Investigating the presence of Wolbachia pipientis in various mosquito species
Yuan Kang and Brian Dempsey
Abstract
Wolbachia pipientis are endosymbionts that infect many insects, arachnids, and nematodes.
By comparing the Wolbachia DNA sequences of different infected species, one can
investigate the evolutionary history of arthropods and the modes of transmission of this
reproductive parasite. We collected and extracted DNA from several mosquito species to test
for the presence of Wolbachia. Positive sequences generated from Culex restuans samples
confirmed the occurrence of lateral transmission of Wolbachia between insects and arachnids
when analyzed with the NCBI BLAST database. However, due to contamination of many of
our samples, we were unable to conclusively compare the Wolbachia sequences between
different mosquito species.
Introduction
Wolbachia pipientis is an α-proteobacterial reproductive parasite that infects a wide
variety of arthropods, including 17-75% of all insects (Huigens 2003). Since it is vertically
transmitted exclusively in the cytoplasm of egg cells, Wolbachia triggers phenotypic changes
in hosts to increase transmission through females. Host manipulations include parthenogenesis,
feminization of males, killing of male embryos, and cytoplasmic incompatibility between
uninfected females and infected males. These effects skew insect populations in favor of
infected females, thus enhancing the parasite’s survival and reproduction (Doran 2001). In
addition, reproductive barriers induced by Wolbachia infection can promote sympatric
speciation within the host population.
Because Wolbachia is a widespread endosymbiont, it can be used to study the
evolutionary history of arthropods (Bordenstein 2006). Comparisons of the 16S rRNA gene
give insight to the relatedness of various infected species. Close Wolbachia gene alignments
also indicate the occurrence of lateral transmission between distantly related insects (Huigens
2003). Further study in identifying a wide range of host species and analyzing their 16S
sequences can make Wolbachia a very useful tool for evolutionary biology and bioinformatics.
Wolbachia infects an estimated 28% of mosquito species (Xi 2006). There are several
potential applications of Wolbachia as a solution to global health issues such as lymphatic
filariasis, malaria, and dengue. Entomologist Steven Dobson hopes to eliminate mosquito
vectors of filariasis in the South Pacific by introducing infected males to the island
populations. Dobson hypothesizes that cytoplasmic incompatibility-induced sterility will
effectively reduce, and even eliminate, the harmful mosquito populations after many
generations of reproduction. This basic principle is applicable to other mosquito-transmitted
diseases as well. More detailed research regarding specific mosquito species and their
Wolbachia will aid in the development of these applications.
1
Materials and Methods
Mosquito larvae were collected from tree holes and discarded tires around the
Worcester area. Several different species were identified in the laboratory using a light
microscope and field guide: Culex restuans, Ochlerotatus japonicus, and Ochlerotatus
triseriatus. Toxoryhnchites rutilus septentrionalis and Orthopodomyia signifera collected in
Princeton, New Jersey, were also used to provide a more diverse range of mosquito species.
Larvae were kept in plastic containers filled with water from their respective collection sites.
Adults were raised in breeding cages at 80% humidity with a 10% sucrose solution food
source. Insects for DNA extraction were preserved in 100% ethanol and stored at -20°C.
The Qiagen DNeasy Kit was used to extract DNA from the collected mosquitoes and
the positive and negative Nasonia control insects provided by the MBL. We initially followed
the Wolbachia Project extraction protocol outlined by George Wolfe (Academy of Science,
Loudon City, VA) but later found that the Qiagen lab manual protocol worked more
effectively. In both procedures, Proteinase K and lysis buffers were added to the macerated
insect tissue. After incubation, ethanol was added to precipitate the DNA. Next, a series of
wash steps using DNeasy spin columns cleaned the DNA. Finally, an elution buffer removed
the clean DNA from the spin column filter. DNA samples were stored at -20°C until PCR.
PuReTaq Ready-To-Go PCR beads were used in the polymerase chain reaction. In
addition to the pre-mixed PCR pellets, each reaction tube also contained 2ul each of wspec
forward primer, wspec reverse primer, and extracted DNA. Positive Wolbachia DNA
previously extracted from a Nasonia specimen served as the control for this step of the
experiment. The thermalcycler was set as follows:
1 cycle
2 min @ 95° C
38 Cycles
30 sec. @ 94° C
45 sec @ 55° C
90 sec @ 72° C
1 cycle
10 min. @72° C
After PCR, samples were kept at 4°C until gel electrophoresis. 5ul of each DNA
sample was mixed with 1.5ul of 6X loading dye and pipetted into a prepared agarose gel. 5ul
of a DNA ladder solution was used in one lane to calculate the size of the unknown fragments.
The gel was run at 80V for 30 minutes and then viewed under an ultraviolet light. Since the
agarose gel contained ethidium bromide, we did not need to perform an additional staining step
in order to see the DNA.
Unknown samples that tested positive for Wolbachia and had good control results were
cleaned with ethanol, resuspended in distilled water, and sent in to be sequenced by the MBL.
The DNA sequences were uploaded onto the NCBI BLAST (Basic Local Alignment Search
Tool) to generate phylogenic trees based on other sequences in the database.
2
Results
Most of our trials yielded unreliable control data and could not be sent in for
sequencing. In many cases, the negative Nasonia control displayed a positive band after gel
electrophoresis, a sign of contaminated samples. Only three Culex restuans samples showed
solid bands around 400 bp long with good controls.
Several other species tested positive for Wolbachia, but their data is questionable due to
poor Nasonia control results.
3
ID
Order
Genus
species
C2
Diptera
Culex
restuans
C3
C4
Diptera
Diptera
Culex
restuans
Culex
restuans
16S Ribosomal DNA
Sequence (wspec F)
*Not clean
16S Ribosomal DNA
Sequence (wspec R)
GACCCGAGACGTATTCACCG
TGGCGTGCTGATCCACGATTA
CTAGCGATTCCAACTTCATGT
ACTCGAGTTGCAGAGTACAA
TCCGAACTGAGATGTCTTTTA
GGGATTAGCTTAAGCTTGCGC
ACCTTGCAACCCATTGTAGAC
ACCATTGTAGCACGTGTGTAG
CCCACTCCATAAAGGCCATG
ATGACTTGACATCATCCCCAC
CTTCCTCCAGCTTATCACTGG
CAGTTTCCTTAAAGTACCCAG
CATTACCTGATGGCAACTAA
GGATGAGGGTTGCGCTCGTT
GCGGGACTTAACCCAACATC
TCACGACACGAGCTGACGAC
AGCCATGCAACACTTGTGTG
AAATCCGGCCGAACCGACCC
TATCCCTTCG
GAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAAGTGT
TGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA
AGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTGCTATC
AGGTAATGCTGAGTACTTTAAGGAAACTGCCAGTGATAAG
CTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCTT
TATGGAGTGGGCTACACACGTGCTACAATGGTGTCTACAA
TGGGTTGCAAGGTGCGCAAGCTTAAGTTAATCCCTAAAAG
ACATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGA
AGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGT
GAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCA
TGGGAATTGGTTTCC
TGTTGCATGGCTGTCGTC
AGCTCGTGTCGTGAGAT
GTTGGGTTAAGTCCCGC
AACGAGCGCAACCCTCA
TCCTTAGTTGCCATCAG
GTAATGCTGAGTACTTT
AAGGAAACTGCCAGTGA
TAAGCTGGAGGAAGGTG
GGGATGATGTCAAGTCA
TCATGGCCTTTATGGAG
TGGGCTACACACGTGCT
ACAATGGTGTCTACAAT
GGGTTGCAAGGTGCGCA
AGCCTAACCTAACCCCT
AAAAGACATCTCAGTTC
GGATTGTACTCTGCAAC
TCGAGTACATGAAGTTG
GAATCGCTAGTAATCGT
GGATCAGCATGCCACGG
TGAATACGTTCTCGGGT
CTTGTACACACTGCCCG
TCACGCCATGGGAATTG
GTTTC
*Not Clean
Genus species
Aligned With
Orders
Aligned With
Pseudolynchia
canariensis,
Trichogramma deion,
Nasonia longicornis,
Diaphorencyrtus
aligarhensis,
Drosophila
pseudoananassae,
Callosobruchus
chinensis,
Metaseiulus
occidentalis,
Tetranychus urticae,
Nasonia giraulti
Diptera,
Pseudolynchia
canariensis,
Nasonia longicornis,
Cnidocampa
flavescens,
Mamestra brassicae,
Helicoverpa armigera,
Ostrinia furnacalis,
Corcyra cephalonica,
Trichogramma
ostriniae,
Bombus lucorum,
Bombus terrestris,
Nasonia giraulti,
Nasonia vitripennis
Diptera,
Trichogramma deion,
Diaphorencyrtus
aligarhensis,
Drosophila
pseudoananassae,
Pseudolynchia
canariensis,
Callosobruchus
chinensis,
Metaseiulus
occidentalis,
Tetranychus urticae,
Cnidocampa
flavescens,
Mamestra brassicae,
Ostrinia furnacalis,
Helicoverpa armigera,
Pieris rapae,
Clostera anachoreta,
Corcyra cephalonica
Hymenoptera,
Hymenoptera,
Coleoptera,
Prostigmata
(Arachnida)
Hymenoptera,
Lepidoptera
Diptera,
Coleoptera,
Prostigmata
(Arachnida),
Lepidoptera
4
ID
Order
Tox Diptera
J11
J12
Diptera
Diptera
Genus species
Toxorynchites
rutilus
septentrionalis
Ochlerotatus
japonicus
Ochlerotatus
japonicus
16S Ribosomal DNA
Sequence (wspec F)
16S Ribosomal DNA
Sequence (wspec R)
ATACCTATTCGAAGGGATAGGGTCGGTTGGCCGGATT
TCACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTC
GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC
CTCATCCTTAGTTGCTATCAGGTAATGCTGAGTACTTT
AAGGAAACTGCCAGTGATAAGCTGGAGGAAGGTGGG
GATGATGTCAAGTCATCATGGCCTTTATGGAGTGGGC
TACACACGTGCTACAATGGTGTCTACAATGGGTTGCA
AGGTGCGCAAGCTTAAGCTAATCCCTAAAAGACATCT
CAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAG
TTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGG
TGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCAC
GCCATGGGAATTGGTTTCACTCGAAGCT
CATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGT
TTCACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTC
GTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACC
CTCATCCTTAGTTGCTATCAGGTAATGCTGAGTACTTT
AAGGAAACTGCCAGTGATAAGCTGGAGGAAGGTGGG
GATGATGTCAAGTCATCATGGCCTTTATGGAGTGGGC
TACACACGTGCTACAATGGTGTCTACAATGGGTTGCA
AGGTGCGCAAGCTTAAGTTAATCCCTAAAAGACATCT
CAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAG
TTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGG
TGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCAC
GCCATGGGAATTGGTTTCACTCGAAGCT
TACCTTTTCGAAGGGATAGGGTCGGTTCGGCCGGATT
CACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTCG
TGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCC
TCATCCTTAGTTGCTATCAGGTAATGCTGAGTACTTTA
AGGAAACTGCCAGTGATAAGCTGGAGGAAGGTGGGG
ATGATGTCAAGTCATCATGGCCTTTATGGAGTGGGCT
ACACACGTGCTACAATGGTGTCTACAATGGGTTGCAA
GGTGCGCAAGCTTAAGCTAATCCCTAAAAGACGTCTC
AGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGT
TGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGT
GAATACGTTCTCGGGTCTTGTACACACTGCCCGTCAC
GCCATGGGAATTGGTTTCACTCGAAG
Genus species
Aligned With
Pseudolynchia
canariensis,
Nasonia longicornis,
Cnidocampa
flavescens,
Mamestra brassicae,
Ostrina furnacalis,
Helicoverpa
armigera,
Pieris rapae,
Corcyra
cephalonica,
Trichogramma
ostriniae
Pseudolynchia
canariensis,
Nasonia longicornis,
Cnidocampa
flavescens,
Mamestra brassicae,
Ostrina furnacalis,
Helicoverpa
armigera,
Pieris rapae,
Corcyra
cephalonica,
Trichogramma
ostriniae
Pseudolynchia
canariensis,
Nasonia longicornis,
Cnidocampa
flavescens,
Mamestra brassicae,
Ostrina furnacalis,
Helicoverpa
armigera,
Pieris rapae,
Corcyra
cephalonica,
Trichogramma
ostriniae
Orders
Aligned With
Diptera,
Hymenoptera,
Lepidoptera,
Coleoptera
Diptera,
Hymenoptera,
Lepidoptera,
Coleoptera
Diptera,
Hymenoptera,
Lepidoptera,
Coleoptera
5
ID
Order
Genus species
O1
Diptera
Orthopodomyia
signifera
O2
Tr1
Diptera
Diptera
Orthopodomyia
signifera
Ochlerotatus
triseriatus
16S Ribosomal DNA
Sequence (wspec F)
*Not clean
16S Ribosomal DNA
Sequence (wspec R)
ACGTATTCACCGTGGCATG
CTGATCCACATTACTAGCG
ATTCCAACTTCATGTACTC
GAGTTGCAGAGTACAATCC
GAACTGAGATGTCTTTTAA
GGAATAACTTAAGCTTGCC
CACCTTGCAACCCATTGTA
GACACCATTGTAGCACGTG
TGTAGCCCACTCCATAAAG
GCCATGATGACTTGACATC
ATCCCCACCTTCCTCCAGC
TTATCACTGGCAGTTTCCTT
AAAGTACTCAGCATTACCT
GATAGCAACTAAGGATGG
GGTTGCGCTCGTTGCGGGA
CTTAACCCAACATCTCACG
ACACGAGCTGACGACAGC
CATGCAACACTTGTGTGAA
ATCCGGCCGAACCGACCCT
AT CCCTT
CATACCTATTCGAAGGGATAGGGTCGGTCGGCCGGATTT
CACACAAGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTG
AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCAT
CCTTAGTTGCTATCAGGTAATGCTGAGTACTTTAAGGAA
ACTGCCAGTGATAAGCTGAGGAAGGTGGGGATGATGTC
AAGTCATCATGGCCTTTATGGAGTGGGCTACACACGTGC
TACAATGGTGTCTACAATGGGTTGCAAGGGGCAGGCTT
AAGTTATTCCTTAAAAGACATCTCAGTTCGGATTGTACT
CTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATC
GTGGATCAGCATGCCACGGTGAATACGTTTCGGGTCTTG
TACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTC
GAAGCTAAAATTTC
GTGTTGCATGGCTGTCAT
CAGCTCGTGTGTGAGAT
GTTGGGTTAAGTCCCGC
AACGAGCGCAACCCTCA
TCCTTAGTTACCATCAGG
TAATGCTGGGGACTTTA
AGGAAACTGCCAGTGAT
AAACTGGAGGAAGGTGG
GGATGATGTCAAGTCAT
CATGGCCCTTATGGAGT
GGGCTACACACGTGCTA
CAATGGTGGCTACATGG
GCTGCAAAGTCGCGAGG
CTAAGCCAATCCCTTAA
AAGCCATCTCAGTTCGG
ATTGTACTCTGCAACTCG
AGTGCATGAAGTTGGAT
CGCTAGTAATCGTGGAT
CAGCACGCCACGGTGAA
TACGTTCTCGGGTCTTGT
ACACACTGCCCGTCACG
CCATGGGAATTGGTTTC
*Not clean
Genus species
Aligned With
Pseudolynchia
canariensis,
Nasonia
longicornis,
Nasonia giraulti,
Cnidocampa
flavescens,
Mamestra
brassicae,
Ostrina furnacalis,
Helicoverpa
armigera,
Pieris rapae,
Corcyra
cephalonica,
Trichogramma
ostriniae
Pseudolynchia
canariensis,
Nasonia
longicornis,
Corcyra
cephalonica,
Trichogramma
deion,
Nasonia giraulti,
Nasonia
vitripennis,
Cnidocampa
flavescens,
Mamestra
brassicae,
Ostrinia
furnacalis,
Helicoverpa
armigera,
Trichogramma
ostriniae
Cnidocampa
flavescens,
Mythimna
separata,
Ascotis selenaria,
Spodoptera exiqua,
Ostrinia
furnacalis,
Trichogramma
ostriniae,
Bombus locurum,
Bombus terrestris
Orders
Aligned With
Diptera,
Hymenoptera,
Lepidoptera,
Coleoptera
Diptera,
Hymenoptera,
Lepidoptera,
Coleoptera
Lepidoptera,
Hymenoptera
6
ID
Order
Tr5
Diptera
Tr6
Diptera
Genus species
Ochlerotatus
triseriatus
Ochlerotatus
triseriatus
16S Ribosomal DNA
Sequence (wspec F)
*Not clean
*Not clean
16S Ribosomal DNA
Sequence (wspec R)
CCCGAGAACGTATTCCC
GTGGCATGCTGATCCAC
GATTACTAGCGATTCCCT
TTCATGTACTCGAGTTGC
AGAGTACAATCCGAACT
GACATGTCTTTTAGGGA
TTAACTTAAGCTTGCCCA
CCTTGCAACCCATTGTA
GACACCATTGTAGCACG
TGTGTAGCCCACTCCAT
AAAGGCCATGATGACTT
GACATCATCCCCACCTTC
CTCCAGCTTATCACTGGC
AGTTTCCTTAAAGTACTC
AGCATTACCTGATAGCA
ACTAAGGTGAGGGTTGC
GCTCGTTGCGGGACTTA
ACCCAACATCTCACGAC
ACGAGCTGACGACAGCC
ATGCACACTTGTGTGAA
ATCCGGCCGAACCGACC
CTACCCTTCGAAAAGGT
ATGTGTATTTT
TATTCACCGTGGCATGCT
GATCCACGATTACTAGC
GATTCCCACTTCATGTAC
TCGAGTTGCAGAGACAA
TCCGAACTGAGATTTCTT
TTAAGGAATAACTTAAG
CTTGCGCACCTTGCAAC
CCATTGTAGACACCATT
GTAGCACGTGTGTAGCC
CACTCCATAAGGCCATG
ATGACTTGACATCATCC
CCACCTTCCTCCAGCTTA
TCACTGGCAGTTTCCTTA
AAGTACTCAGCATTACC
TGATAGCAACTAAGGAT
GAGGGTTGCGCTCGTTG
CGGGACTTAACCCAACA
TCTCACGACACGAGCTG
ACGACAGCCATGCAACA
CTTGTGTGAAATCCGGC
CGAACCGACCCTATACC
TTTTAAAATAGATG
Genus species
Aligned With
Orders
Aligned With
Nasonia longicornis,
Pseudolynchia
canariensis,
Nasonia giraulti,
Cnidocampa
flavescens,
Mamestra brassicae,
Ostrinia furnacalis,
Helicoverpa
armigera,
Pieris rapae,
Corcyra
cephalonica,
Trichogramma
ostriniae,
Bombus locurum
Diptera,
Pseudolynchia
canariensis,
Nasonia longicornis,
Nasonia giraulti,
Cnidocampa
flavescnes,
Mamestra brassicae,
Ostrina furnacalis,
Helicoverpa
armigera,
Pieris rapae,
Corcyra
cephalonica,
Trichogramma
ostriniae
Diptera,
Hymenoptera,
Lepidoptera,
Coleoptera
Hymenoptera,
Lepidoptera,
Coleoptera
7
Discussion
Several Culex restuans specimens tested positive for Wolbachia with reliable Nasonia
control bands. BLAST results showed close alignment with other Diptera organisms, as
expected, as well as some Hymenoptera, Lepidoptera, Coleoptera, and Prostigmata
(arachnids). The latter supports the hypothesis that Wolbachia was transmitted both vertically
and horizontally throughout evolutionary history, since insects and arachnids are fairly distant
relatives. The DNA sequences from other mosquito species showed very little variation in
BLAST results. This suggests that the 16S gene is not significantly variable among the
mosquito species sampled. However, due to faulty controls in these later trials, the DNA
sequences generated may be contaminated and unreliable.
The extraction phase of this lab has the most potential for error. The Qiagen protocol
proved to be more reliable at extracting DNA than the procedure provided by the Wolbachia
Project website. The directions in the Qiagen kit called for the use of a tissue lysis (ATL)
buffer in addition to the cell lysis (AL) buffer and a three-hour incubation period at 56° C. An
additional lysis reagent and more heating time may have enhanced the release of cellular
contents from the mosquito cells, including intracellular Wolbachia DNA. We also found it
helpful to wash insects in distilled water before macerating them, especially if the specimens
had been preserved in ethanol for a long time. Centrifuging each insect in 400ul distilled water
prevented any ethanol or external debris from interfering with the lysis buffers or
contaminating the experiment. This procedure took more time but resulted in a better DNA
yield.
Contamination of the negative Nasonia control was also a major problem during the
extraction step. While we were careful to change pipette tips for each sample tube, minute
amounts of Wolbachia DNA still could have been transferred from one tube to another in other
ways and amplified during PCR. For instance, the gloves used to open and close the
microcentrifuge tube lids could have transferred some liquid between the samples. Since the
controls were very inconsistent, another possible error is unreliable Nasonia specimens. Some
of our positive controls lacked Wolbachia DNA, and some negative controls displayed false
positive results. In both cases, the unknown mosquito DNA results could not be used because
the entire trial was not reliable. In the future, it would be beneficial to amplify a portion of
insect DNA in addition to the Wolbachia sequence to determine whether or not the DNA
extraction step was successful.
One early source of error for our first couple trials was during the polymerase chain
reaction. We did not set the thermalcycler for a heated lid, which caused uneven heating of the
reaction tubes. This resulted in no visible bands once the PCR product was run through a gel.
After correcting this mistake, DNA bands appeared on subsequent gels.
The bioinformatics analysis of our Wolbachia sequences was very vague, and there
were no close matches to any mosquito species for the Culex samples or other species. In fact,
many of the closest matches were to Nasonia and Trichogramma, suggesting possible
contamination between the wasp controls and the unknown mosquito specimens. Because the
Culex DNA sequences were not completely clean, editing for ambiguous base pairs was
difficult. Only the C3 sample yielded decent forward and reverse sequences which could be
manipulated and combined to form one clear sequence. The other two samples, C2 and C4,
8
showed contamination on either the forward or reverse sequences, so they could not be edited.
While one sequence for these two samples was still usable for BLAST, we did not have the
opportunity to correct misalignments based on the complementary strand. We did not receive
the chromatograms for the other sequences, since the MBL staff compiled them for us. This
experiment would be more conclusive with a larger sampling pool, more reliable results, and
cleaner DNA sequences.
It is important to keep in mind that the phylogenic tree generated by BLAST depends
on the breadth of sequences in the NCBI database. Our DNA may not have generated matches
to mosquito Wolbachia because those sequences simply have not been thoroughly studied and
uploaded yet. In the future, as Wolbachia research expands to cover more species, the database
will be able to produce better DNA sequence matches and more complete phylogenies.
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