplified DNA fragments. One hundred kilobyte ladder (Promega, Madison, WI) was used to confirm presence of wAlbA (363 bp) and wAlbB (508 bp) genes[12] (Figure 1).
Figure 1. Gel electrophoresis result image. Lane 1 is the 100 kB ladder. Lane 2-6 are my samples.
The upper row are wAlbB amplified gene at 508 bp and the lower row bands are wAlbA amplified gene at 363 bp.
2.3. Tetracycline treatment
All Wolbachia removal studies were conducted on strains with confirmed wAlbA and wAlbB superinfection. Studies were conducted as stated in Table 1. Treatment 1 was conducted as previously described by Otsuka and Takaoka in 1997[3]. Treatment 5 and 6 were conducted as described previously by Dobson and Rattanadechakul in 2001[11]. Treatments 2, 3, 4 and 7 consisted of a modified technique were conducted by this group. Larvae after the treatment period in treatment 1, 2, 3 and 4 were transferred back into water without tetracycline and reared to adulthood. In each treatment, randomly caught 25 blood fed adult mosquitoes were allowed to lay eggs first and then tested for presence of Wolbachia using PCR method as mentioned above. If no Wolbachia infection was found in all tested mosquitoes, the eggs obtained were hatched. Larvae after 24 h treatment in treatment 5 was transferred back into water without tetracycline and reared to adulthood.
Table 1. Tetracycline treatment design.
| Treatment | Life cycle stage | Treatment period (h) | Concentration of tetracycline |
| 1[a] | 24th-48th hour larvae | 24 | 5.00 mg/mL in 2 L overnight water |
| 2[c] | 24th-72nd hour larvae | 72 | 1.25 mg/mL in 2 L overnight water |
| 3[c] | 48th-120th hour larvae | 72 | 1.25 mg/mL in 2 L overnight water |
| 4[c] | 48th-72th hour larvae | 24 | 1.25 mg/mL in 2 L overnight water |
| 5[b] | 48th-72th hour larvae | 24 | 1.25 mg/mL in 2 L overnight water |
| Newly emerged adult mosquito | Continuous | 0.50 mg/mL in 10% sucrose solution with 100 mg B-Complex | |
| 6[b] | Newly emerged adult mosquito | Continuous | 1.00 mg/mL in 10% sucrose solution with 100 mg B-Complex |
| 7[c] | Newly emerged adult mosquito | Continuous | 1.25 mg/mL in 10% sucrose solution with 100 mg B-Complex |
Adult mosquitoes in treatment 5, 6 and 7 were blood fed after two weeks and one month for egg collection. Twenty five mosquitoes from which eggs were collected were tested for presence of Wolbachia using PCR method as mentioned above. Eggs collected from the treatment 5, 6 and 7 were allowed to hatch in 2 L overnight water. Egg hatching rate were calculated to determine egg viability for each treatment. Once the F1 generation mosquitoes were obtained, 25 blood fed adult mosquitoes were randomly caught from each colony, allowed to lay egg first and then tested for presence of Wolbachia using PCR method as mentioned above. Average Wolbachia infectivity of F1 for treatment 6 and 7 was obtained by calculating the mean infected mosquito numbers for three replicates of Wolbachia testing. The same calculation was done for average Wolbachia infectivity of F2 for treatment 7.
3. Results
The strain of Ae. albopictus used in this study had 100% both wAlbA and wAlbB infection. Figure 1 shows the result of PCR amplification when both wAlbA and wAlbB is present. The F4 eggs were used in this Wolbachia removal study.
Percentage of eggs hatched that survived to pupation, percentage of adult mosquitoes emerged, percentage of Wolbachia infection status of the F0 and percentage of F1 eggs hatched were calculated for all treatments 1-7. Results are shown in Table 2.
Table 2. Percentage eggs hatch in F0 and F1 tetracycline treated strains.
| Treatment | No. of eggs (F0) | Pupae after 5 d | Adult mosquito (F0) | Wolbachia infection status | No. of eggs obtained (F1) | Eggs hatch |
| 1 | 132 | 4 (3.00%) | 4 (100.00%) | All 4 Wolbachia free (100.0%) | 0 | NA |
| 2 | 111 | 6 (5.41%) | 5 (83.30%) | All 5 Wolbachia free (100.0%) | 0 | NA |
| 3 | 122 | 42 (34.43%) | 32 (76.19%) | All 25 tested Wolbachia free (100.0%) | 5 | 0 (0.00%) |
| 4 | 142 | 88 (61.97%) | 83 (94.32%) | 13 out of 25 Wolbachia free (52.0%) | 29 | 0 (0.00%) |
| 5[x] | 145 | 82 (56.55%) | 78 (95.12%) | All 25 tested Wolbachia free (100.0%) | 230 | 41 (17.83%) |
| 6[y] | 153 | 107 (70.00%) | 105 (98.13%) | All 25 tested Wolbachia free (100.0%) | 249 | 142 (57.03%) |
| 7[z] | 143 | 92 (64.34%) | 88 (95.65%) | All 25 tested Wolbachia free (100.0%) | 189 | 98 (51.85%) |
[x]: Emerged mosquitoes were treated with 0.5 mg/mL tetracycline treated sucrose solution. F0 adult mosquitoes were only Wolbachia free after 1 month of treatment.
[y]: Emerged mosquitoes were treated with 1.0 mg/mL tetracycline treated sucrose solution. F0 adult mosquitoes were only Wolbachia free after 1 month of treatment.
[z]: Emerged mosquitoes were treated with 1.25 mg/mL tetracycline treated sucrose solution. F0 adult mosquitoes were Wolbachia free after 2 weeks of treatment. NA: Not applicable.
Treatment 5, 6 and 7 had F1 eggs therefore studies were continued to obtain the percentage of F1 adult mosquitoes, Wolbachia infectivity status of F1 colony and Wolbachia infectivity status of F2 colony (only treatment 7). Results for this continuation studies are shown in Table 3.
Table 3. Wolbachia infectivity status of F1 and F2 tetracycline treated strains.
| Treatment | No. of larvae | No. of adults (F1) | Average Wolbachia infectivity of F1 | Average Wolbachia infectivity of F2 |
| 5 | 41 | 32 (78.05%) | All 25 tested Wolbachia free (100.0%) | Not applicable because no eggs was obtained |
| 6 | 142 | 130 (91.55%) | 18 out of 25 Wolbachia free (72.0%) | Not applicable |
| 7 | 98 | 92 (93.88%) | All 25 tested Wolbachia free (100.0%) | All 25 tested Wolbachia free (100.0%) |
4. Discussion
Tetracycline is a group of broad-spectrum antibiotics. Its overall usage has been reduced with the increasing bacterial resistance[13]. Since Wolbachia is an endosymbiotic bacteria, tetracycline at the right concentration and delivery method should be able to remove them from their respective hosts. This concurs with previous studies conducted[11].
Treatment 1 which was conducted based on Otsuka method was not effective in this study as it caused low egg viability, high larval mortality and sterile adult mosquitoes[3]. Same issue have been reported by Dobson and Rattanadechakul in 2001[11]. This may have been due to the high concentration of the tetracycline used to treat the larvae.
Similar larval treatments were carried out with reduced concentration to 1.25 mg/mL in treatment 2, 3 and 4 at different exposure periods.
High larval mortality was observed when larvae were treated for more than 24 h. However, improved larval mortality was observed when the 48 h larvae were treated instead of the 24 h larvae. This may be because 24 h larvae are too young to withstand the tetracycline treatment.
Treatment 4 was designed to expose 48 h larvae for 24 h which gave lower larval mortality and a higher percentage of adults.
Although low larval mortality was observed, the treatment failed to remove Wolbachia completely from all surviving adults. Therefore it can be concluded that perhaps the period of treatment or tetracycline concentration was not sufficient.
Treatment 5 was conducted based on Dobson report in 2001 which subjects both larvae and adult mosquitoes tetracycline[11]. This method had low larval mortality and was able to completely remove Wolbachia from all surviving F0 adults. A good number of F1 eggs were obtained but the hatching rate of the F1 eggs was very low compared to untreated strains.
Treatment 6 was conducted based on the final method from Dobson paper in 2001 which treats only the adult with 1.0 mg/mL[11]. No alternative food source was provided for the mosquitoes. F0 Adult mosquitoes were tested for Wolbachia after 2 weeks exposure to tetracycline sucrose treatment. Mosquitoes were not found to be completely free of Wolbachia. F0 adult mosquitoes were again tested for Wolbachia after 1 month tetracycline treatment and all were Wolbachia free. Eggs were collected and F1 mosquitoes were obtained. Although the experiment was repeated three times, we failed to obtain entirely Wolbachia free F1 adult mosquitoes. Therefore treatment 6 as proposed by Dobson was not effective in this study.
Treatment 7 was designed exactly as treatment 6 with a slight increment of the concentration of tetracycline in the sucrose solution. Complete Wolbachia removal from the F0 adult mosquitoes was observed in two weeks tetracycline treated mosquitoes. This was confirmed with two replicates. Egg hatching rate was slightly lower than treatment 6 and 93.88% became F1 adults. In contrast to treatment 6, F1 adults were 100% Wolbachia free. Average was obtained from three replicates. All F2 adults was also found to be Wolbachia free.
Tetracycline treatment of only adult mosquitoes simplifies the process, improves the egg hatchability, reduces larval mortality and increases adult fecundity. The best concentration for the adult treatment is concluded to be 1.25 mg/mL in sucrose solution with no alternative food source. This method is able to remove both wAlbA and wAlbB completely in just two weeks and gives subsequent generations free of Wolbachia.
This self-sustaining Wolbachia free Ae. albopictus colony developed can be used to study the effect of Wolbachia on Malaysian Ae. albopictus. Future research may be conducted to develop a singly infected Ae. albopictus strain with a modified antibiotic treatment as none has been established so far.
Acknowledgments
This project have been financially supported by University Malaya Research Grant (UMRG) with grant NO.: RG372/11HTM.
Comments
Background
There is increasing interest on Wolbachia endosymbionts in Aedes vectors as they are related to fecundity and to dengue transmission. There is a need to obtain strains of Ae. albopictus free from endosymbionts but existing methods through treatment of larvae with tetracycline have not been satisfactory.
Research frontiers
An improved method to obtained Wolbachia free and viable Ae. albopictus mosquitoes for further studies.
Related reports
Although the use of tetracycline to obtain Wolbachia free Aedes has been previously studied, the dosage and methods used did not produce satisfactory results.
Innovations and breakthroughs
An improved method of using tetracycline in obtaining subsequent generations of Wolbachia free Ae. albopictus.
Applications
This study is important for research on Wolbachia and dengue susceptibility.
Peer review
The study has provided a viable method to produce Wolbachia free Ae. albopictus which is increasingly implicated as a vector of dengue transmission in many endemic countries. Susceptibility studies with dengue serotypes in Wolbachia positive or negative mosquitoes will be easily available now.
Footnotes
Foundation Project: Supported by University Malaya Research Grant (UMRG) with grant NO.: RG372/11HTM.
Conflict of interest statement: We declare that we have no conflict of interest.
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