Transmission rates of the gene driver, Wolbachia, under different
temperature regimes
Abstract. The prevalence of insect-borne diseases, such as malaria, stresses the need to
develop effective pest-control methods. Much potential lies in gene drivers which
facilitate the introduction of novel genes into a population. The maternally-inherited,
endosymbiotic bacterium Wolbachia is being investigated as a gene driver because
infected female hosts acquire a reproductive advantage over uninfected female hosts and
spread Wolbachia through the population. This study examined how temperature affected
the transmission rates of Wolbachia in populations of Drosophila melanogaster.
Experimental vials were established with 25% Wolbachia-infected flies: 75% uninfected
flies and held at either 24°C or 26°C. Transmission rates were measured using PCR
analysis of the fly offspring which determined the presence or absence of the Wolbachia
specific wsp gene. The results show that at 24°C the percent infected was 11%,
significantly lower than 43% infected at 26°C; transmission rates (change in percent
infected after one generation) corresponding to each temperature were -14% and 18%,
respectively. Clearly temperature could be an important factor in the implementation of
Wolbachia as a gene driver. Further research on the dynamics of Wolbachia transmission
as affected by environmental conditions is central to assessing its feasibility as a gene
driver.
Keywords: temperature, Wolbachia, gene driver
1
Introduction
The limitations of pesticides in combating diseases like malaria and dengue stress the
need to develop new control strategies. Researchers have increasingly sought the use of
transgenic insect vectors that are modified to prevent disease transmission to humans
(Kittayapong et al. 2002). Since it is economically impractical to release large numbers of
genetically modified insects, a drive mechanism is preferable to ensure spread of the gene
throughout the host population. Successful population replacement systems must drive effector
genes to fixation within an appropriate time frame, induce no destructive impacts on either the
host or bystander species, and counteract the possible development of resistance over time
(Sinkins and Gould 2006).
The bacterium Wolbachia is being studied as a gene driver in insect host populations.
Wolbachia are maternally-inherited, bacterial endosymbionts that induce cytoplasmic
incompatibility (CI) in the host; CI significantly reduces the number of eggs hatched from
matings between infected males and uninfected females (Rasgon and Scott 2003). Infected
females produce viable progeny when mated with either infected or uninfected males due to a
rescue mechanism that allows fertilization (Sinkins and Gould 2006). Subsequently females
harboring Wolbachia have a direct reproductive advantage over uninfected females which allows
the infection to spread overtime (Kittayapong et al. 2002). The inherited bacterium shows wide
host diversity, occurring in 16-22% of insect species (Werren and Windsor 2000), and has been
found within the insect order Diptera (Dyson et al, 2002), which includes mosquitoes and fruit
flies.
2
In order for Wolbachia to serve successfully as a gene driver, the penetrance of CI must
be high to confer a reproductive advantage to infected females. Various studies have shown a
decrease in CI effectiveness due to host genotype interactions with Wolbachia. A study on
Drosophila melanogaster done by Yamada et al (2007) concluded that males that require longer
developmental time expressed weak CI while neither male age nor Wolbachia densities in testes
affected strength of CI. In Culex pipiens, Wolbachia will spread to fixation within the population
if initial infection exceeds 1.4 percent, however, parameters such as strong CI and female
fecundity may change according to species-specific host interactions (Rasgon and Scott, 2003).
Environmental factors such as temperature directly affect the host-symbiont associations
of Wolbachia (Mouton 2006). Temperature serves as a critical determinant in that it impacts
nearly all biological processes, including generation time. Temperature criteria in transmission of
Wolbachia have not been adequately analyzed, partly because varied temperature effects were
shown in several different experiments (Mouton 2006; Hurst et al 2000). In this study, the effects
of temperature on the transmission rate of Wolbachia were examined in populations of D.
melanogaster as tested in two temperature regimes.
Materials and Methods
Yellow-body white-eye Drosophila melanogaster colonies
A university lab provided the Wolbachia pipientis strain wMel infected yellow-white
flies and uninfected yellow-white flies were obtained from Carolina Biological. All laboratory
Drosophila colonies were reared in fly vials and fed Carolina Biological fly diet. All colonies
were initially reared in 24 °C in an environmental chamber on a 14:10-hr light: dark cycle. The
3
infected and control flies were kept in separate vials on opposite sides of the chamber. The flies
were maintained for two days in the chamber after which they were separated into four
categories: 1) W+ 24 °C - colonies that contained 25 percent infected flies at room temperature
(24 °C), 2) W- 24 °C - colonies that contained only uninfected (control) flies at room
temperature, 3) W+ 26 °C - colonies that contained 25 percent infected flies at 26 °C, and 4) W26 °C colonies that contained only uninfected (control) flies at 26 °C. Each category included
four separate vials, each filled with 16 adults, 8 male and 8 female. Care was taken to place vials
with infected flies away from vials with only control flies and a separate set of equipment
(paintbrushes, etc.) was used to avoid cross contamination. After 24 hours, adult mortality rate
was observed and more vials were added to each category to substitute those that had more than
2 dead flies. New vials were added; two to W- 24 °C; one to W- 26 °C; two to W+ 26 °C. Adult
mortality was again observed after the second day but no additional vials were set up. Four vials
with the lowest adult mortality as previously recorded from each experimental group were
chosen for DNA extraction while only two vials from each control group were chosen.
DNA extraction
DNA was extracted from 20 randomly chosen individual flies from each vial using
Promega Wizard animal tissue DNA extraction kit (the manufacture’s protocol can be found at
http://www.promega.com/tbs/tm050/tm050.html).
Flies
were
placed
individually
into
microcentrifuge tubes, frozen with liquid nitrogen, and then ground by hand before digesting
with proteinase K. The DNA was reconstituted in standard TE buffer and stored at -20 °C until
used for PCR amplification.
4
Polymerase Chain Reaction and DNA Analysis
Polymerase chain reaction analysis was carried out for 20 randomly selected flies from
each of eight vials in the experimental groups and four vials in the control groups for a total of
240 flies. The PCR reaction was multiplexed with wsp gene primers to amplify a 610 bp
fragment and COI housekeeping gene (cytochrome oxidase) primers to amplify a 658 bp
fragment. A single-copy gene wsp that encodes a specific surface protein of Wolbachia was used
to detect the parasite’s presence. The cytochrome oxidase gene ubiquitous in all arthropod
mitochondria was used as a negative control to test for successful amplification. Primers were
specifically designed to amplify regions of the wsp gene and CO1 gene (see Table 1). PCR was
performed under the following conditions: Step 1 - 95 °C for 2 min, Step 2 - 94°C for 1 min,
Step 3 - 53 °C for 1 min, Step 4 - 72 for 1 min, repeat from Step 2 for 34 cycles, Step 5 - 72 °C
for 5 min, and then held at 4°C. The PCR included 12.5 ul of GoTaq Master Mix (Promega), 1 ul
of each 10 uM wsp primers, 2 ul of each 5 uM CO1 primers, and 2.5 ul of template DNA (<250
ng) brought up to a 25 ul volume with nuclease-free water.
After three hours of PCR, samples were frozen at -20 °C until DNA gel electrophoresis.
Forty PCR samples were dry loaded into a 50-well D3-14 Owl gel rig at a time along with one
BenchTop DNA ladder (Promega) at each end. Ten ul of each sample was loaded; 5 ul of ladder
was loaded. A 0.5 cm thick 2% agarose gel was made from 10.00 g of agar dissolved in 500 ml
1x TBE buffer from which 161 ml of the solution was poured to make the gel. Approximately
800 ml of 1x TBE buffer was poured on to the loaded gel. The gel rig ran for approximately 3
hours at 72 volts. Gels were scored by observing size of bands relative to the ladder. Lanes that
had one band for CO1 were scored as uninfected, lanes that had two bands (one for wsp and one
5
for CO1) were scored as infected, and lanes that had only one band for wsp were also scored as
infected. Lanes that had no bands were dropped from the analysis because of lack of
amplification.
Table 1 Fly primers. Sequences are written 5’ to 3’.
Primer
Sequence
wsp81 F
TGG TCC AAT AAG TGA TGA AGA AAC
wsp691 R
AAA AAT TAA ACG CTA CTC CA
LCO1490
GGT CAA CAA ATC ATA AAG ATA TTG G
HCO2198
TAA ACT TCA GGG TGA CCA AAA AAT CA
Data Analysis
A chi-square test was performed to determine if the number infected was different
between the experimental treatments under 24°C and 26°C. The expected number infected was
based on the original 25 percent infected.
Results
Fly mortality for each vial was recorded one day after the flies were separated into their
treatments. Mortality rates were similar for each vial: averages were obtained for each
temperature treatment and were used to calculate percent survival as shown in Table 2.
6
Table 2 Fly Mortality. Vials were monitored for dead flies after 24 hours from experimental setup. +/indicate presence or absence of Wolbachia, respectively. Means are shown with one standard error.
Temperature (°C)
24
26
Wolbachia
+
+
-
Avg. # dead/vial
0.75 ± 0.48
3.00 ± 1.41
2.50 ± 1.04
1.25 ± 0.75
% Survival
95.3
81.2
84.4
92.2
The number of infected flies and uninfected flies were determined by scoring the gels, on which
each lane contained DNA extracted from one individual fly (Figure 1). The different sized bands
for wsp and CO1 are clearly separated on the gel. Empty lanes indicate lack of amplification and
were dropped from the analysis.
603 bp
Figure 1 Representative gel. The gel is from a 26 °C Wolbachia treatment. Each lane represents an
individual fly. The ladder has a 603 bp middle band. The wsp gene is 610 bp while the CO1 gene is 658
bp.
The percentage of Wolbachia infected flies was calculated from the total amplified flies
per treatment. Based on the data in Table 3, the results of the chi-square indicate that the number
of wsp flies in the two temperature treatments were significantly different (Χ2 = 11.54, 1 df,
p < 0.01). All W+ vials had a starting infection rate of 25% in generation 1. Had the transmission
7
rate remained constant, then the same 25 percent infected is expected in generation 2 in both
temperature regimes, but in fact the rate increased in the 26 °C and decreased in the 24 °C (see
Figure 2). Transmission rate was calculated by dividing the change in percent infected between
the two generations by the change in the number of generations, in this case one. Transmission
rate in the 24 °C treatment was -14 percent as compared to an 18 percent transmission rate in the
26 °C treatment.
Table 3 Percent infected. Number of flies that were infected with Wolbachia was calculated from the
number of flies that amplified successfully.
Temperature (°C)
24
26
Wolbachia
+
+
-
# of wsp flies
5
0
26
0
# of flies amplified
45
20
60
7
% infected
11
0
43
0
50
Percent Infected
45
40
35
30
25
24C
20
26C
15
10
5
0
1
2
Generation
Figure 2 Transmission Rates. The graph displays transmission rates at two different
temperatures from populations initially inoculated with 25 percent infected flies.
8
Discussion
In this experiment, a strong effect of temperature on transmission was detected.
Regardless of optimal environmental conditions, population displacement by infected individuals
will not occur until a threshold release rate is met while one that exceeds the threshold increases
the displacement rate (Dobson, 2002). Interestingly, a study done by Hurst et al (2000) showed
that elevated temperature at 26 °C decreased both the bacterial density and penetrance of the
male-killing trait associated with Wolbachia in D. bifasciata compared to that of 18 °C: males
produced at 26 °C harbored Wolbachia but demonstrated weakened CI. It is important to note the
possibility of individuals to acquire Wolbachia but lack the gene driver effect due to low
penetrance of CI. Detailed analysis has yet to be made to affirm that male death requires high
density of Wolbachia; therefore a density threshold has not been established and analyzed under
elevated temperatures. A possible explanation for the altered CI is that perhaps the heat-shock
modified the bacteria’s molecular construct within the cell but results from high electron
microscopic analysis refute this, instead they show that 26 °C temperatures do not change the
structural interaction between the bacteria and ovarian fly cells since Wolbachia retained its
morphology and localization (Zhukova 2008). In another instance, elevated temperature (37°)
decreased bacterial density in Aedes albopictus (Wiwatanaratanabutr & Kittayapong 2009) while
crowding only reduced density if nutrients were restricted (Dutton, 2004). In contrast, no such
impact on density was evident in the wasp Leptopilina heterotoma that harbors three strains of
Wolbachia; in fact, density was highest at 26°C and did not influence CI (Mouton, 2006). The
effects of temperature vary by host species and have significant implications for population
dynamics.
9
The complexity associated with temperature effects on the transmission rates of
Wolbachia in insect vectors stresses the need to evaluate how temperature and transmission
together affect Wolbachia as a gene driver. The temperature effects were entered into the Dobson
model (Dobson et al 2002) as an interpretation of maternal transmission failure rate, which was
calculated by dividing the change in percent of uninfected flies between the two generations by
the change in the number of generations, in this case one. The treatment under 24 °C had a
failure rate of 14 percent whereas the treatment under 26 °C had a transmission failure rate of -18
percent, which was entered as 0 percent in the model. The decrease in transmission rate under
the 24 °C treatment as manifested by a negative Wolbachia-infected population growth (Figure 3)
annuls Wolbachia as a gene driver under low temperatures. In contrast, the Wolbachia-infected
population under the 26 °C treatment initially increases then reaches equilibrium (Figure 3) and
establishes itself as a gene driver within the whole population. Note that although a temperature
effect on CI rather than maternal transmission rate cannot be ruled out, the Dobson model does
accurately represent Wolbachia as a gene driver in terms of infected population growth and
transmission rates under the two temperatures. The loss of the 24°C W+ gene driver resulting
from a negative transmission rate suggests the infeasibility of releasing infected vectors under
low temperatures. This study only compared transmission differences between the two
temperatures 24 °C and 26°C: an optimal temperature range for Wolbachia transmission has yet
to be established.
The complex interactions between host, environmental conditions, and the presence of
different bacterial strains make fitness parameters difficult to predict. In studies of Wolbachia as
a gene driver in insects, much emphasis has been placed on host variables such as male age and
female fecundity. Actual implementation of Wolbachia as a gene driver will be subject to
10
environmental conditions, notably temperature. Before Wolbachia can be utilized as a control
method against insect hosts, temperature effects must be fully analyzed to predict how
introduced infections may behave.
70
Adults
60
50
X26°C W+
40
Y24°C W+
30
WW
20
Total alive
10
0
0
2
4
6
8
10
12
14
Figure 3 Dobson population model. 26°C W+ had a maternal transmission failure rate of 0
percent while 24°C W+ had a transmission rate failure of 14 percent.
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