Rachel Martin
Marine Biological Laboratory
Summer Envisionship 2008
July 30, 2008
Wolbachia transmission patterns in different species of Nasonia
Abstract
The bacterium, Wolbachia, infects 60-85% of the world’s insect populations by harboring
itself in insect gonads. It traditionally has a maternal inheritance pattern. The purpose of this
project is to determine if Wolbachia from an infected male was being horizontally transferred to
an uninfected female during mating. A cross between an infected male and uninfected female
usually results in embryonic death and remains incompatible. This theory was tested in three
different species of Nasonia: Nasonia vitripennis, Nasonia longicornis, and Nasonia giraulti.
Each species harbors a different type of Wolbachia ranging from high density to low density.
Also, offspring from int13.2 (uninfected) females and int12.1 (infected) males, a cross that has
previously displayed a horizontal transmission of Wolbachia, were screened to determine if the
transmission of Wolbachia was being inherited by the next generation.
Uninfected and infected Nasonia were collected before they emerge and separated into tubes
to ensure each Nasonia would only mate once. The uninfected females were mated to infected
males and then stored in an -80°C freezer until DNA extraction and amplification could proceed.
Wsp primers were used in all PCR reactions.
The results from these experiments suggest that transmission of Wolbachia does not occur
with Nasonia giraulti or Nasonia longicornis. A small rate of transmission was seen in Nasonia
vitripennis. The transmission of Wolbachia seen in the cross between int13.2 females and int12.1
males is not inherited in the next generation, as examined in the embryos and diapause.
More experimentation will be conducted to expand on these results. Similar methods will be
used with Drosophila melanogaster to determine if these results are seen in a different species.
Introduction
Wolbachia are bacteria that infect a wide range of mainly arthropod hosts. The bacteria are
maternally inherited and manipulate host reproduction in order to increase transmission rates in
the population. The bacteria cause feminization, male killing, parthenogenesis and cytoplasmic
incompatibility (CI) as means of manipulation. More specifically, CI is a form of postfertilization reproductive failure and results in an incompatible cross (Clark et al. 2002).
Not all strains of Wolbachia can induce CI. But, the mechanism by which Wolbachia
manipulates their host’s reproductive potential, successfully, is unknown. It has been proposed
that the bacteria could modify infected sperm during the maturation process. This modification
renders them unable to successfully fertilize uninfected eggs (Riparbelli et al. 2007).
Three strains of Nasonia have been used to study Wolbachia-induced CI: Nasonia
vitripennis, Nasonia longicornis, and Nasonia giraulti. N. giraulti and N. longicornis mostly
show embryonic death. In the CI embryos of these two species, the paternal genome does not
properly divide to the two daughter nuclei. The severe abnormalities in this segregation result in
defects in other mitotic divisions (Tram et al. 2006).
In contrast, N. vitripennis show a conversion of the CI embryos to developing males. This is
due to the fact that unfertilized eggs normally develop into males. The exclusion of the paternal
genome leads to the development of all male offspring (Tram et al. 2006).
According to research conducted by Lassy and Karr in 1996, the sperm successfully enters
the egg in an incompatible cross. The paternal chromosomes do not properly condense and fuse
with the chromosomes of the mother before the first division during mitosis (Clark et al. 2002).
In this experiment to determine whether Wolbachia is horizontally transmitted from infected
males to uninfected females, I think that the three species of Nasonia will show a Wolbachia
infection when mated.
The research is beneficial in order to contribute to the knowledge of the biology of
Wolbachia. It is helpful to realize how Wolbachia manipulates its hosts. There is not much
known about the mechanisms of how Wolbachia cause CI or does not cause CI. Various research
can be done to understand more about Wolbachia, which harbors itself in most of the arthropods
that inhabit our planet.
Methods
Mating female Nasonia for DNA Extractions:
1. Crack open hosts a day or two before the Nasonia are due to emerge.
2. Collect black pupae from inside the host.
3. Examine pupae under a microscope to determine the gender of the Nasonia.
4. Separate males and females into different, labeled, glass tubes.
5. Wait for Nasonia to emerge.
6. Put one male and one female into an upside-down glass tube.
7. Watch to guarantee that the Nasonia courted and mated.
8. Remove the female and place into a 1.5ml microcentrifuge tube.
9. Repeat steps 6-8 until all crosses are completed.
10. Place all tubes into a cardboard freezer box and put into -80°C freezer.
DNA Extraction of Nasonia Tissue using the Gentra Puregene Tissue Kit:
1. Preheat water baths to 55°C and 65°C.
2. Remove Nasonia from -80°C freezer and put in bench top cooler.
3. Freeze Nasonia in liquid nitrogen.
4. Grind frozen tissue with a pestle. Work quickly and keep tissue on ice at all times.
5. Dispense 300l of Cell Lysis Solution into the 1.5ml grinder tube with the ground tissue.
6. Heat at 65°C for 1 hour.
7. Incubate for 1 minute on ice to quickly cool the sample.
8. Add 100l Protein Precipitation Solution.
9. Vortex vigorously for 20s at high speed.
10. Centrifuge for 10 minutes at 16,000 g. The precipitated proteins should form a tight pellet.
If the protein pellet is not tight, incubate on ice for 5 minutes and repeat the centrifugation.
11. Pipet 300l 100% isopropanol into a clean 1.5ml microcentrifuge tube.
12. Add the supernatant from the previous step by pouring carefully. Be careful the protein
pellet is not dislodged during pouring.
13. Mix by inverting gently 50 times.
14. Centrifuge 1 min at 16,000g.
15. Carefully discard the supernatant, and drain the tube by inverting on a clean paper towel,
taking care that the pellet remains in the tube.
16. Add 300l 70% ethanol and invert several times to wash the DNA pellet.
17. Centrifuge for 1 minute at 16,000g.
18. Carefully discard the supernatant. Drain the tube on a clean paper towel, taking care that
the pellet remains in the tube. Allow to air dry for up to 15 minutes. The pellet might be soft
and easily dislodged.
19. Add 50l DNA Hydration Solution and vortex for 5 seconds at medium speed to mix.
20. Incubate at 65°C for 1 hour to dissolve the DNA. Samples can be incubated at room
temperature overnight with gentle shaking, instead. Ensure tube cap is tightly closed to
avoid leakage.
DNA Extraction of Nasonia Embryo Tissue using the Gentra Puregene Tissue Kit:
1. Preheat water bath to 65°C.
2. Remove ethanol from embryos using a P20. Be careful not to dislodge embryos from the
sides of the tube. They will fit up the P20 tip.
3. Rinse with distilled water.
4. Remove the water.
5. Crush embryos with a pestle.
6. Add 100l Cell Lysis Solution to the embryos and grind gently with a pestle.
7. Incubate at 65°C for 30 minutes.
8. Cool samples to room temperature by placing on ice or putting in fridge for 1 minute.
9. Add 33l Protein Precipitation Solution to cell lysate.
10. Vortex vigorously at high speed for 20 seconds to mix the Protein Precipitation Solution
uniformly with the cell lysate.
11. Centrifuge at 16000g for 3 minutes. The precipitated proteins should form a tight pellet. If
a tight pellet does not form, repeat sep 3 followed by incubation on ice for 5 minutes and
repeat step 4.
12. Pour the supernatant containing the DNA into a clean 1.5ml tube containing 100l 100%
Isopropanol.
13. Mix sample gently by inverting several times.
14. Centrifuge at 16000g for 1 minute. The DNA should be visible as a small white pellet.
15. Pour off supernatant and drain the tube on a clean paper towel. Add 100l of 70% ethanol.
Invert tube 10 times to wash the DNA.
16. Centrifuge at 16000g for 1 minute. Carefully pour off the supernatant. Pellet may be loose
so watch the pellet carefully and pour slowly.
17. Invert and drain on clean paper towel for 10 minutes.
18. Add 20l DNA Hydration Solution to all samples.
19. Rehydrate DNA by incubating sample at 65°C for 1 hour or overnight at room temperature.
20. Store at 4°C.
Polymerase Chain Reaction (PCR):
1. Pick a primer to use. This primer should amplify a region specific to what you are looking
for. In this case, a Wolbachia surface protein (wsp) primer was used to amplify a region that
is unique to Wolbachia.
2. Remove reagents from freezer and place on ice to thaw.
3. Place PCR (.2ml) tubes in a PCR tube tray holder. Label PCR tubes.
4. Create PCR template on Microsoft Excel.
5. Vortex reagents to mix thoroughly. Aliquot reagents into a 1.5l microcentrifuge tube. This
mixture is called the “Master Mix.”
6. Place PCR tray with tubes on ice and pipet 9l “ Master Mix” into each PCR tube. When
setting up PCR, use specified PCR pipets and filtered pipet tips.
8. Pipet 1l template DNA into the specified PCR tube.
9. Centrifuge PCR tubes and place PCR tubes into a thermocycler.
10. Run program for specific primer on thermocycler.
Gel Electrophoresis:
1. Add 1g of UltraPure™ Agarose to 100ml 1X TAE buffer.
2. Microwave for 1 minute or until melted. Caution that the mixture does not boil. Let the
agarose cool, but not solidify.
3. Tape the open sides of the gel tray. Place combs into their slots.
4. Pour agarose into the tray and let solidify.
5. Peel the tape off the sides and remove combs.
6. Place the tray into a gel rig filled with 1X TAE buffer.
7. Aliquot 2l blue/yellow loading dye for every PCR sample onto a piece of Parafilm.
8. Pipet 8l PCR sample onto a dot of loading dye and mix by pipetting.
9. Pipet mixture into a well in the gel.
10. Repeat steps 8-9 until every PCR sample has been loaded into a well.
11. Replace cover on gel rig. Plug anode and cathode into the power source. Turn on (100
volts).
12. Set a timer for 50 minutes.
13. Turn off power source and unplug anode and cathode. Remove rig cover.
14. Remove gel from buffer and place in an ethidium bromide bath. CAUTION: Follow all
important safety procedures when using ethidium bromide.
15. Set a timer for 20 minutes.
16. Remove gel from ethidium bromide bath.
17. Take a picture of the gel under UV light.
Results
Previous experimentation led to
positive results of a positive horizontal
Diapause - DNA from diapuse from a int13.2 female mated with
an int12.1 male were extracted to determine if a Wolbachia
transmission between int13.2 females
transmission is seen in the offspring of an uninfected female
and int12.1 males. Diapause DNA was
horizontally by a male.
screened to determine if this
Sample #
Type of Cross
# of diapuase
Result
A35
4
transmission was being transferred to
A12
4
the offspring of the host. Using a wsp
A13
5
A22
6
primer, results suggest that Wolbachia
A21
9
int12.1 Male x
is not being inherited by the offspring
A23
4
int13.2 Female
A37
10
of the host, specifically in diapause.
A46
4
A25
7
Positive and negative results are shown
A27
3
in Figure 1. All experimental samples
A18
4
B5
int13.2 xint13.2
6
from the cross between an int12.1 male
C5
int12.1 x int12.1
1
+
and an int13.2 female are negative for
the presence of Wolbachia. The
negative control crosses between an int13.2 male and an int13.2 female yields a negative result
for the presence of Wolbachia. The positive control crosses between an int12.1 male and am
int12.1 female yields a positive result.
Figure 1
Figure 2
Embryo
s from
int13.2
Sample #
Type of Cross
# of embryos Result Sample #
Type of Cross
# of embryos Result
females
A6
7
A1
12
mated
A8
9
A27
8
A9
4
A40
1
int12.1 Male x
with
A10
10
A23
int13.2 Female
7
Figure
1
A11
7
A44
5
int12.1
A14
6
A18
8
males
A15
7
B1
5
int12.1 Male x
int13.2 Male x
A17
4
B3
10
were
int13.2 Female
int13.2 Female
A19
11
B4
6
also
A20
6
C4
2
A33
9
C6
3
+
screene
int12.1 Male x
A42
10
C8
6
int12.1 Female
d to
A30
14
C9
6
A36
2
C5
2
determi
A7
6
ne if the
horizontal transmission seen in the cross was being inherited by the offspring of the host. DNA
concentrations were determined because positive controls were not testing positive.
Concentrations were really low, but similar to DNA from a positive embryo controls. 1l and
3l template DNA was used in PCR for the samples. An int12.1 DNA and an embryo DNA
containing Wolbachia were used to compare the strength of bands in the tests. Experimental
samples from the int13.2 female mated with the int12.1 male did not show any results suggesting
the presence of Wolbachia. Negative control samples from the int13.2 female mated with the
int13.2 male did not suggest a presence of Wolbachia either. Only 20% of the positive control
samples suggested a presence of Wolbachia. The gel electrophoresis signal from the positive
control was weak. If there was Wolbachia in any of the embryos, then the small signal of the
sample was unlikely to show up.
Embryos - DNA from embryos from an int13.2 female mated with an int12.1 male were extracted to
determine if a Wolbachia transmission is seen in the offspring of an uninfected female transferred
horizontally by an infected male.
Figure 3
N. vitripennis - DNA from 13.2 females mated with 4.9 males to see if
there is a transfer of Wolbachia from an infected male to an uninfected
female
Sample # Type of Cross Result
A1
A2
A3
A4
A5
A6
A7
A8
+
4.9 Male x 13.2
A9
Female
A10
A11
A12
A13
A14
A15
A16
A17
-
Sample # Type of Cross Result
A18
A19
A20
A21
4.9
Male
x
A22
13.2 Female
A23
A24
A25
B1
+
4.9 Male x 4.9
B2
+
Male
B3
+
C1
C2
13.2 Male x
C3
13.2 Female
C4
C5
-
The positive results from the
int13.2 females mated with the
int12.1 males suggested the same
patterns might be observed in
other species of Nasonia with
different strains of Wolbachia.
Nasonia vitripennis 13.2 females
were mated to 4,9 males and
extracted to determine if the
Wolbachia from the male was
being transferred to the female.
Experimental crosses between the
13.2 females and 4.9 males
suggest that 4% of females
become infected with Wolbachia
(Figure 3). The mechanism of infection is unknown. Positive control crosses between 4.9 males
and females suggest 100% infection rate. Negative control crosses between 13.2 males and
females suggest 0% of samples were infected with Wolbachia.
Figure 4
N. vitripennis - DNA from 13.2
females mated with 4.9 males was
screened to determine if there is a
horizontal
transmission
of
Wolbachia from the male to the
female. Each male was mated to 3
different females.
Sample # Type of Cross Result
1.1
1.2
1.3
2.1
4.9 Male x 13.2
2.2
Female
2.3
3.1
3.2
3.3
1.4
+
4.9 Male x 4.9
1.5
+
Female
1.6
7.1
13.2 Male x
7.2
13.2 Female
7.3
-
Attempts to replicate Nasonia
vitripennis data from Figure 3
proved unsuccessful. Males were
mated to three different females
to determine if Wolbachia would
digress with each female. No
Wolbachia was seen in 13.2
females mated with 4.9 males
(Figure 4). Positive control
crosses suggest a 66%
transmission rate between 4.9
females mated with 4.9 males.
Negative control crosses suggest
a 0% transmission rate of
Wolbachia between 13.2 females
mated with 13.2 males.
Another attempt to replicate data
from Figure 3 will proceed in August/September 2008.
Figure 5
N. giraulti
- DNA from RV2R
females mated with 16.2 males
was screened to determine if there
is a horizontal transmission of
Wolbachia from the male to the
female.
Sample # Type of Cross Result
A1
A2
A3
A4
A5
16.2 Male x
A6
RV2R Female
A7
A8
A9
A10
A11
B1
+
16.2 Male x
B2
+
16.2 Female
B3
+
C1
RV2R Male x
C2
RV2R Female
C3
-
Nasonia giraulti were also mated to determine if there was a transmission of Wolbachia from
16.2 males to RV2R females. Results, as shown in Figure 5, suggest that Wolbachia from a 16.2
male is not transferred to a RV2R female. Positive control crosses between a 16.2 male and
female suggest 100% infection rate. Negative control crosses between a RV2R male and female
suggest 0% infection rate.
Figure 6
N. longicornis
- DNA from females
mated with males was screened to
determine
if there is a horizontal
transmission of Wolbachia
from the
male to the female. Cross types cannot
be determined at this date. Large
amounts
of unsuccessful courtships
between Nasonia rose susupiscion about
the male Nasonia strain. Virgin females
were put on hosts to determine Nasonia
strain and Wolbachia infection.
Sample #
A1
A2
A3
A4
A5
A6
A7
A8
A9
A10
A11
A12
A13
A14
A15
A16
A17
A18
A19
Result
-
Sample #
A20
A21
A22
A23
A24
A25
A26
A27
A28
B1
B2
B3
B4
B5
C1
C2
C3
C4
C5
Result
+
-
Nasonia longicornis females were mated to males. The
type and infection status of the males is unknown. The
positive strain was supposedly 2.1. The negative strain
was supposedly IV7R3-1b. Many samples of the Nasonia
longicornis were not mating with each other. Suspicion of
the species of Nasonia was brought to attention. Wing
size of the “2.1” males seemed too short to be Nasonia
longicornis. In order to make a definitive conclusion,
“2.1” virgin females were put on 2 hosts and honey.
Wing size of these male offspring will be recorded once
they emerge. Also, pupae from the hosts will be collected
and stored in 95% ethanol until an extraction and
amplification can be performed. These tests will conclude
the strain of Wolbachia in the Nasonia.
Results, as shown in Figure 6, suggest no Wolbachia was
either transferred or transferred in the cross between the
“2.1” male and the IV7R3-1b female. Results suggest that
there may have been Wolbachia present in either the male
or the female in the positive control crosses. All negative
control crosses yielded results that suggest there is no
Wolbachia present.
Discussion
Experimentation results suggest that Wolbachia is not inherited by the offspring, neither
diapause nor embryos, of int13.2 females mated with int12.1 males, even though the Wolbachia
is transferred from the infected male to the uninfected female. Also, horizontal transmission of
Wolbachia has not been seen in N. giraulti or N. longicornis. A small infection rate was seen in
N. vitripennis, but this could be due to human error. A definite conclusion will need to be drawn
after more experimentation.
Continued experimentation will consist of attempting to replicate results of the horizontal
infection of Wolbachia in Nasonia vitripennis (13.2) females. Also, DNA from N. longicornis
offspring will be analyzed to determine if they are infected with Wolbachia. These male
offspring’s wing size will be analyzed to determine if they are in fact N. longicornis. Aside from
the research of Wolbachia horizontal transmission in Nasonia, similar experiments will be
conducted using Drosophila.
References
Clark, Michael E., Veneti, Z., Bourtzis, K., Karr, T.L. “The distribution and proliferation of the
intracellular bacteria Wolbachia during spermatogenesis in Drosophila.” Mechanisms of
Development. 111 (2002): 3-15.
Riparbelli, M.G., Giordano, R., Callaini, G. “Effects of Wolbachia on sperm maturation and
architecture in Drosophila simulans Riverside.” Mechanisms of Development. 124
(2007): 699-714.
Tram, U., Fredrick, K., Werren, J.H., Sullivan, W. “Paternal chromosome segregation during the
first mitotic division determines Wolbachia-induced cytoplasmic incompatibility
phenotype.” Journal of Cell Science. 119 (2006): 3655-3663.