1
Culture independent analysis of microbiota in the gut of
pine weevils
KTH Biotechnology
2013-January-13
Diploma work by:
Tobias B. Ölander
Environmental Microbiology, KTH
Supervisor: Associate prof. Gunaratna K. Rajarao
Examinator: Prof. Stefan Ståhl
2
Abstract
In Sweden, the pine weevil causes damages for several hundreds of millions kronor annually.
The discouraged use of insecticides has resulted in that other methods to prevent pine weevil
feeding needs to be found. Antifeedants found in the pine weevil own feces is one such
alternative. The source of the most active antifeedants in the feces is probably from bacterial
or fungal lignin degrading symbionts in the pine weevil gut. The aim of the project was to
analyze the pine weevil gut microbiota with the help of culture independent methods. DNA
(including bacterial DNA) was extracted from both midgut and egg cells. The extracted DNA
was amplified with PCR. A clone library was created by cloning the amplified DNA into
plasmid vectors and transforming the vector constructs with chemically competent cells. The
clones were amplified again with either colony PCR or plasmid extraction followed by PCR,
and used for RFLP (Restriction Fragment Length Polymorphism) and sequencing. Species
found in the midgut sample included Acinetobacter sp., Ramlibacter sp., Chryseobacterium
sp., Flavisolibacter sp. and Wolbachia sp. Species found in the egg sample included
Wolbachia sp. and Halomonas sp. Wolbachia sp. and Halomonas sp. were found to be the
dominant members of the midgut and egg cells respectively.
Abbreviations
PCR
Polymerase Chain Reaction
RFLP
Restriction Fragment Length Polymorphism
T-RFLP
Terminal Restriction Fragment Length Polymorphism
DGGE
Denaturing Gradient Gel Electrophoresis
TGGE
Temperature Gradient Gel Electrophoresis
D-HPLC
Denaturing High-Performance Liquid Chromatography
RISA
Ribosomal Intergenic Spapcer Analysis
TAE
Tris base, acetic acid and EDTA
TBE
Tris base, boric acid and EDTA
3
Table
of Contents
Abstract ... 2
Abbreviations ... 2
1
Introduction ... 5
1.1
Aim ... 5
2
Background ... 6
2.1
Pine Weevil ... 6
2.2 Insecticides ... 7
2.3
Antifeedants ... 8
2.3.1
Antifeedant activity of pine weevil feces ... 8
2.4
Community analysis ... 9
2.4.1
Genetic fingerprinting ... 10
2.4.2
Sequencing ... 11
2.4.3
16S rRNA gene ... 12
2.4.4
Community analysis of insect gut ... 12
3
Materials & Methods ... 13
3.1
Flowchart ... 13
3.2
DNA extraction ... 13
3.3
PCR amplification ... 14
3.4
Cloning and transformation ... 15
3.5
Plasmid extraction ... 15
3.6
Colony PCR ... 16
3.7 RFLP ... 17
3.8
Agarose gel electrophoresis ... 17
3.9
Sequencing ... 17
3.10
Phylogenetic analysis ... 17
4
Results ... 18
4.1
DNA extraction ... 18
4.2
PCR amplification ... 19
4.3
Cloning and transformation ... 20
4.4
Plasmid DNA extraction ... 21
4.4.1
PCR with plasmid DNA ... 22
4.5
Colony PCR ... 22
4.6
RFLP ... 23
4.7
Sequencing ... 25
4.8
Phylogenetic analysis ... 28
4
5.1
PCR amplification ... 31
5.2
Colony PCR ... 32
5.3
RFLP ... 32
5.4
Phylogenetic analysis ... 33
6
Conclusions ... 33
7
Further Studies ... 34
8
Acknowledgments ... 34
9
References ... 34
10
Appendices ... 40
I.
PCR Amplification – midgut, hindgut & egg sample ... 40
II.
PCR (plasmid template) – midgut sample ... 43
III.
Colony PCR – midgut sample ... 44
IV. Colony PCR – egg sample ... 61
V.
Good’s Method ... 69
5
1 Introduction
From an economical perspective, the pine weevil is the most important forest pest in Sweden,
as well as for major parts of the rest of Europe [1]. The insect is a serious threat to the
regeneration of newly planted conifers (i.e. pines and spruces). Just in Sweden, the pine
weevil’s feeding on young conifer plants causes damages for several hundreds of millions
kronor annually [1] [2].
Damages caused by the pine weevil is an issue recognized as early as the middle of the 19th
century, but the problem with pine weevil feeding increased significantly in Sweden during
the 1950s, due to the more and more prevalent forestry practice of clearcutting [2].
Due to the increasing pressure, to abolish the use of traditional insecticides in the forest
industry, alternative means for fighting forest pests, like the pine weevil, are required.
One alternative would be to search for a more eco-friendly insect repellant or antifeedant to
use against the weevils. A study published in 2006 has shown that several organic
compounds, found in the pine weevil’s own excrement, have antifeedants activity against the
pine weevil [10].
The organic compounds with the highest antifeedants activity were structurally related to
lignin and therefore probably the result of lignin degrading bacteria or fungal symbionts in the
pine weevil gut [10]. Many bacteria found within the gut of arthropods (invertebrate animals
having an exoskeleton and a segmented body, i.e. insects like the pine weevil) are important
in the breakdown, mineralization and cycling of many organic compounds [47].
Gut bacteria might not only be the natural source of the antifeedants, but may also be utilized
as small “factories” to produce the sought-after compounds. A proper analysis of the pine
weevil gut microbiota is therefore an important step in identifying and developing a new
effective insect repellant.
1.1 Aim
To characterize the composition of the microbiota in the pine weevil midgut, culture
independent approaches were applied.
The primary method to determine the composition of the microbiota was to extract bacterial
DNA from the pine weevil midgut, amplify the DNA with PCR (polymerase chain reaction)
and to create a clone library. The clones were then again amplified, with either colony PCR or
plasmid extraction followed by PCR, and used for RFLP (restriction fragment length
polymorphism) and sequencing of gene16S rRNA.
Additionally, the same method as used to determine the microbiota in the midgut was also
used to determine the composition of the microbiota in hindgut and egg cells extracted from
the ovaries of the same female pine weevil sample.
6
2 Background
2.1 Pine Weevil
Pine weevil is the common name for several beetle species belonging to the genus Hylobius
[3]. In Scandinavia the most common Hylobius species is H. abietis and the species most
often referred to when using the common name pine weevil [3]. There are also three other
Hylobius species in Scandinavia, of which two species (H. pinastri and H. piceus) also feed
on conifers plants, but to a lesser extent than H. abietis [3]. If not stated otherwise, the
common name pine weevil will refer to H. abietis in this report.
Adult pine weevils are 8-14 mm in length, dark-dark brown in color with patches of yellow
hair on their neck shields and wing covers (see figure 1). The pine weevil has two guts, the
midgut and hindgut. The males and females look pretty similar, but can be distinguished by
features on the abdomen [3]. Female pine weevils can lay up to 1000 eggs during their life
[10].
Pine weevils feed on the inner bark of the stem of young conifer plants, but also on the bark
from the roots, stems and branches of young conifer trees [2]. While the feeding on young
trees causes no known significant damage, the feeding on plants can cause severe damage by
girdling (also called ring barking) [2]. Girdling results in the removal of the cambium (bark),
which includes the xylem and phloem. The phloem is largely responsible for transportation of
carbohydrates and the xylem is largely responsible for transportation of water. When severing
just the phloem layer, death might take several years. Severing the xylem layer as well results
in a quicker death [4] [5] [9].
Every spring flying pine weevils of both sexes and in large numbers migrates, sometimes tens
of kilometers, to new clearcuttings for the purpose of reproduction. The pine weevils are
attracted to the new regions of clearcuttings by degradation products (including ethanol,
α-pinene and monoterpenes) omitted by the fresh stumps [10].
Pine weevil larvae are yellow white, lack legs and have broad brown heads [3]. The pine
weevil larva develops under the bark or near the bark of recently dead conifer roots. For
managed forests, such as clearcuttings, this would usually be in the roots of fresh stumps. The
female pine weevils lay their eggs either in cavities that they gnaw into the root bark with
their snouts or in the soil next to the roots. Hatched pine weevil larvae feed on the inner stem
of the roots their eggs were placed in. Larvae from newly hatched eggs placed outside of the
roots, in the soil, are attracted to the inner stem by the scent of the degradation product
α-pinene. Older larvae may also need to search for roots to feed on. The older larvae are
attracted to new roots by the degradation products ethanol and
α-pinene [3].
7
2.2 Insecticides
The most common method, to protect conifer seedlings from pine weevil feeding, has so far
been to treat the seedlings with insecticides [6] [14]. However, the use of insecticides is now
discouraged and criticized due to the insecticides impact on the environment and especially
the work environment for the workers in the forest industry [2].
Three insecticides products are currently available on the Swedish market - Hylobi Forest
(active substance lambda cyhalothrin), Forester (cypermethrin) and Merit Forest WG
(imidacloprid) [3]. The Swedish Chemicals Agency’s (Kemikalieinspektionen) current
approval of these substances reaches until the end of 2015 for Hylobi Forest and Forester, and
until the end of 2014 for Merit Forest WG [15].
Today, about 11 millions hectare of the Swedish woodland is FSC certified. That is equivalent
to approximately half of the productive forest area in Sweden [7]. As stated on the Forest
Stewardship Council’s website ”FSC is an independent, non-governmental, not-for-profit
organization established to promote the responsible management of the world’s forests” [7].
Companies on the Swedish market certified to FSC standards are only allowed to use the
insecticide Merit Forest WG and only with one-year dispensations [3] [7].
Both the active ingredient in Hylobi Forest and in Forester belong to a group of chemicals
called pyrethroids, which is a class of synthetic organic compounds similar to the natural
substances pyrethrins. Pyrethrins, natural neurotoxins, are produced from the flowers of
pyrethrums. Pyrethroids are toxic to a broad range of insects, both pests and beneficial insects.
Pyrethroids are also very toxic towards aquatic wildlife (including fishes). Pyrethroids are
only toxic towards humans and other mammals at extremely high concentrations, but may still
cause some health problems at lower concentrations when repeated exposure. Pyrethroids are
skin irritants, but cases of stuffy noses, sneezing, running eyes and nosebleeds have also been
8
reported [16]. Resistance towards pyrethroids amongst insects is an increasing issue and has
been reported for example for bed bugs and malaria mosquitoes [17] [18].
Imidacloprid is the active ingredient of Merit Forest WG. Imidacloprid belongs to a class of
organic compounds called neonicotinoids that are modeled after the natural insecticide
nicotine. Neonicotinoids act by interfering the transmission of stimuli in a type of neuronal
pathway that is more abundant in insects than in warm-blooded animals. The insecticide is
therefore more selectively toxic towards insects than humans and other warm-blooded
animals. Imidacloprid is said to cause minor eye reddening in humans, but is not irritating to
the skin. Data indicate that imidacloprid is less toxic when absorbed through the skin or
inhaled, compared to ingestion. Signs of toxicity in rats include for instance lethargy,
respiratory disturbances and spasms [19]. No accounts of human poisoning are recorded, but
the signs and symptoms of poisoning are expected to be similar to those shown in rats.
Imidacloprid is toxic to birds and fish and highly toxic to honeybees [19].
There is an ongoing debate regarding how strong the link is between the usage of
neonicotinoid insecticides and the increasing numbers of abandoned honey beehives (reported
in for instance France and Germany) during the last two decades [20].
2.3 Antifeedants
The definition of an antifeedant may vary depending on the cited source material. Two
definitions are ”a naturally occurring substance in certain plants which adversely affects
insects or other animals which eat them” or a compound that ”inhibits normal feeding
behaviour” [8] [12]. In this report, the latter definition is used.
Furthermore, an optimal antifeedant should also be, citing Månsson et al. (2005), ”an
environmentally friendly compound with long-term stability to the conditions it experiences
in the field. Thus, the compound should have low volatility and not decompose or be washed
away under the influence of environmental factors such as oxygen, UV light, variation in
temperature, and rainfall” [48].
The rapidly developing resistance to conventional insecticides and the need to replace
insecticides with ecologically acceptable compounds has led to an increasing interest in
behaviour modifying chemicals – antifeedants - that will deter insects from feeding. Much
effort is now placed into better understanding the feeding mechanism of insects, as a means to
design simple chemicals that mimic the antifeedant activities of naturally occurring
compounds, such as plant-derived compounds [11].
2.3.1
Antifeedant activity of pine weevil feces
The female pine weevil’s habit of placing their eggs into the host plant tissues with the aid of
their snout is an ancestral trait of the weevil family. The females chew through the outer bark
(into the phloem tissue), about as far as they can reach with their snout. The females then
deposit their egg in the chewed out cavities together with some of their feces and seals the
cavity with a plug made out of bark. Similar ovipositioning behavior has also been noted in
other Hylobius species [10].
9
Feeding bioassay experiments done by Borg-Karlsson et al. (2006, [10]) clearly display an
antifeedant activity towards the methanol extracts of pine weevil feces, for pine weevils of
both sexes. The feeding bioassays also displays that feces from both male and female pine
weevils has antifeedant activity (figure 2) [10].
Figure 2. Feeding bioassay experiments conducted with 20 pine weevils of both sexes. Choosing between
feeding on twigs treated with either methanol extracts or hexane extracts of feces or a control twig treated with
only the corresponding solvent (methanol or hexane). White column – control; black column – methanol extract;
and hatched column – hexane extract. Bars denote SE – standard error [10]
In the article by Borg-Karlsson et al. (2006), the authors note that the most active
antifeedants, in the methanolic extract from the pine weevil feces, are structurally related to
the building blocks of lignin and that the antifeedants are probably the result of lignin
degradation. The authors suggest that the lignin degradation is accomplished, in the gut of the
pine weevil, either by bacteria or fungal symbionts [10].
2.4 Community analysis
Historically, the characterization of microbial community composition was much limited due
to the fact that it was not possible to cultivate a major fraction of the microorganisms in the
biosphere in a laboratory environment (estimations show that the microbial community in 1
gram of soil may contain over one thousand different bacterial species, but less than 1% of
these may be culturable) [23] [24]. Although the culture-dependent methods provided great
insight into the microbial community and its individual members, the limitations meant
difficulties in fully understanding the microbial diversity, and the functionality and
importance of unculturable species in a specific environment [25].
10
The development of molecular biology tools, including culture-independent methods, over the
last two decades has led to the emerge of a new discipline termed molecular microbial
ecology. The use of these culture-independent methods has greatly increased our
understanding and the potential for understanding the microbiota around us [26].
Initially, fatty acids profiling was used as the culture-independent method to analyze
microbial communities, but gradually DNA-based techniques has taken over as the method of
choice [23] [25]. The fatty acids profiling is based on phospholipids (PLFA) of the cell
membranes in living cells (phospholipids degrades quickly upon cell death). The lipid
composition of living cells change based upon the environmental conditions, thus making the
fatty acids a useful biomarker tool for assessments of the current community structure and
physiological state [23]. Fatty acids can also be used for phylogenetic studies. However, due
to the limited complexity of the fatty acids profiling, this method is now often used together
with other profiling methods [23].
Most culture-independent methods used nowadays are DNA-based techniques. Most studies
are done using the 16S ribosomal RNA gene as the molecular marker, but other genetic
markers are also used [23] [25] [27].
2.4.1 Genetic fingerprinting
There are several ways of categorizing the different DNA-based methods, but one subset of
the DNA-based techniques could be said to be the genetic fingerprinting (or DNA profiling,
DNA typing, etc.) techniques. The genetic fingerprinting methods include RFLP, T-RFLP,
DGGE, TGGE, RISA and D-HPLC [25]. RFLP, T-RFLP, DGGE and TGGE all belong to the
more commonly utilized fingerprinting methods [28].
RFLP (restriction fragment length polymorphism) is a method based on the digestion of
amplified DNA sequences (i.e. the 16S rRNA gene) with one or more restriction enzymes.
The fragments are separated and visualized with agarose gel electrophoresis. The idea is that
every unique sequence should be represented by a unique pattern (a restriction pattern) on the
agarose gel. However, the restriction pattern for a specific sequence will look different,
depending on the restriction enzyme(s) used. One difficulty with RFLP is the selection of
restriction enzyme(s) to use, especially for microorganisms with unknown genomes. RFLP is
a simple, but time-consuming method, good for detecting structural changes is more simple
microbial communities, but not so useful for detecting diversity or specific phylogenetic
groups [23] [25].
T-RFLP (terminal restriction fragment length polymorphism) is a modification of RFLP;
more automated, high-throughput and with higher sensitivity than the regular RFLP. The
5’-end DNA fragments are labeled with fluorescent dye. The fragments are separated with
high-resolution gel electrophoresis on an automated DNA sequencer and detected using a laser to
produce an electropherogram. The optimization of the restriction enzyme(s) used remains an
issue, but compared to RFLP, the method can be used for profiling of microbial communities
of higher complexity [23] [25].
For DGGE (denaturing gradient gel electrophoresis) and TGGE (temperature gradient gel
electrophoresis) small PCR products (approximately 200 – 700 bp) are separated on
acrylamide gels, with either a chemical denaturation gradient or temperature denaturation
gradient respectively. High GC content or GC clamp is needed for the sequence. The method
11
is affordable and good for i.e. intracommunity structural changes, but time-consuming and has
suboptimal reproducibility [23] [25].
In D-HPLC (denaturing high-performance liquid chromatography), the DNA is denatured
both chemically and with temperature and separated in a liquid chromatography cartridge. An
UV detector records the different fractions of eluted DNA as absorbance over time in an
electropherogram. The method is quite new and promising for microbial ecology work, but
the separation parameters need to be optimized for each unique sample. More investigation is
also required to fully establish its use [23] [25].
Unlike the other methods described above, where the target sequence usually is the 16S rRNA
gene, the target sequence for RISA (ribosomal intergenic spacer analysis) is the intergenic
space between the 16S and 23S rRNA genes. RISA allows for resolution of closely related
strains. The variability of the sequence may be too great for environmental samples (higher
variability than 16S rRNA gene) [23] [25].
2.4.2 Sequencing
Sequencing methods (determination of the nucleotide order in DNA) is another subset of the
DNA-based techniques. Sequencing the targeted DNA is the community analysis method that
offers the highest phylogenetic resolution [25]. The first sequencing method, Sanger
sequencing, was developed during the 1970s. Sanger sequencing has since then developed
into a high-quality and high-throughput method [29]. However, the cost is still too high to
fully replace other community analysis methods, like fingerprinting techniques, for many
laboratories [29] [33].
Pyrosequencing is another sequencing method, developed during the 1990s. Pyrosequencing
is less suitable than Sanger sequencing for sequencing of long fragments, but is reliable,
quantitative, fast and cheap for sequencing of short to medium range fragments [29].
Pyrosequencing of the 16S rRNA gene pools is currently replacing other sequencing methods
and even genetic fingerprinting methods as the method of choice for community analysis [30].
However, the debate is still ongoing regarding the reproducibility of pyrosequencing and if
the method can adequately recover relative species abundances in the microbial communities
[30].
The main advantage of sequencing compared to fingerprinting is the possibility to categorize
sequences according to taxonomy and function. Results from different studies can be
compared. The sequences can be used for phylogenetics (the study of the evolutionary
relationships between different organisms) and combined into phylogenetic trees showing that
evolutionary relationship [33].
Some high-throughput sequencing methods, like 454 Pyrotag Sequencing, use parallel
sequencing systems that can sequence approximately 400-600 megabases of DNA per
10-hour, but have a limit of 400-500 base pair read length [13]. Genomic DNA can be split into
smaller fragments and ligated with adaptor sequence for which matching primers are
provided. That additional preparation step is not always efficient or justified (due to the extra
cost, time, etc.) for smaller sequences like the 16S rRNA gene. However, currently there is no
consensus regarding which region of the 16S rRNA that is best to sequence, for example for
phylogenetic studies [33]. Different research groups sequence different regions of the gene
[33].
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2.4.3 16S rRNA gene
The 16S ribosomal gene has been used since the mid 1980s for phylogenetic studies of the
microbial community [32]. One advantage of the 16S rRNA gene for community analysis is
that it contains both hypervariable and highly conserved regions (figure 3) [33]. The
conserved regions allow for designing primers that bind to DNA of many different bacteria
and archaea species, and even eukaryotic species. The hypervariable regions on the other hand
are used to distinguish different species from each other [33].
2.4.4 Community analysis of insect gut
Culture independent methods have been used for bacteria community analysis of the gut of
other insect species. The insects investigated include: sawflies species, honey bee, desert
locust, gypsy moth larval and pine beetle [47] [49]. Total DNA (including bacterial DNA)
extracted from gypsy moth larval and pine beetle were for examples amplified with same
universal primers (27f and 1492r) used for this report [49].
13
3 Materials & Methods
The pine weevil samples used for the project were collected in Boda, Dalarna on 26-27 May
2009. The guts and eggs were dissected in a dissection bowl with sterile water using scissors
and tweezers.
The midgut (T8BAM), hindgut (T8BAH) and egg (T8BAegg) sample were collected from the
same five pine weevil females.
3.1 Flowchart
Displayed in figure 4 is the flowchart over the culture-independent methods used for the
bacterial community analysis.
3.2 DNA extraction
DNeasy Blood & Tissue kit (Qiagen) was used for the DNA extraction. The pine weevil
midgut, hindgut and egg samples were stored in a -20°C freezer prior to DNA extraction. The
samples were thawed on ice, but all other extraction steps were carried out at room
temperature. The protocol for Purification of Total DNA from Animal Blood or Cells
(pretreatment for Gram-positive bacteria) was followed.
The samples were suspended in 180
μl of the enzymatic lysis buffer, grounded with a sterile
wooden toothpick into a fine pulp and vortexed thoroughly. The samples were incubated at
37°C for 30 minutes
, after which 25 μl of proteinase K and 200 μl of lysis buffer AL were
added to the samples. The samples were vortexed and incubated at 56°C for 30 minutes. 200
μl of 95% ethanol was then added, the samples were vortexed immediately and thoroughly,
Figure 4. Flowchart over the culture-independent methods used for bacterial
community analysis in this report.
14
and centrifuged at 12 000 rpm for 10 minutes. The supernatants were salvaged (the
centrifugation step was repeated when there was tissue debris remaining in any supernatant)
and pipetted onto the DNeasy minispin columns, placed in 2 ml collection tubes.
The tubes were centrifuged at 6 000 rpm for 1 minute and the flow-through discarded.
Furthermore,
500 μl of washing buffer AW1 was added to each sample and the samples were
centrifuged at 6 000 rpm for 1 minute. The flow-through was discarded and
500 μl of washing
buffer AW2 was added to the columns. The samples were centrifuged at 12 000 rpm for 3
minutes. The flow-through was discarded and the samples were centrifuged again at 12 000
rpm for 3 minutes, after which the minispin columns were placed in clean 1.5 ml
microcentrifuge tubes.
For the elution step,
50 μl of elution buffer AE was pipetted onto the center of each of the
minispin columns. The samples were incubated at room temperature for 1-2 minutes, then
centrifuged at 6 000 rpm for 1 minute. The eluted DNA was stored at -20°C until further
analysis. The eluted DNA could potentially contain DNA from both the pine weevils’
microbiota and from the pine weevil itself.
3.3 PCR amplification
The extracted DNA samples, sterile nuclease free H
2O, High-Fidelity buffer 5X (NEB),
universal primers 27f and 1492r, the dNTP mix (NEB), MgCl
2(NEB) and Phusion
polymerase (NEB) were thawed completely on ice before use. Where possible, all subsequent
preparation steps were also carried out on ice. Pre-labeled PCR tubes were used for the PCR
reaction. Sterile H
2O was used as a negative control. The PCR resulted in blunt-ended PCR
products.
The total reaction
volume for each PCR tube was always 50 μl, but the volume and
concentration for each of the reagents varied slightly between different PCR runs, PCR tubes
and whether midgut, hindgut or egg sample. The volumes and concentrations stated below are
those used for the PCR tube, with midgut sample DNA, that produced the PCR products that
were used for further analysis steps (see appendix I, for the exact volumes and concentrations
used for other PCR tubes and PCR runs, both midgut, hindgut and egg sample).
After thawing the reagents, 26.
6 μl sterile nuclease free H
2O, then 1
μl of a 1:10 dilution
(diluted with sterile nuclease free H
2O) of DNA template was added directly into the PCR
tube. A mastermix was then prepared and the reagents were added to the mastermix in the
specified order: 10
μl/PCR tube of HF buffer; 5 μl/tube each of primer 27f (2 μM) and primer
1492r (2
μM); 1.2 μl dNTP mix (10mM); 0.7 μl MgCl
2(0.7 mM); and last
0.5 μl Phusion
polymerase (0.
02U/μl).
The primers were vortexed and the HF buffer, dNTP mix and polymerase were tapped gently
before added to the mastermix. The mastermix was thoroughly mixed by pipetting the mixture
slowly up and down six times. 22.4
μl of the mastermix was added directly to each PCR tube.
The solution in each PCR tube was thoroughly mixed by pipetting the solution slowly up and
down six times.
Thermocycler PTC-200 (MJ Research) was used for the PCR program. Initial denaturation
was at 98°C for 2 minutes, followed by denaturation at 98°C for 10 seconds. The annealing
time was always 30 seconds, but for the midgut sample the annealing temperature started at
15
60°C, decreasing one degree for each PCR cycle until reaching 50°C (touchdown annealing).
The annealing temperature was 50°C for the following 19 cycles of the program. The
annealing temperature for the egg sample was 55°C and the PCR program ran for 30 cycles.
The extension step was always at 72°C for 25 seconds and the final extension always at 72°C
for 10 minutes. The PCR products were stored in -20°C until further analysis.
3.4 Cloning and transformation
Zero Blunt TOPO PCR Cloning kit for Sequencing (Invitrogen) was used for the cloning and
transformation. The blunt-ended PCR products were cloned into the plasmid vector, supplied
with the kit, and transformed with One Shot TOP10 chemically competent E. coli.
(Invitrogen).
The PCR products, sterile nuclease free H
2O, salt solution (provided in the kit) and the TOPO
vectors were thawed completely on ice before use. Where possible, all subsequent preparation
steps were also carried out on ice.
Cloning reaction - midgut sample:
PCR product (2
μl); 1 μl of salt dilution; 2 μl of sterile H
2O; and 1
μl of the TOPO
vector (total volume 6
μl) were mixed together and incubated at room temperature for 10
minutes.
Cloning reaction - egg sample:
PCR product (3.5
μl); 1 μl of salt dilution; 0.5 μl of sterile H
2O; and 1
μl of the TOPO
vector (total volume 6
μl) were mixed together and incubated at room temperature for 10
minutes.
Transforming chemically competent cells – midgut & egg sample:
2
μl of the cloning reaction was added to one vial of TOP10 chemically competent cells (the
cells were thawed on ice for 2-5 minutes before use). The solution was mixed gently with a
pipette tip and incubated on ice for 10 - 20 minutes. The cells were heat-shocked at 42°C in a
water-bath for 45 seconds, then immediately placed on ice. 250
μl of room tempered S.O.C.
medium was added to the mixture. The mixture was incubated at 37°C for 1 hour in a
horizontally shaking incubator (180 rpm).
After incubation, 10 - 50
μl of the transformation mixture was spread out on Kanamycin (50
μg/ml) Low Salt LB plates, pre-warmed at 37°C for 30 minutes. The plates were incubated at
37°C overnight. The plates were stored at 4 - 8°C and re-plated on new Kanamycin Low Salt
LB plates every second week.
3.5 Plasmid extraction
QIAprep spin miniprep kit (Qiagen) was used for the extraction of the plasmids from the
transformants in the clone library.
Single colonies were picked with a sterile wooden toothpick and suspended in 3 ml of Low
Salt LB medium (containing 50
μg/ml Kanamycin) and incubated overnight (not more than 16
hours) at 37°C in a horizontally shaking incubator (180 rpm).
16
All plasmid extraction steps were carried out at room temperature. The overnight culture was
divided into two microcentrifuge tubes and centrifuged at 9 000 rpm for 3 minutes. The
supernatant from both tubes was discarded. The resulting pelleted bacterial cells in one of the
microcentrifuge tubes were completely resuspended with 250
μl buffer P1. The whole
resuspension from the first tube was transferred over (the empty tube discarded) to the second
tube and the pelleted bacterial cells in that tube were also resuspended. 250
μl of buffer P2
was added to the tube and mixed thoroughly by inverting the tube 4 – 6 times or until the
solution became viscous and slightly clear. The mixture was not allowed to stand for more
than 5 minutes, before adding the next buffer.
Next, 350
μl buffer N3 was added, and mixed immediately and thoroughly by inverting the
tube 4 – 6 times (or until the solution became cloudy). The solution was centrifuged at 13 000
rpm for 10 minutes. The resulting supernatant was pipetted onto a QIAprep spin column and
centrifuged at 13 000 rpm for 45 seconds. The flow-through was discarded. The spin column
was then washed by adding 500
μl of buffer PB and centrifuged at 13 000 rpm for 45 seconds.
The flow-through discarded. In a second washing step, 750
μl of buffer PE was added onto
the spin column and the spin column was centrifuged at 13 000 rpm for 45 seconds. The
flow-through discarded. The spin column was centrifuged an additional time at 13 000 rpm for 1
minute to remove residual washing buffer. The flow-through discarded.
The spin column was then placed in a clean 1.5 ml microcentrifuge tube. 50
μl of elution
buffer EB was added directly onto the center of the spin column and the solution was
incubated at room temperature for 1 minute. The plasmid DNA was eluted from the spin
column into the microcentrifuge tube by centrifuging the solution at 13 000 rpm for 1 minute.
The plasmid DNA was stored at -20°C until further analysis.
The plasmid DNA was analyzed with agarose gel electrophoresis.
3.6 Colony PCR
Colonies from the clone library were picked with a sterile wooden toothpick or with a sterile
pipette tip (a 1
μl – 100 μl pipette tip). The colonies were suspended in 50 μl of sterile
nuclease free H
2O.
After thawing the reagents, 25.3
μl sterile nuclease free H
2O, then 3
μl of the suspended DNA
template was added directly into each PCR tube. A mastermix was prepared and the reagents
were added to the mastermix in the specified order: 10
μl/PCR tube of HF buffer; 5 μl/tube
each of primer 27f (2
μM) and primer 1492r (2 μM); 1.2 μl dNTP mix (10mM); and last 0.5
μl Phusion polymerase (0.02U/μl). The PCR protocol as described earlier in section 3.3 was
then followed.
Thermocycler PTC-200 (MJ Research) was used for the PCR program. The PCR program ran
for 30 cycles. Cell breakage/initial denaturation was at 95°C for 10 minutes, followed by
denaturation at 98°C for 10 seconds. The annealing time was 30 seconds and the annealing
temperature 55°C. The extension step was at 72°C for 25 seconds and the final extension at
72°C for 10 minutes. The PCR products were stored at -20°C until further analysis.
17
3.7 RFLP
Restriction enzymes RsaI (Promega) and HaeIII (Takara) (Fig 5) were used for the DNA
digestion. 14.5
μl sterile H
2O; 2
μl M Buffer 10X (Takara); 4 μl PCR product; 1 μl RsaI; and
0.5
μl HaeIII were mixed together (in the mentioned order) and stirred gently with a pipette
tip. The samples were digested at 37°C for 1 hour. The restriction enzymes were inactivated
by adding 4
μl of loading dye 10X (Takara) to the mixture. The digested PCR products were
stored at -20°C until further analysis.
The agarose gel electrophoresis of the digested samples was accomplished with a sub-cell
system from Bio-Rad. The samples were separated on a 1.5 % agarose gel in buffer TBE.
After casting the gel and transferring the gel to the buffer tank, 7
μl of the samples were
loaded into each well. DNA marker Generuler plus 100 bp ready-to-use (Fermentas) was
loaded on the left side of the samples and DNA marker 50 bp step ladder (Promega) was
loaded on the right side of the samples. Run voltage was 80V
RsaI
HaeIII
5’….GT
▼AC…3’
5’…GG
▼CC…3’
3’…CA
▲TG…5’
3’…CC
▲GG…5’
3.8 Agarose gel electrophoresis
The agarose gel electrophoresis of the DNA extraction, plasmid DNA and PCR samples were
accomplished with a sub-cell system from Bio-Rad. 2
μl of loading dye 6X were added to 5 μl
(less volume for the plasmid DNA) of each sample. The samples were separated on a 1.0 %
agarose gel in buffer TAE or TBE. After casting the gel and transferring the gel to the buffer
tank, the samples were loaded into each well. DNA marker was loaded on the left and/or right
side of the samples. Run voltage was 100V.
3.9 Sequencing
The sequencing was done in four batches. The first, third and fourth batch were submitted for
commercial sequencing, and the second batch was sequenced at the laboratory in Alba Nova,
level 3.
The methods used for sequencing were considered as out of scope for this project and were
not investigated.
Batch 1 to 3 was sequenced with primers 27f and 1492r.
Batch 4 was sequenced using plasmid DNA extracted from the clones. The plasmid extraction
was done by associate professor Olle Terenius (SLU). Batch 4 was sequenced with primers
M13f and M13r.
3.10 Phylogenetic analysis
Sequenced midgut and egg clones were aligned with multiple sequence alignment tool
ClustalW. The aligned clones were then used to construct a maximum likelihood tree (using
18
the Tamura-Nei model for DNA sequence evolution and 500 bootstrap replications for testing
the reliability of the phylogenetic tree). Both the multiple sequence alignment and tree
construct was done in the computer program Mega version 5.05.
The percentage of coverage of the sequence analysis was calculated with Good’s method,
using the formula [1 - (n/N)] x 100 (where n is the number of sequences represented by one
OTU - operational taxonomic unit - and N is the total number of sequences) [60].
For this project an OTU was counted as sequences with 97 percent or higher similarity.
4 Results
4.1 DNA extraction
DNA extracted from the midgut and hindgut sample yielded clear bands on the agarose gel
image (figure 6). The putative concentration of the eluted DNA was higher for the midgut
sample than the hindgut sample. The agenda was to elute bacterial DNA from the host cells,
but the eluted DNA samples might have also included other microbial DNA (i.e. fungal DNA)
and DNA from the host itself.
The DNA extraction from the egg sample resulted in a faint, but visible band on the agarose
gel electrophoresis (figure 7). The estimated concentration of the eluted DNA was less than
42 ng/
μl. The agenda was to elute bacterial DNA from the host cells, but the eluted DNA
samples might have also included other microbial DNA (i.e. fungal DNA) and DNA from the
host itself.
Figure 6. DNA extraction: T8BAM & T8BAH. Lane M – DNA
marker (100 bp exACTGene, Fischer). Lane T8M – T8BAM
midgut sample. Lane T8H – T8BAH hindgut sample.
19
4.2 PCR amplification
Eluted DNA from the T8BAM (midgut) and T8BAegg sample were successfully amplified
using PCR, as described in section 3.3. The PCR products were approximately 1.5 kb in
length (as shown in figure 8 and 9), which corresponds to the expected amplicon length when
using universal primers 27f and 1492r.
Several PCR runs were made for T8BAM, but PCR product was only found for one PCR run
and only in one PCR tube (tube 5 corresponding to lane 3 in the agarose gel electrophoresis
image, figure 8, shown below). The amount of DNA template used for that tube 5 was 0.1
μl.
The conditions used for each PCR tube (i.e. amount of DNA template) can be found in
appendix I.
Figure 7. DNA extraction: T8BAegg. Lane M – DNA
marker. Lane T8egg – T8BAegg sample (1 kb
Quick-load, NEB).
Figure 8. PCR amplification: T8BAM. Lane 1 – DNA
marker (1 kb Generuler, Fermentas). Lane 2 – PCR tube 5
(negative control). Lane 3 – PCR tube 5 (0.1
μl DNA
template). Lane 4 – PCR tube 4 (0.5
μl DNA template).
20
Only one PCR run was done with T8BAegg. PCR products were found in three of the PCR
tubes (see figure 9). Tube 6 (corresponding to lane 1), containing 0.1
μl DNA template was
used for further analysis. The conditions used for each PCR tube (i.e. amount of DNA
template) can be found in appendix I.
All attempts to amplify the DNA eluted from the T8BAH hindgut sample were unsuccessful.
The conditions used for each PCR tube from one PCR run can be found in appendix I (no
agarose gel image).
4.3 Cloning and transformation
The petri plates incubated with 10 – 20
μl transformation mixture contained < 50 to < 100
transformed E. coli colonies and the plates incubated with 20 – 40
μl transformation mixture
contained < 100 to < 200 transformed E. coli colonies. 60 midgut clones and 30 egg clones
(figure 10) were randomly selected for a clone library and further analysis.
Figure 9. PCR amplification: T8Begg. Lane 1 – PCR tube 6 (0.1
μl DNA
template). Lane 2 – PCR tube 5 (0.5
μl DNA template). Lane 3 – PCR
tube 4 (1
μl DNA template). Lane 4 – PCR tube 3 (3 μl DNA template).
Lane 5 – PCR tube 2 (5
μl DNA template). Lane 6 – DNA ladder (1kb
Generuler, Fermentas).
21
4.4 Plasmid DNA extraction
Plasmid DNA was extracted from T8BAM clone 1 - 4, to assess if the competent E. coli cells
were transformed properly and contained recombinants of the right size. The extracted
plasmids were also used for downstream analysis (PCR amplification and sequencing). The
agarose gel image showed 2 clear bands for each clone: one band at approximately 5 - 6 kb
(open circular plasmid) and another band at approximately 3 kb (supercoiled plasmid). See
figure 11.
Figure 10. Clone library for T8BAM: clone 1 – 12.
Figure 11. Plasmid extraction: T8BAM clones. Lane 1 – DNA
ladder (1 kb Quick-load, NEB). Lane 2 – clone 1. Lane 3 – clone
2. Lane 4 – clone 3. Lane 5 – clone 4.
22
4.4.1 PCR with plasmid DNA
PCR was successful when using the extracted T8BAM plasmid DNA as templates (clone 1 –
4). See figure 12.
4.5 Colony PCR
Colony PCR resulted in PCR products of the appropriate length (approx. 1.5 kb, figure 13) for
49 out of the 60 selected midgut clones. However, the DNA concentration for 7 out of the 49
successfully amplified clones was assessed to be too low to be used for sequencing.
Colony PCR for the egg clones resulted in PCR product for all 30 clones. However, 8 out of
the 30 clones had amplicons of incorrect length or resulted in several amplicons of different
lengths.
Figure 12. PCR with plasmid DNA templates:
T8BAM clones. Lane 1 – DNA ladder (1 kb
Quick-load, NEB). Lane 2 – clone 1. Lane 3 – clone 2.
Lane 4 – clone 3. Lane 5 – clone 4.
23
Figure 13 and 14 display the agarose gel electrophoresis images for midgut PCR products and
egg PCR products respectively. See appendix III and IV for all agarose gel electrophoresis
images.
4.6 RFLP
The agarose gel electrophoresis images for several double digested midgut clones are shown
in figure 15 and 16. Based on the restriction patterns on the gel images, an attempt was made
to divide the clones into different RFLP groups (shown in the figures). However, assigning
the restriction patterns into different RFLP groupings was difficult, since many bands were
either faint or distorted or both. Furthermore, all restriction patterns in i.e. RFLP group A are
not completely identical and should perhaps be divided into several RFLP groups. For
example; E. coli (clone 41), Shigella sp. (clone 45), Chryseobacterium sp. (clone 46) and
Figure 13. Colony PCR: T8BAM (midgut). Lane 1 –
clone 14. Lane 2 – clone 13. Lane 3 – clone 12. Lane
4 – clone 11. Lane 5 – clone 10. Lane 6 – clone 9.
Lane 7 – DNA ladder (1kb Quick-load, NEB).
Figure 14. Colony PCR: T8BAegg. Lane 1 – clone 10.
Lane 2 – clone 9. Lane 3 – clone 8. Lane 4 – clone 7.
Lane 5 – clone 6. Lane 6 – clone 5. Lane 7 – DNA
ladder (1kb Generuler, Fermentas).
24
Flavisolibacter ginsengisoli (clone 8) are all heterologous species, but assigned to RFLP
group A.
Chimeric sequences clone 16 (Acinetobacter sp. and Wolbachia sp.) and clone 26
(Ramlibacter sp. and Wolbachia sp.), are also assigned to group A.
Some Wolbachia clones (i.e. clone 10; RFLP group E and 21; RFLP group F) are assigned to
different RFLP groups than the majority of the Wolbachia clones. Most Wolbachia clones are
assigned to group A.
RFLP was not done on the egg clone sequences due to time constraints.
Figure 15. RFLP: T8BAM (midgut). Lane 1 corresponds to clone 1 and so on. Lane M1 – DNA
ladder (50 bp Step Ladder, Promega). Lane M2 – DNA ladder (100 bp Quick-load, NEB).
25
4.7 Sequencing
The three tables below list the BLAST result for each sequenced clone.
Sequencing results of sufficient quality were found for all 39 midgut clones submitted for
sequencing. 31 out of 39 sequenced clones had BLAST results that matched known species
with 98 percent or higher.
One egg clone was never submitted for sequencing. Sequencing results of sufficient quality
were found for 27 out of 29 egg clones submitted for sequencing. 22 out of 29 sequenced
clones had BLAST results that matched known species with 98 percent or higher.
The sequenced single-strains were assembled into double-strains with CodonCode Aligner
version 4.0.3. It was not possible to assemble the single-strains for all clones (see table 1 – 3
for further details).
The sequenced clones were checked for chimeric sequences with DECIPHER’s Find
Chimeras web tool and with USEARCH’s UCHIME version 5.0 [21] [22]. Clone16_midgut
and Clone26_midgut were identified as chimeric with both Find Chimeras and UCHIME,
Clone6_midgut was identified as chimeric with just UCHIME. Midgut clones 48 and 60, and
egg clone 25 were marked as indecipherable by Find Chimeras (meaning that “the clones
could not be properly evaluated for chimeric sequences”) [22].
Figure 16. RFLP: T8BAM (midgut). Lane 39 corresponds to clone 39 and so on. Lane M1 – DNA ladder
(50 bp Step Ladder, Promega). Lane M2 – DNA ladder (100 bp Quick-load, NEB).
26
Table 1. Sequenced midgut clones (clone 1 – 30).
MIDGUT CLONES
BLAST RESULT MAX.
ID. [%] TAXONOMY CLASS: ORDER RFLP GROUP BATCH NO. IN TREE
1 PCR product not submitted for sequencing N/A NA A N/A N/A 2 Wolbachia endosymbiont of D. pinicola 99 Alphaproteobacteria; Rickettsiales B First Y 3 Wolbachia endosymbiont of D. pinicola 99 Alphaproteobacteria; Rickettsiales B First Y 4 PCR product not submitted for sequencing N/A N/A A N/A N/A 5 PCR product not submitted for sequencing N/A N/A C N/A N/A 6a Wolbachia sec. endosymbiont of C. okumai 99 Alphaproteobacteria; Rickettsiales D First N 6b Conserved? Agrobacterium/Rhizobium/Shinella 99 N/A D First N 7 Wolbachia sec. endosymbiont of C. okumai 99 Alphaproteobacteria; Rickettsiales A Third Y 8 Uncultured bacterium/Flavisolibacter ginsengisoli 98/97 Sphingobacteria; Sphingobacteriales A/A Fourth Y 9 Wolbachia sec. endosymbiont of C. okumai 99 Alphaproteobacteria; Rickettsiales A Third Y 10 (27f) Wolbachia endosymbiont of D. pinicola 98 Alphaproteobacteria; Rickettsiales E Third Y 11 No PCR product, not sequenced N/A N/A N/A N/A N/A 12 Too low DNA concentration for sequencing N/A N/A N/A N/A N/A 13 No PCR product, not sequenced N/A N/A N/A N/A N/A 14 (1492r) Wolbachia endosymbiont of Glossina austeni 99 Alphaproteobacteria; Rickettsiales A/I Third N
15 No PCR product, not sequenced N/A N/A N/A N/A N/A 16a Acinetobacter sp. 99 Gammaproteobacteria; Pseudomonadales A/A Fourth Y 16b Uncultured bacterium/Wolbachia pipientis 100/99 Alphaproteobacteria; Rickettsiales A/A Fourth N 17 Too low DNA concentration for sequencing N/A N/A N/A N/A N/A 18 Too low DNA concentration for sequencing N/A N/A N/A N/A N/A 19 Too low DNA concentration for sequencing N/A N/A N/A N/A N/A 20 No PCR product, not sequenced N/A N/A N/A N/A N/A 21 Wolbachia endosymbiont of P. longiceps 96 Alphaproteobacteria; Rickettsiales F Second N 22 (1492r) Uncultured alphaproteobacterium/Wolbachia sp. 96/96 Alphaproteobacteria; Rickettsiales A Second N 22 (27f) Wolbachia endosymbiont of P. longiceps 96 Alphaproteobacteria; Rickettsiales A Second N 23 No PCR product, not sequenced N/A N/A N/A N/A N/A 24 (1492r) Wolbachia sec. endosymbiont of C. okumai 96 Alphaproteobacteria; Rickettsiales G Second N
25 Wolbachia sec. endosymbiont of C. okumai 98 Alphaproteobacteria; Rickettsiales A Third Y 26a Uncultured soil bacterium/Ramlibacter sp. 99/99 Betaproteobacteria; Burkholderiales A Fourth Y 26b Wolbachia pipientis 99 Alphaproteobacteria; Rickettsiales A Fourth N 27 Uncultured bacterium /E. coli/Shigella sp. 95/95/95 Gammaproteobacteria; Enterobacteriales A Second N 28 (27f) Wolbachia endosymbiont of P. longiceps 95 Alphaproteobacteria; Rickettsiales A Second N 29 (1492r) Wolbachia sp. 92 Alphaproteobacteria; Rickettsiales A Second N 30 No PCR product, not sequenced N/A N/A N/A N/A N/A
27
Table 2. Sequenced midgut clones (clone 31 – 60).
MIDGUT CLONES
BLAST RESULT MAX.
ID. [%] TAXONOMY CLASS; ORDER RFLP GROUP BATCH NO. IN TREE
31 No PCR product, not sequenced N/A N/A N/A N/A N/A 32 (1492r) Wolbachia sp. 87 Alphaproteobacteria; Rickettsiales A Second N
33 Too low DNA concentration for sequencing N/A N/A N/A N/A N/A 34 Wolbachia sec. endosymbiont of C. hilgendorfi 99 Alphaproteobacteria; Rickettsiales J Third Y 35 No PCR product, not sequenced N/A N/A N/A N/A N/A 36 No PCR product, not sequenced N/A N/A N/A N/A N/A 37 Wolbachia sec. endosymbiont of Curculio sp. 98 Alphaproteobacteria; Rickettsiales A Second N 38 No PCR product, not sequenced N/A N/A N/A N/A N/A 39 Wolbachia secondary endosymbiont 99 Alphaproteobacteria; Rickettsiales C Third Y 40 Wolbachia secondary endosymbiont 99 Alphaproteobacteria; Rickettsiales A Third Y 41 Escherichia coli 99 Gammaproteobacteria; Enterobacteriales A Third Y 42 Too low DNA concentration for sequencing N/A N/A N/A N/A N/A 43 Uncultured bacterium clone/Wolbachia sp. 99/99 Alphaproteobacteria; Rickettsiales A Fourth Y 44 Too low DNA concentration for sequencing N/A N/A N/A N/A N/A 45 Shigella sp. 99 Gammaproteobacteria; Enterobacteriales A Third Y 46 Unidentified bacterium/Chryseobacterium sp. 98/98 Flavobacteria; Flavobacteriales A Third Y 47 Wolbachia pipientis 99 Alphaproteobacteria; Rickettsiales A Fourth Y 48 (1492r) Enterobacter sp. 78 Gammaproteobacteria; Enterobacteriales A Third N 49 Wolbachia pipientis 99 Alphaproteobacteria; Rickettsiales A Fourth Y 50 Uncultured bacterium clone/Wolbachia sp. 99/99 ND/Alphaproteobacteria; Rickettsiales A Fourth Y 51 Wolbachia sp. 99 Alphaproteobacteria; Rickettsiales A Third N 52 Wolbachia sec. endosymbiont of C. okumai 99 Alphaproteobacteria; Rickettsiales H Third Y 53 (1492r) Wolbachia sec. endosymbiont of C. okumai 99 Alphaproteobacteria; Rickettsiales A Third Y 54 (1492r) Wolbachia sec. endosymbiont of D. nikananu 99 Alphaproteobacteria; Rickettsiales H Third Y 55 Wolbachia sec. endosymbiont of C. okumai 98 Alphaproteobacteria; Rickettsiales C Third Y 56 Wolbachia sp. 99 Alphaproteobacteria; Rickettsiales A Fourth Y 57 Uncultured bacterium clone/Wolbachia sp. 99/99 Alphaproteobacteria; Rickettsiales C Fourth Y 58 No PCR product, not sequenced N/A N/A N/A N/A N/A 59 (1492r) Wolbachia sec. endosymbiont of C. hilgendorfi 99 Alphaproteobacteria; Rickettsiales A Third Y
28
Table 3. Sequenced egg clones (clone 1 – 30).
EGG CLONES
BLAST RESULT MAX.
ID. [%] TAXONOMY CLASS; ORDER BATCH NO. IN TRE E
1 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Fourth Y 2 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 3 Uncultured bacterium clone/Wolbachia pipientis 99/99 Alphaproteobacteria; Rickettsiales Third N 4 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 5 Wolbachia sp. 100 Alphaproteobacteria; Rickettsiales Third N 6 (1492r) Halomonas phoceae 98 Gammaproteobacteria; Oceanospirillales Third Y 7 Wolbachia sec. endosymbiont of Curculio okumai 99 Alphaproteobacteria; Rickettsiales Third Y 8 Halomonas phoceae 98 Gammaproteobacteria; Oceanospirillales Third Y 9 Halomonas phoceae 97 Gammaproteobacteria; Oceanospirillales Third N 10 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 11 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 12 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 13 Shewanella sp. 100 Gammaproteobacteria; Alteromonadales Fourth Y 14 Sequencing data of insufficient quality N/A N/A Third N/A 15 (1492r) Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y
16 (27f) Shewanella haliotis 99 Gammaproteobacteria; Alteromonadales Third Y 17 PCR product not submitted for sequencing N/A N/A N/A N/A 18 Sequencing data of insufficient quality N/A N/A Third N/A 19 Uncultured bacterium clone/Wolbachia pipientis 93/93 Alphaproteobacteria; Rickettsiales Third N 20 Wolbachia sec. endosymbiont of Curculio okumai 99 Alphaproteobacteria; Rickettsiales Third Y 21 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 22 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 23 (1492r) Streptococcus mitis 91 Bacilli; Lactobacillales Third N 24 Wolbachia pipientis 100 Alphaproteobacteria; Rickettsiales Third N 25 (1492r) Persephonella sp. 90 Aquificae; Aquificales Third N
26 Wolbachia endosymbiont of Sogatella furcifera 99 Alphaproteobacteria; Rickettsiales Third N 27 Uncultured organism clone/ Escherichia coli 96/96 Gammaproteobacteria; Enterobacteriales Third N 28 Halomonas phoceae 99 Gammaproteobacteria; Oceanospirillales Third Y 29 (1492r) Expression vector pOT-RA 99 N/A Third N/A 30 (1492r) Expression vector pOT-RA 100 N/A Third N/A
