Culture Independent Analysis of Microbiota in the Gut of Pine Weevils

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

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

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 5 Discussion ...... 29

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 VI. Sequence for clones & sample species ...... 71

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.

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].

Figure 1. Pine weevil.

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

7 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].

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].

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

10 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].

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].

Figure 3. Predicted 16S rRNA Secondary Structure [31].

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].

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.

Figure 4. Flowchart over the culture-independent methods used for bacterial community analysis in this report.

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,

13 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 H2O, High-Fidelity buffer 5X (NEB), universal primers 27f and 1492r, the dNTP mix (NEB), MgCl2 (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 H2O 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 H2O, then 1 μl of a 1:10 dilution (diluted with sterile nuclease free H2O) 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 MgCl2 (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

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 H2O, 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 H2O; 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 H2O; 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).

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 H2O.

After thawing the reagents, 25.3 μl sterile nuclease free H2O, 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.

3.7 RFLP

Restriction enzymes RsaI (Promega) and HaeIII (Takara) (Fig 5) were used for the DNA digestion. 14.5 μl sterile H2O; 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’ Figure 5. Restriction site for restriction enzyme RsaI and HaeIII.

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

17 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.

Figure 6. DNA extraction: T8BAM & T8BAH. Lane M – DNA marker (100 bp exACTGene, Fischer). Lane T8M – T8BAM midgut sample. Lane T8H – T8BAH hindgut sample.

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 7. DNA extraction: T8BAegg. Lane M – DNA marker. Lane T8egg – T8BAegg sample (1 kb Quick- load, NEB).

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 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).

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.

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).

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 10. Clone library for T8BAM: clone 1 – 12.

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 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.

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.

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.

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 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 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.

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).

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

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).

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).

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].

Table 1. Sequenced midgut clones (clone 1 – 30). MIDGUT BLAST RESULT MAX. TAXONOMY RFLP BATCH IN CLONES ID. [%] CLASS: ORDER GROUP NO. 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

Table 2. Sequenced midgut clones (clone 31 – 60). MIDGUT BLAST RESULT MAX. TAXONOMY RFLP BATCH IN CLONES ID. [%] CLASS; ORDER GROUP NO. 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 60 Moss transformation vector pLGZ2 83 N/A A Fourth N/A

Table 3. Sequenced egg clones (clone 1 – 30). EGG BLAST RESULT MAX. TAXONOMY BATCH IN CLONES ID. [%] CLASS; ORDER NO. 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

4.8 Phylogenetic analysis

Figure 17 displays the phylogenetic tree with sequenced midgut and egg clones.

It was not possible to fit all sequenced clones into the phylogenetic tree, since Mega considered some of the clones to be too divergent from the other sequences. Also see table 1 – 3 for information regarding which clones are represented in the phylogenetic tree.

The coverage (Good’s method) for the midgut clones was 80.6% and the coverage for the egg clones was 78.3%. This means that the probability of the next cloned sequence falling in a novel OTU was 19.4% and 21.7% respectively.

Midgut clones: 31 were used for N and 6 were used for n. Some clones were excluded from the calculations. See appendix V for more details.

Midgut clones: 23 were used for N and 5 were used for n. Some clones were excluded from the calculations. See appendix V for more details.

Figure 17. Phylogenetic tree for sequenced midgut and egg clones.

5 Discussion

The microbial community analysis of the pine weevil gut identified Wolbachia spp. as an abundant member of the midgut community. For the egg sample both Wolbachia spp. and Halomonas phoceae were identified as abundant. Wolbachia was the only bacteria found in

29 both the midgut and egg sample. Wolbachia was also found during a previous project done at the institution. The genus Janthinobacterium that was also found during the previous project was not identified here.

• Wolbachia is a diverse bacterial genus capable of infecting an exceptionally broad range of animals (including arthropods) [39]. Studies show that 25 – 70% of all insect species are potential hosts for the bacteria [41]. Wolbachia commonly infects the reproductive tissue of the host and are passed on to new generations through the host’s egg, which would explain Wolbachia was identified both in the midgut and egg sample [41]. Wolbachia are Gram-negative.

• Halomonas spp. are halophiles, commonly found in aquatic surroundings, but have also been reported as found in insect gut [53] [54]. Halomonas species can survive in a very broad range of temperature, pH and osmotic pressure, thus making them useful in industrial applications [55]. Halomonas are Gram-negative.

Other bacteria found in midgut sample community were Acinetobacter sp. (clone 16, chimeric), Ramlibacter sp. (clone 26, chimeric), E. coli (clone 41), Shigella sp. (clone 45), Chryseobacterium sp. (clone 46) and Flavisolibacter ginsengisoli (clone 8).

• Acinetobacter spp. Gram-negative genus found in soil, water and living organisms, including insect gut [49] [56]. The genus has several useful features for biotechnology application i.e. metabolic versatility and robustness [56]. Only one midgut clone found.

• Chryseobacterium spp. Gram-negative bacilli, ubiquitously found in soil and water. Only one midgut clone found [43].

• Ramlibacter spp. are Gram-negative, aerobic and chemoorganotrophic, found as either non-flagellated rods or cysts (a dormant stage that helps the organism to survive in unfavorable environmental conditions) [57]. Only one midgut clone found.

• Flavisolibacter ginsengisoli is a Gram-negative, aerobic, non-motile, chemoheterotrophic, rod-shaped bacterium. Normally found in soil [42]. Only one midgut clone found.

Midgut clone 41 highest BLAST match was for E. coli and midgut clone 45 highest BLAST match was for Shigella sp. Shigella is closely related to E. coli [44]. Some are even claiming that Shigella is actually just an extremely diverse strain of E. coli [45]. The relatedness can be seen by the fact that clone 41 only had a slightly higher BLAST for E. coli than for Shigella, whereas the BLAST results were vice versa for clone 45. The relatedness of species could also been seen by their grouping in the maximum likelihood tree created with Mega.

• Shigella spp. are Gram-negative, rod-shaped, nonmotile, non-spore forming [44].

• Escherichia coli (E. coli) are Gram-negative rods that live in the intestinal tracts of animal, including insect gut [46].

In the egg sample bacterium Shewanella haliotis was also found (clone 13 and 16):

• Shewanella halitos has previously been discovered in the gut microflora of abalones (sea snails from the family Haliotidae). The bacterium is Gram-negative, facultatively anaerob and rod-shaped [58].

The chemically competent cells used for the cloning step were E. coli cells, thus making it difficult to determine if the midgut clones with high BLAST results for E. coli/Shigella are natural members of the midgut microbiota. The 16S rRNA sequences, with BLAST result matching E. coli/Shigella, could have come from the competent cells’ chromosomal DNA instead of the introduced plasmid vectors. To investigate that further it would have been useful to know the 16S rRNA sequence for the competent cells, unfortunately Invitrogen has not sequenced the full genome for the competent cells and did not know the sequence for the 16 rRNA gene. Another option or complement would have been to isolate the plasmid vector from the corresponding clones and investigated if the plasmids had any DNA insert at all and, if so, sequence the insert. This was not done due to time constraints.

All identified species were Gram-negative. Interesting to note is that the pretreatment used for the DNA extraction was for Gram-positive bacteria, showing that the protocol will also work well for Gram-negative bacteria. All identified clones, except clone 8 (Flavisolibacter ginsengisoli) belong to the proteobacteria phylum. Proteobacteria is a phylum with great diversity.

5.1 PCR amplification

The optimal conditions for PCR of total DNA extracted from the pine weevil gut microbiota had already been investigated and a protocol established in an earlier degree project done at the same institution. However, many of the amplification reactions failed during this project. No issues were identified with the PCR machine or with the primers, magnesium, dNTP mix, PCR buffer or polymerase used. The PCR programs and substrate concentrations that were taken from the protocol and that would work in same cases would fail at other times. More amplification attempts failed in the beginning of the project than in the end, which would indicate that at least part of the error was in the manual labor, not the protocol itself.

Also worth to note is that the DNA template concentration is an important factor for the performance of an amplification reaction. Too high concentration of DNA template (here, especially bacterial DNA) may interfere with the amplification reaction. Considerations need to be made for that the concentration of total DNA extracted from pine weevil can vary from sample to sample, therefore several concentrations of template was tried. The concentration of the extracted DNA was not measured, but would have probably been of limited used. The extracted DNA includes the sought-after bacterial DNA, but also other DNA from the pine weevil tissues, thus making difficult to determine the concentration of the bacterial DNA.

There might also have been something from the original sample or introduced during the DNA extraction that inhibited the amplification reaction. For instance, excess salts or ionic detergents. The DNA extracted with the DNeasy Blood & Tissue kit should in theory be pure enough for PCR, but it might have been useful to further purify the extracted DNA before use.

The extract from the hindgut sample contained DNA. This is evident from the clear band on the agarose gel electrophoresis (see figure 6). However it is not known how much, if any, of the DNA that was from the gut microbiota. The enviroment in the midgut and hindgut could be quite different; this has been shown for other insect species [50]. The protocol optimized in

31 the earlier degree project at the institution was for midgut and so might not be optimal for the hindgut. This might also be the reason why none of the PCR runs worked for the hindgut sample. However, a more reasonable explanation would be that the protocol was not properly executed.

5.2 Colony PCR

Colony PCR is a widely used method for fast screening of large numbers of bacterial colonies, for the correct DNA vector constructs or for a gene of interest [35] [36]. The rationale for mainly using colony PCR in this project was that more colonies could hopefully be screened and successfully sequenced with colony PCR than with plasmid extraction and at less cost. An additional PCR step, after the plasmid extraction, might also have been needed before the sequencing; linear DNA are usually better templates than circular DNA and PCR products are less likely to contain DNA polymerase inhibitors than plasmid extracts [59]. The optimal conditions for colony PCR of the pine weevil gut microbiota had also already been investigated in a degree project done at the same institution. An advantage with plasmid extraction would have been the possibility to sequence a larger fragment of the 16S rRNA sequence and more possibilities for the design of the sequence primer(s). Using plasmids would also had made it easier to determine if the BLAST results for E. coli/Shigella came from competent cell chromosomal DNA or from bacteria in the pine weevil gut.

One issue with the used colony PCR protocol and colony PCR protocols in general is the uncertainty of the concentration of DNA template used. The DNA colonies were initially collected with toothpicks, making the number of bacteria used hard to control. Using too many bacteria can inhibit the PCR [38]. A slight improvement made in the followed protocol was to use pipette tips instead, making it bit easier to control how many bacteria that were used. It has been reported that Taq polymerase can be inhibited by cell debris and component of the culture media. However, if this is also the case for the Phusion polymerase, used in the applied colony PCR protocol is not known by the author of this project [37].

5.3 RFLP

The putative RFLP grouping did not correspond completely with the BLAST result of the sequencing. There could be several reasons for this.

Closely related species are not always distinguishable with RFLP, their restriction patterns looking the same or almost the same since they share all or many restriction sites. Wolbachia was mostly assigned to RFLP group A, but RFLP group A was also assigned for some other species that might share the same restriction sites as Wolbachia.

Some of the Wolbachia clones were not assigned to RFLP group A, which the majority of Wolbachia clones belonged to (i.e. clone 10; RFLP group E and clone 21; RFLP group F). This could be because they were not cut in a proper way by the restriction enzymes.

The restriction enzymes might also have cut the sequences in too many short fragments, making the restriction patterns hard to interpret. Another set of the restriction enzymes might have provided better resolution by (1) cutting the sequences in fewer short fragments and (2) cutting different species at different locations in their DNA.

There might also have been some issues with the separation of DNA fragments on the agarose gels. The same double digested sample of clone 8 was loaded onto both agarose gels shown in figure 15 and 16. However, the restriction pattern for clone 8 (Flavisolibacter ginsengisoli) is slightly different for the two agarose gels. Migration of DNA can be retarded and bands distorted when too much buffer covers the gels [61]. This would not explain why heterologous sequences separated on the same gel seemingly have the same restriction pattern, but could explain why the restriction patterns for clone 8 are not identical.

Some of the bands on the agarose gels were quite fuzzy and spread out, while other bands were too faint. Loading too much DNA on a gel can result fuzzy bands, while loading too little can result in faint bands. A dilemma with RFLP is that the optimal DNA loading volume is higher for small sequence fragments than for larger fragments.

5.4 Phylogenetic analysis

It is important to note when doing phylogenetic analyses that the presumed grouping and relationship between different species is dependent on several factors, including reference database and PCR primers use and targeted 16S rRNA region.

There are many reference databases in use, but the coverage for the species and how well the databases are maintained or curated may vary. Therefore it might have been beneficial to also match the sequenced clones to another reference (i.e. the database kept by the Ribosomal Database Project) besides those used by the BLAST engine.

The choice of region sequenced and design of primer is vital [33]. No primer is truly universal; the sequences amplified from an environmental sample will depend on the primers used [32]. Commonly utilized primers 27f and 1492r were used for this project. 27f and 1492r have been known to amplify both prokaryotic and eukaryotic rRNA genes [51]. To be certain to find all the microbiota in the pine weevil gut and egg sample it might have been good to use another primer pair in addition to 27f and 1492r.

An advantage with the 16S rRNA gene is that it contains both fast and slow evolving regions, allowing a high resolution of phylogenetic relationship. Targeting only shorter regions of the gene can lead to lower resolution of phylogenetic relationship. When using primer pair 27f and 1492r almost the full gene is sequenced, both the fast and slow evolving regions. On the other hand, smaller sequences have the advantages that they are less subject to heterogeneity biases and chimera formation during the amplification step [32]. Some studies have also shown that sequencing and using only small fragment of the 16S rRNA gene can be sufficient for community analysis, including phylogenetic studies [32] [33].

6 Conclusions

• Wolbachia is most likely a dominant member of the pine weevil midgut. Halomonas might not be part of the midgut bacterial community, but is probably a dominant bacterial community member of the pine weevil egg cells. • Further investigations are required to be certain which bacterial species are commonly part of the midgut microbiota. • Since the number of clones screened is rather limited, plasmid extraction might be a better approach than colony PCR.

• RFLP might be of limited use as a screening tool, before properly validated for more pine weevil gut samples.

7 Further Studies

• Determine if E. coli or Shigella sp. or both is part of the midgut microbiota.

• Determine if any of the found gut bacteria can (1) metabolize lignin (2) produce the organic compounds shown to have antifeedants activity. • Determining if Halomonas is also part of the midgut microbiota is of extra interest, because of its promising usefulness in biotechnical applications. Acinetobacter and E. coli are also of extra interest because of that reason.

• Further investigation needed regarding the optimal PCR condition for amplification of hindgut bacterial DNA.

• Originally planned as secondary method to characterize the gut microbiota: separate amplified extracted gut DNA with D-HPLC (denaturing high-performance liquid chromatography). This to determine if D-HPLC (1) could be used as a profiling tool, (2) to separate amplicons sufficiently for direct sequencing.

• Select a PCR primer pair for capture of fungus. Fungal symbionts in the gut may also be the origin of antifeedants [10].

• It might be worth to investigate if another primer pair than 27f and 1492r would make the PCR amplification more stable. Another primer pair might also capture bacterial species not found with 27f and 1492r. One or more sets of primer pairs could be used in addition to 27f and 1492r.

8 Acknowledgments

I wish to thank my supervisor, associate professor prof. Gunaratna K. Rajarao for all of her help and patience during this project. I would also like to thank associate professor Olle Terenius (SLU) for long hours, explaining how to use various sequence alignment and phylogenetics tools and for providing valuable data for the project.

9 References

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10 Appendices

I. PCR Amplification – midgut, hindgut & egg sample

Table 1A. T8BAM (midgut) sample. PCR on 22-nov-2011: samples.

PCR tube Mastermix volume H2O volume Sample volume PCR Product No.

2 22.4 μl 24.6 μl 3 μl No 3 22.4 μl 26.6 μl 1 μl No 4 22.4 μl 22.6 μl 5 μl of 1:10 dilution (0.5 μl) No 5 22.4 μl 26.6 μl 1 μl of 1:10 dilution (0.1 μl) Yes

6 22.4 μl 24.6 μl 3 μl H2O – negative control No

Table 1B. T8BAM (midgut) sample. PCR on 22-nov-2011: mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

HF Buffer 5X 10 μl 6 60 μl Primer 27f 2 μM 5 μl 6 30 μl Primer 1492r 2 μM 5 μl 6 30 μl dNTP mix 10 mM 1.2 μl 6 7.2 μl

MgCl2 0.7 mM 0.7 μl 6 4.2 μl Phusion enzyme 0.02 U/μl 0.5 μl 6 3 μl

Total volume 22.4 μl 134.4 μl

Table 1C. T8BAM (midgut) sample. PCR on 22-nov-2011: PCR program. Step Temperature Time Comment

Initial denaturation 98°C 2 min Denaturation 98°C 10 sec Touchdown annealing 60°C - 51°C 30 sec Annealing 50°C 30 sec 20 cycles Extension 72°C 30 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 1. 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).

Table 2A. T8BH (hindgut) sample. PCR on 25-nov-2012: samples.

PCR tube Mastermix volume H2O volume Sample volume PCR product No.

2 22.4 μl 24.6 μl 3 μl No 3 22.4 μl 26.6 μl 1 μl No 4 22.4 μl 26.6 μl 1 μl of 1:10 dilution (0.1 μl) No

5 22.4 μl 24.6 μl 3 μl H2O – negative control No

Table 2B. T8BAH (hindgut) sample. PCR on 25-nov-2012: mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

HF Buffer 5X 10 μl 5 60 μl Primer 27f 2 μM 5 μl 5 30 μl Primer 1492r 2 μM 5 μl 5 30 μl dNTP mix 10 mM 1.2 μl 5 7.2 μl 0.7 mM 0.7 μl 5 3.5 μl Phusion enzyme 0.02 U/μl 0.5 μl 5 2.5 μl

Total volume 22.4 μl 112 μl

Table 2C. T8BAH (hindgut) sample. PCR on 25-nov-2012: PCR program. Step Temperature Time Comment

Initial denaturation 98°C 2 min

Denaturation 98°C 10 sec Touchdown annealing 60°C - 51°C 30 sec Annealing 50°C 30 sec 20 cycles Extension 72°C 30 sec Final extension 72°C 10 min Hold 4°C ∞

Table 3A. T8BAegg sample. PCR on 25-jan-2012: samples.

PCR tube Mastermix volume H2O volume Sample volume PCR product No.

2 21.7 μl 23.3 μl 5 μl No 3 21.7 μl 25.3 μl 3 μl Yes 4 21.7 μl 27.3 μl 1 μl No 5 21.7 μl 23.3 μl 5 μl of 1:10 dilution (0.5 μl) Yes 6 21.7 μl 27.3 μl 1 μl of 1:10 dilution (0.1 μl) Yes

Table 3B. T8BAegg sample. PCR on 25-jan-2012: mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

HF Buffer 5X 10 μl 6 60 μl Primer 27f 2 μM 5 μl 6 30 μl Primer 1492r 2 μM 5 μl 6 30 μl dNTP mix 10 mM 1.2 μl 6 7.2 μl Phusion enzyme 0.02 U/μl 0.5 μl 6 3 μl

Total volume 21.7 μl 130.2 μl

Table 3C. T8BAegg sample. PCR on 25-jan-2012: PCR program (30 cycles). Step Temperature Time

Initial denaturation 98°C 2 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 3. 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).

II. PCR (plasmid template) – midgut sample

Table 4A. T8BAM (midgut) sample. Plasmid template. PCR on 17-jan-2012: clone 1 - 4.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

1 15.7 μl 33.8 μl 0.5 μl Toothpick Yes 2 15.7 μl 33.8 μl 0.5 μl Toothpick Yes 3 15.7 μl 33.8 μl 0.5 μl Toothpick Yes 4 15.7 μl 33.8 μl 0.5 μl Toothpick Yes

Table 4B. T8BAM (midgut) sample. PCR on 17-jan-2012 (clone 1 - 4): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

HF Buffer 5X 10 μl 5 50 μl Primer 27f 5 μM 2 μl 5 10 μl Primer 1492r 5 μM 2 μl 5 10 μl dNTP mix 10 mM 1.2 μl 5 6 μl Phusion enzyme 0.02 U/μl 0.5 μl 5 2.5 μl

Total volume 15.7 μl 75.8 μl

Table 4C. T8BAM (midgut) sample. PCR on 17-jan-2012 (clone 1 - 4): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 4. 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.

III. Colony PCR – midgut sample

Table 5A. T8BAM (midgut) sample. Colony PCR on 18-jan-2012: clone 5 - 8.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

5 15.7 μl 31.3 μl 3 μl Toothpick Yes 6 15.7 μl 31.3 μl 3 μl Toothpick Yes 7 15.7 μl 31.3 μl 3 μl Toothpick Yes 8 15.7 μl 31.3 μl 3 μl Toothpick Yes

Table 5B. T8BAM (midgut) sample. Colony PCR on 18-jan-2012 (clone 5 - 8): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

HF Buffer 5X 10 μl 5 50 μl Primer 27f 5 μM 2 μl 5 10 μl Primer 1492r 5 μM 2 μl 5 10 μl dNTP mix 10 mM 1.2 μl 5 6 μl Phusion enzyme 0.02 U/μl 0.5 μl 5 2.5 μl

Total volume 15.7 μl 78.5 μl

Table 5C. T8BAM (midgut) sample. Colony PCR on 18-jan-2012 (clone 5 - 8): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 5. Colony PCR. T8BAM (midgut). Clone 5 – 8: Lane 1 – clone 8. Lane 2 – clone 7. Lane 3 – clone 6. Lane 4 – clone 5. Lane 5 – ladder (1kb Quick-load, NEB).

Table 6A. T8BAM (midgut) sample. Colony PCR on 24-jan-2012: clone 9 – 20.

Clone Mastermix H2O volume Sample volume Toothpick / Pipette PCR volume tip Product 9 21.7 μl 25.3 μl 3 μl Toothpick Yes 10 21.7 μl 25.3 μl 3 μl Toothpick Yes

11 21.7 μl 25.3 μl 3 μl Toothpick No 12 21.7 μl 25.3 μl 3 μl Toothpick Too weak 13 21.7 μl 25.3 μl 3 μl Toothpick No 14 21.7 μl 25.3 μl 3 μl Toothpick Yes 15 21.7 μl 25.3 μl 3 μl Toothpick No 16 21.7 μl 25.3 μl 3 μl Toothpick Yes 17 21.7 μl 25.3 μl 3 μl Toothpick Too weak 18 21.7 μl 25.3 μl 3 μl Toothpick Too weak 19 21.7 μl 25.3 μl 3 μl Toothpick Too weak 20 21.7 μl 25.3 μl 3 μl Toothpick Too weak

Table 6B. T8BAM (midgut) sample. Colony PCR on 24-jan-2012 (clone 9 – 20): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

HF Buffer 5X 10 μl 14 140 μl Primer 27f 2 μM 5 μl 14 70 μl Primer 1492r 2 μM 5 μl 14 70 μl dNTP mix 10 mM 1.2 μl 14 16.8 μl Phusion enzyme 0.02 U/μl 0.5 μl 14 7 μl

Total volume 21.7 μl 303.8 μl

Table 6C. T8BAM (midgut) sample. Colony PCR on 24-jan-2012 (clone 9 - 20): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 6.1. Colony PCR. T8BAM (midgut). Clone 9 – 14: 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 6.2. Colony PCR. T8BAM (midgut). Clone 15 – 20: Lane 1 – clone 20. Lane 2 – clone 19. Lane 3 – clone 18. Lane 4 – clone 17. Lane 5 – clone 16. Lane 6 – clone 15. Lane 7 – DNA ladder (1kb Quick-load, NEB).

Table 7A. T8BAM (midgut) sample. Colony PCR on 26-jan-2012: clone 21 - 26.

Clone Mastermix H2O volume Sample volume Toothpick / Pipette PCR volume tip Product

21 21.7 μl 25.3 μl 3 μl Toothpick Yes 22 21.7 μl 25.3 μl 3 μl Toothpick Yes 23 21.7 μl 25.3 μl 3 μl Toothpick No

24 21.7 μl 25.3 μl 3 μl Toothpick Yes 25 21.7 μl 25.3 μl 3 μl Toothpick No 26 21.7 μl 25.3 μl 3 μl Toothpick No

Table 7B. T8BAM (midgut) sample. Colony PCR on 26-jan-2012 (clone 21 – 26): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

HF Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 7C. T8BAM (midgut) sample. Colony PCR on 26-jan-2012 (clone 21 - 26): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 7. Colony PCR. T8BAM (midgut). Clone 21 – 26: Lane 1 – clone 26. Lane 2 – clone 25. Lane 3 – clone 24. Lane 4 – clone 23. Lane 5 – clone 22. Lane 6 – clone 21. Lane 7 – DNA ladder (1kb Quick-load, NEB).

Table 8A. T8BAM (midgut) sample. Colony PCR on 2-mar-2012: clone 27 - 38.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

27 21.7 μl 25.3 μl 3 μl Tip Yes 28 21.7 μl 25.3 μl 3 μl Tip Yes 29 21.7 μl 25.3 μl 3 μl Tip Yes 30 21.7 μl 25.3 μl 3 μl Tip No 31 21.7 μl 25.3 μl 3 μl Tip No 32 21.7 μl 25.3 μl 3 μl Tip Yes 33 21.7 μl 25.3 μl 3 μl Tip Too weak 34 21.7 μl 25.3 μl 3 μl Tip No 35 21.7 μl 25.3 μl 3 μl Tip No 36 21.7 μl 25.3 μl 3 μl Tip No 37 21.7 μl 25.3 μl 3 μl Tip Yes 38 21.7 μl 25.3 μl 3 μl Tip No

Table 8B. T8BAM (midgut) sample. Colony PCR on 2-mar-2012 (clone 27 – 38): mastermix Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 14 140 μl Primer 27f 2 μM 5 μl 14 70 μl Primer 1492r 2 μM 5 μl 14 70 μl dNTP mix 10 mM 1.2 μl 14 16.8 μl Phusion enzyme 0.02 U/μl 0.5 μl 14 7 μl

Total volume 21.7 μl 303.8 μl

Table 8C. T8BAM (midgut) sample. Colony PCR on 2-mar-2012 (clone 27 - 38): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 8.1. Colony PCR. T8BAM (midgut). Clone 27 – 32: Lane 1 – clone 32. Lane 2 – clone 31. Lane 3 – clone 30. Lane 4 – clone 29. Lane 5 – clone 28. Lane 6 – clone 27. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Figure 8.2. Colony PCR. T8BAM (midgut). Clone 33 – 38: Lane 1 – clone 38. Lane 2 – clone 37. Lane 3 – clone 36. Lane 4 – clone 35. Lane 5 – clone 34. Lane 6 – clone 33. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 9A. T8BAM (midgut) sample. Colony PCR on 3-mar-2012: clone 39 - 44.

Clone Mastermix H2O volume Sample volume Toothpick / PCR Product volume Pipette tip

39 21.7 μl 25.3 μl 3 μl Tip Yes 40 21.7 μl 25.3 μl 3 μl Tip Yes 41 21.7 μl 25.3 μl 3 μl Tip Yes

42 21.7 μl 25.3 μl 3 μl Tip Too weak 43 21.7 μl 25.3 μl 3 μl Tip Yes 44 21.7 μl 25.3 μl 3 μl Tip Too weak

Table 9B. T8BAM (midgut) sample. Colony PCR on 3-mar-2012 (clone 39 - 44): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 9C. T8BAM (midgut) sample. Colony PCR on 3-mar-2012 (clone 39 - 44): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 9. Colony PCR. T8BAM (midgut). Clone 39 – 44: Lane 1 – clone 44. Lane 2 – clone 43. Lane 3 – clone 42. Lane 4 – clone 41. Lane 5 – clone 40. Lane 6 – clone 39. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 10A. T8BAM (midgut) sample. Colony PCR on 4-mar-2012: clone 45 - 50.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

45 21.7 μl 25.3 μl 3 μl Tip No 46 21.7 μl 25.3 μl 3 μl Tip No 47 21.7 μl 25.3 μl 3 μl Tip No 48 21.7 μl 25.3 μl 3 μl Tip No 49 21.7 μl 25.3 μl 3 μl Tip Weak 50 21.7 μl 25.3 μl 3 μl Tip Weak

Table 10B. T8BAM (midgut) sample. Colony PCR on 4-mar-2012 (clone 45 – 50): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 10C. T8BAM (midgut) sample. Colony PCR on 4-mar-2012 (clone 45 - 50): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 10. Colony PCR. T8BAM (midgut). Clone 45 – 50: Lane 1 – clone 50. Lane 2 – clone 49. Lane 3 – clone 48. Lane 4 – clone 47. Lane 5 – clone 46. Lane 6 – clone 45. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 11A. T8BAM (midgut) sample. Colony PCR on 5-mar-2012: clone 51 – 52, 54 – 55, 59 - 60.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

51 21.7 μl 25.3 μl 3 μl Tip Yes 52 21.7 μl 25.3 μl 3 μl Tip Yes 54 21.7 μl 25.3 μl 3 μl Tip Yes 55 21.7 μl 25.3 μl 3 μl Tip Yes 59 21.7 μl 25.3 μl 3 μl Tip Yes 60 21.7 μl 25.3 μl 3 μl Tip Yes

Table 11B. T8BAM (midgut) sample. Colony PCR on 5-mar-2012 (clone 51 – 52, 54 – 55, 59 – 60): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 8 80 μl Primer 27f 2 μM 5 μl 8 40 μl Primer 1492r 2 μM 5 μl 8 40 μl dNTP mix 10 mM 1.2 μl 8 9.6 μl Phusion enzyme 0.02 U/μl 0.5 μl 8 4 μl

Total volume 21.7 μl 173.6 μl

Table 11C. T8BAM (midgut) sample. Colony PCR on 5-mar-2012 (clone 51 – 52, 54 – 55, 59 – 60): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 11. Colony PCR. T8BAM (midgut). Clone 51 – 52, 54 – 55, 59 - 60: Lane 1 – clone 60. Lane 2 – clone 59. Lane 3 – clone 55. Lane 4 – clone 54. Lane 5 – clone 52. Lane 6 – clone 51. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 12A. T8BAM (midgut) sample. Colony PCR on 10-mar-2012: clone 14 - 18.

Clone Mastermix H2O volume Sample volume Toothpick / Pipette PCR Product volume tip

14 21.7 μl 26.3 μl 2 μl Tip Yes 14 21.7 μl 23.3 μl 5 μl Tip No 16 21.7 μl 26.3 μl 2 μl Tip Yes 16 21.7 μl 23.3 μl 5 μl Tip No 17 21.7 μl 24.3 μl 3 μl Tip No 18 21.7 μl 24.3 μl 3 μl Tip No

Table 12B. T8BAM (midgut) sample. Colony PCR on 10-mar-2012 (clone 14 - 18): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 8 80 μl Primer 27f 2 μM 5 μl 8 40 μl Primer 1492r 2 μM 5 μl 8 40 μl dNTP mix 10 mM 1.2 μl 8 9.6 μl Phusion enzyme 0.02 U/μl 0.5 μl 8 4 μl

Total volume 21.7 μl 173.6 μl

Table 12C. T8BAM (midgut) sample. Colony PCR on 10-mar-2012 (clone 14 - 18): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 12. Colony PCR. T8BAM (midgut). Clone 14 – 18: Lane 1 – clone 18. Lane 2 – clone 17. Lane 3 – clone 16. Lane 4 – clone 16. Lane 5 – clone 14. Lane 6 – clone 14. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 13A. T8BAM (midgut) sample. Colony PCR on 11-mar-2012: clone 53, 56 - 58.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

53 21.7 μl 25.3 μl 1 μl Tip Yes 53 21.7 μl 25.3 μl 3 μl Tip Yes 53 21.7 μl 25.3 μl 5 μl Tip Yes 56 21.7 μl 25.3 μl 3 μl Tip Yes 57 21.7 μl 25.3 μl 3 μl Tip Yes 58 21.7 μl 25.3 μl 3 μl Tip No

Table 13B. T8BAM (midgut) sample. Colony PCR on 11-mar-2012 (clone 53, 56 – 58): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 13C. T8BAM (midgut) sample. Colony PCR on 11-mar-2012 (clone 53, 56 - 58): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 13. Colony PCR. T8BAM (midgut). Clone 53, 56 – 58: Lane 1 – clone 53. Lane 2 – clone 53. Lane 3 – clone 58. Lane 4 – clone 57. Lane 5 – clone 56. Lane 6 – clone 53. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 14A. T8BAM (midgut) sample. Colony PCR on 12-mar-2012: clone 8.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

8 21.7 μl 26.3 μl 3 μl Tip No 8 21.7 μl 26.3 μl 2 μl Tip Yes

Table 14B. T8BAM (midgut) sample. Colony PCR on 12-mar-2012 (clone 8): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 14C. T8BAM (midgut) sample. Colony PCR on 12-mar-2012 (clone 8): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec

Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 14. Colony PCR. T8BAM (midgut). Clone 8: Lane 1 – clone 8. Lane 2 – clone 8. Lane 3 – DNA ladder (1kb Generuler, Fermentas).

Table 15A. T8BAM (midgut) sample. Colony PCR on 12-mar-2012: clone 45 - 48.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

45 21.7 μl 26.3 μl 2 μl Tip Yes 46 21.7 μl 26.3 μl 2 μl Tip Yes 47 21.7 μl 26.3 μl 2 μl Tip Yes 48 21.7 μl 26.3 μl 2 μl Tip Yes

Table 15B. T8BAM (midgut) sample. Colony PCR on 12-mar-2012 (clone 45 – 48): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl

Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 15C. T8BAM (midgut) sample. Colony PCR on 12-mar-2012 (clone 45 - 48): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 15. Colony PCR. T8BAM (midgut). Clone 45 – 48: Lane 1 – clone 48. Lane 2 – clone 47. Lane 3 – clone 46. Lane 4 – clone 45. Lane 5 – T8BAegg clone 12. Lane 6 – T8BAegg clone 11. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 16A. T8BAM (midgut) sample. Colony PCR on 14-mar-2012: clone 25 – 26, 30 – 31 & 33 - 34.

Clone Mastermix H2O volume Sample volume Toothpick / PCR volume Pipette tip Product

25 21.7 μl 26.3 μl 2 μl Tip Yes 26 21.7 μl 26.3 μl 2 μl Tip Yes 30 21.7 μl 26.3 μl 2 μl Tip No 31 21.7 μl 26.3 μl 2 μl Tip No

33 21.7 μl 26.3 μl 2 μl Tip No 34 21.7 μl 26.3 μl 2 μl Tip Yes

Table 16B. T8BAM (midgut) sample. Colony PCR on 14-mar-2012 (clone 25 – 26, 30 – 31 & 33 – 34): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 8 80 μl Primer 27f 2 μM 5 μl 8 40 μl Primer 1492r 2 μM 5 μl 8 40 μl dNTP mix 10 mM 1.2 μl 8 9.6 μl Phusion enzyme 0.02 U/μl 0.5 μl 8 4 μl

Total volume 21.7 μl 173.6 μl

Table 16C. T8BAM (midgut) sample. Colony PCR on 14-mar-2012 (clone 25 – 26, 30 – 31 & 33 - 34): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 16. Colony PCR. T8BAM (midgut). Clone 25 – 26, 30 – 31 & 33 - 34: Lane 1 – clone 34. Lane 2 – clone 33. Lane 3 – clone 31. Lane 4 – clone 30. Lane 5 – clone 26. Lane 6 – clone 25. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

IV. Colony PCR – egg sample

Table 17A. T8BAegg sample. Colony PCR on 12-mar-2012: clone 1 – 4.

Clone Mastermix H2O volume Sample volume Toothpick / PCR Product volume Pipette tip

1 21.7 μl 26.3 μl 2 μl Tip Yes 2 21.7 μl 26.3 μl 2 μl Tip Yes 3 21.7 μl 26.3 μl 2 μl Tip Wrong size 4 21.7 μl 26.3 μl 2 μl Tip Yes

Table 17B. T8BAegg sample. Colony PCR on 12-mar-2012 (clone 1 – 4): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 17C. T8BAegg sample. Colony PCR on 12-mar-2012 (clone 1 – 4): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 17. Colony PCR. T8BAegg. Clone 1 – 4: Lane 1 – clone 4. Lane 2 – clone 3. Lane 3 – clone 2. Lane 4 – clone 1. Lane 5 – T8BAM (midgut) clone 8. Lane 6 – T8BAM (midgut) clone 8. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 18A. T8BAegg sample. Colony PCR on 12-mar-2012: clone 5 – 10.

Clone Mastermix H2O volume Sample volume Toothpick / PCR Product volume Pipette tip

5 21.7 μl 26.3 μl 2 μl Tip Wrong size 6 21.7 μl 26.3 μl 2 μl Tip Yes 7 21.7 μl 26.3 μl 2 μl Tip Yes 8 21.7 μl 26.3 μl 2 μl Tip Yes 9 21.7 μl 26.3 μl 2 μl Tip Yes 10 21.7 μl 26.3 μl 2 μl Tip Yes

Table 18B. T8BAegg sample. Colony PCR on 12-mar-2012 (clone 5 – 10): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 18C. T8BAegg sample. Colony PCR on 12-mar-2012 (clone 5 – 10): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 18. Colony PCR. T8BAegg. Clone 5 – 10: 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).

Table 19A. T8BAegg sample. Colony PCR on 12-mar-2012: clone 11 – 12.

Clone Mastermix H2O volume Sample volume Toothpick / PCR Product volume Pipette tip

11 21.7 μl 26.3 μl 2 μl Tip Yes 12 21.7 μl 26.3 μl 2 μl Tip Yes

Table 19B. T8BAegg sample. Colony PCR on 12-mar-2012 (clone 11 – 12): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 7 70 μl Primer 27f 2 μM 5 μl 7 35 μl Primer 1492r 2 μM 5 μl 7 35 μl dNTP mix 10 mM 1.2 μl 7 8.4 μl Phusion enzyme 0.02 U/μl 0.5 μl 7 3.5 μl

Total volume 21.7 μl 151.9 μl

Table 19C. T8BAegg sample. Colony PCR on 12-mar-2012 (clone 11 – 12): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 19. Colony PCR. T8BAegg. Clone 11 – 12: Lane 1 – T8BAM (midgut) clone 48. Lane 2 – T8BAM (midgut) clone 47. Lane 3 – T8BAM (midgut) clone 46. Lane 4 – T8BAM midgut) clone 45. Lane 5 – clone 12. Lane 6 – clone 11. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 20A. T8BAegg sample. Colony PCR on 13-mar-2012: clone 13 – 18.

Clone Mastermix H2O volume Sample volume Toothpick / PCR Product volume Pipette tip 13 21.7 μl 26.3 μl 2 μl Tip Yes 14 21.7 μl 26.3 μl 2 μl Tip Wrong size 15 21.7 μl 26.3 μl 2 μl Tip Yes 16 21.7 μl 26.3 μl 2 μl Tip Yes 17 21.7 μl 26.3 μl 2 μl Tip Weak

18 21.7 μl 26.3 μl 2 μl Tip Yes

Table 20B. T8BAegg sample. Colony PCR on 13-mar-2012 (clone 13 – 18): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 8 80 μl Primer 27f 2 μM 5 μl 8 40 μl Primer 1492r 2 μM 5 μl 8 40 μl dNTP mix 10 mM 1.2 μl 8 9.6 μl Phusion enzyme 0.02 U/μl 0.5 μl 8 4.0 μl

Total volume 21.7 μl 173.6 μl

Table 20C. T8BAegg sample. Colony PCR on 13-mar-2012 (clone 13 – 18): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 20. Colony PCR. T8BAegg. Clone 13 – 18: Lane 1 – Negative control (H2O): Lane 2 – clone 18. Lane 3 – clone 17. Lane 4 – clone 16. Lane 5 – clone 15. Lane 6 – clone 14. Lane 7 – clone 13. Lane 8 – DNA ladder (1kb Generuler, Fermentas).

Table 21A. T8BAegg sample. Colony PCR on 13-mar-2012: clone 19 – 24.

Clone Mastermix H2O volume Sample volume Toothpick / PCR Product volume Pipette tip

19 21.7 μl 26.3 μl 2 μl Tip Yes 20 21.7 μl 26.3 μl 2 μl Tip Yes 21 21.7 μl 26.3 μl 2 μl Tip Yes 22 21.7 μl 26.3 μl 2 μl Tip Yes 23 21.7 μl 26.3 μl 2 μl Tip Yes 24 21.7 μl 26.3 μl 2 μl Tip Wrong size

Table 21B. T8BAegg sample. Colony PCR on 13-mar-2012 (clone 19 – 24): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 8 80 μl Primer 27f 2 μM 5 μl 8 40 μl Primer 1492r 2 μM 5 μl 8 40 μl dNTP mix 10 mM 1.2 μl 8 9.6 μl Phusion enzyme 0.02 U/μl 0.5 μl 8 4.0 μl

Total volume 21.7 μl 173.6 μl

Table 21C. T8BAegg sample. Colony PCR on 13-mar-2012 (clone 19 – 24): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 21. Colony PCR. T8BAegg. Clone 19 – 24: Lane 1 – clone 24. Lane 2 – clone 23. Lane 3 – clone 22. Lane 4 – clone 21. Lane 5 – clone 20. Lane 6 – clone 19. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

Table 22A. T8BAegg sample. Colony PCR on 14-mar-2012: clone 25 – 30.

Clone Mastermix H2O volume Sample volume Toothpick / PCR Product volume Pipette tip

25 21.7 μl 26.3 μl 2 μl Tip Wrong size 26 21.7 μl 26.3 μl 2 μl Tip Wrong size 27 21.7 μl 26.3 μl 2 μl Tip Yes 28 21.7 μl 26.3 μl 2 μl Tip Yes 29 21.7 μl 26.3 μl 2 μl Tip Wrong size 30 21.7 μl 26.3 μl 2 μl Tip Wrong size

Table 22B. T8BAegg sample. Colony PCR on 14-mar-2012 (clone 25 – 30): mastermix. Reagents Concentration Volume/tube Tubes+overfill Total volume

GC Buffer 5X 10 μl 8 80 μl Primer 27f 2 μM 5 μl 8 40 μl Primer 1492r 2 μM 5 μl 8 40 μl dNTP mix 10 mM 1.2 μl 8 9.6 μl Phusion enzyme 0.02 U/μl 0.5 μl 8 4.0 μl

Total volume 21.7 μl 173.6 μl

Table 22C. T8BAegg sample. Colony PCR on 14-mar-2012 (clone 25 – 30): PCR program (30 cycles). Step Temperature Time

Initial denaturation 95°C 10 min

Denaturation 98°C 10 sec Annealing 55°C 30 sec Extension 72°C 25 sec Final extension 72°C 10 min Hold 4°C ∞

Figure 22. Colony PCR. T8BAegg. Clone 25 – 30: Lane 1 – clone 30. Lane 2 – clone 29. Lane 3 – clone 28. Lane 4 – clone 27. Lane 5 – clone 26. Lane 6 – clone 25. Lane 7 – DNA ladder (1kb Generuler, Fermentas).

V. Good’s Method

Table V.1. OTU - Midgut clones MIDGUT BLAST RESULT OTU MIDGUT BLAST RESULT OTU CLONES GROUP CLONES GROUP 1 PCR product not submitted for sequencing N/A 31 No PCR product, not sequenced N/A 2 Wolbachia endosymbiont of D. pinicola 1 32 (1492r) Wolbachia sp. 8 3 Wolbachia endosymbiont of D. pinicola 1 33 Too low DNA concentration for sequencing N/A 4 PCR product not submitted for sequencing N/A 34 Wolbachia sec. endosymbiont of C. hilgendorfi 1 5 PCR product not submitted for sequencing N/A 35 No PCR product, not sequenced N/A 6a Wolbachia secondary endosymbiont of C. okumai 1 36 No PCR product, not sequenced N/A

6b Conserved? Agrobacterium/Rhizobium/Shinella N/A 37 Wolbachia secondary endosymbiont of Curculio sp. 1

7 Wolbachia secondary endosymbiont of C. okumai 1 38 No PCR product, not sequenced N/A

8 Uncultured bacterium/Flavisolibacter ginsengisoli 2 39 Wolbachia secondary endosymbiont 1

9 Wolbachia secondary endosymbiont of C. okumai 1 40 Wolbachia secondary endosymbiont 1

10 (27f) Wolbachia endosymbiont of D. pinicola 1 41 Escherichia coli 6 11 No PCR product, not sequenced N/A 42 Too low DNA concentration for sequencing N/A 12 Too low DNA concentration for sequencing N/A 43 Uncultured bacterium clone/Wolbachia sp. 1 13 No PCR product, not sequenced N/A 44 Too low DNA concentration for sequencing N/A 14 (1492r) Wolbachia endosymbiont of Glossina austeni 1 45 Shigella sp. 6 15 No PCR product, not sequenced N/A 46 Unidentified bacterium/Chryseobacterium sp. 3 16a Acinetobacter sp. 9 47 Wolbachia pipientis 1 16b Uncultured bacterium/Wolbachia pipientis 1 48 (1492r) Enterobacter sp. 4 17 Too low DNA concentration for sequencing N/A 49 Wolbachia pipientis 1 18 Too low DNA concentration for sequencing N/A 50 Uncultured bacterium clone/Wolbachia sp. 1 19 Too low DNA concentration for sequencing N/A 51 Wolbachia sp. 1 20 No PCR product, not sequenced N/A 52 Wolbachia secondary endosymbiont of C. okumai 1

21 Wolbachia endosymbiont of P. longiceps 8 53 (1492r) Wolbachia secondary endosymbiont of C. okumai 1

22 (1492r) Uncultured alphaproteobacterium/Wolbachia sp. 8 54 (1492r) Wolbachia sec. endosymbiont of D. nikananu 1 22 (27f) Wolbachia endosymbiont of P. longiceps 8 55 Wolbachia secondary endosymbiont of C. okumai 1

23 No PCR product, not sequenced N/A 56 Wolbachia sp. 1 24 (1492r) Wolbachia secondary endosymbiont of C. okumai 8 57 Uncultured bacterium clone/Wolbachia sp. 1

25 Wolbachia secondary endosymbiont of C. okumai 1 58 No PCR product, not sequenced N/A

26a Uncultured soil bacterium/Ramlibacter sp. 10 59 (1492r) Wolbachia sec. endosymbiont of C. hilgendorfi 1 26b Wolbachia pipientis 1 60 Moss transformation vector pLGZ2 5 27 Uncultured bacterium /E. coli/Shigella sp. 7 28 (27f) Wolbachia endosymbiont of P. longiceps 8 29 (1492r) Wolbachia sp. 8 30 No PCR product, not sequenced N/A

Table V.2. OTU - Midgut clones OTU DESCRIPTION AMOUNT INCLUDED/EXCLUDED GROUP IN CALCULATION 1 Wolbachia spp. 26 INCLUDED 2 Flavisolibacter ginsengisoli 1 INCLUDED 3 Chryseobacterium sp. 1 INCLUDED 4 “Enterobacter sp.” (≤96 with Blast) 1 INCLUDED 5 Transformation vector (no clone) 1 EXCLUDED

6 E. coli & Shigella sp. (possible contamination) 2 EXCLUDED 7 “E. coli & Shigella sp.” (≤96 with Blast & possible contamination) 1 EXCLUDED 8 “Wolbachia spp.” (≤96 with Blast) 7 EXCLUDED 9 Acinetobacter sp. 1 INCLUDED 10 Ramlibacter sp. 1 INCLUDED N/A Not sequenced 22 EXCLUDED TOTAL 64

N is 31 n is 6

Table V.3. OTU - Egg clones EGG BLAST RESULT OTU MIDGUT BLAST RESULT OTU CLONES GROUP CLONES GROUP 1 Halomonas phoceae 1 16 (27f) Shewanella haliotis 4 2 Halomonas phoceae 1 17 PCR product not submitted for sequencing N/A 3 Uncultured bacterium clone/Wolbachia pipientis 2 18 Sequencing data of insufficient quality N/A 4 Halomonas phoceae 1 19 Uncultured bacterium clone/Wolbachia pipientis 8 5 Wolbachia sp. 2 20 Wolbachia sec. endosymbiont of C. okumai 2

6 (1492r) Halomonas phoceae 1 21 Halomonas phoceae 1 7 Wolbachia sec. endosymbiont of C. okumai 2 22 Halomonas phoceae 1

8 Halomonas phoceae 1 23 (1492r) Streptococcus mitis 5 9 Halomonas phoceae 1 24 Wolbachia pipientis 2 10 Halomonas phoceae 1 25 (1492r) Persephonella sp. 6 11 Halomonas phoceae 1 26 Wolbachia endosymbiont of Sogatella furcifera 7 12 Halomonas phoceae 1 27 Uncultured organism clone/ Escherichia coli 7 13 Shewanella sp. 4 28 Halomonas phoceae 1 14 Sequencing data of insufficient quality N/A 29 (1492r) Expression vector pOT-RA 3 15 (1492r) Halomonas phoceae 1 30 (1492r) Expression vector pOT-RA 3

Table V.4. OTU - Egg clones OTU DESCRIPTION AMOUNT INCLUDED/EXCLUDED GROUP IN CALCULATION 1 Halomonas phoceae 13 INCLUDED 2 Wolbachia spp. 6 INCLUDED 3 Transformation vector (no clone) 2 EXCLUDED

4 Shewanella haliotis 2 INCLUDED 5 “Streptococcus mitis” (≤96 with Blast) 1 INCLUDED 6 “Persephonella sp” (≤96 with Blast) 1 INCLUDED 7 “E. coli.” (≤96 with Blast & possible contamination) 1 EXCLUDED 8 “Wolbachia spp.” (≤96 with Blast) 1 EXCLUDED N/A Not sequenced 3 EXCLUDED 1 TOTAL 30

N is 23 n is 5

VI. Sequence for clones & sample species

>JF728926_Chryseobacterium_sp. TCTTCGGAACAGAGAGAGCGGCGTACGGGTGCGTAACACGTGTGCAACCTACCTTTATCAGGGGAATAGCCTTTCGAAAGGA AGATTAATACTCCATAATATATGAACAGGCATCTGTTTATATTGAAAACTCCGGTGGATAAAGATGGGCACGCGCAAGATTA GATAGTTGGTGAGGTAACGGCTCACCAAGTCAATGATCTTTAGGGGTCCTGAGAGGGAGATCCCCCACACTGGTACTGAGAC ACGGACCAGACTCCTACGGGAGGCAGCAGTGAGGAATATTGGACAATGGGTGAGAGCCTGATCCAGCCATCCCGCGTGAAG GATGACGGTCCTATGGATTGTAAACTTCTTTTGTATAGGGATAAACCTTTCTACGTGTAGAAAGCTGAAGGTACTATACGAAT AAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTATCCGGATTTATTGGGTTTAAAGGGTC CGTAGGCGGACCGATAAGTCAGTGGTGAAATCTCATAGCTTAACTATGAAACTGCCATTGATACTGTCGGTCTTGAGTAAATT AGAGGTAGCTGGAATAAGTAGTGTAGCGGTGAAATGCATAGATATTACTTAGAACACCAATTGCGAAGGCAGGTTACCATGA TTTAACTGACGCTGAGGGACGAAAGCGTGGGTAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGCTAAC TCGTTTTTGGAGCGCAAGCTTCAGAGACCAAGCGAAAGTGATAAGTTAGCCACCTGGGGAGTACGTTCGCAAGAATGAAACT CAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGATTATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCAAGGCTTA AATGGGAATTGACAGGTTTAGAAATAGACCCTTCTTCGGACAATTTTCAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTG AGGTGTTAGGTTAAGTCCTGCAACGAGCGCAACCCCTGTCACTAGTTGCTAACATTAAGTTGAGGACTCTAGTGAGACTGCCT ACGCAAGTAGAGAGGAAGGTGGGGATGACGTCAAATCATCACGGCCCTTAC GCCTTGGGCCACACACGTAATACAATGGCCGGTACAGAGGGCAGCTACACAGCGATGTGATGCGAATCTCGAAAGCCGGTCT CAGTTCGGATTGGAGTCTGCAACTC >JF496543_Shigella_sp. GAAAGGTAGCTGGCGGCATGCCTAACACATGCAAGTCGAACGAGTGAGCAACGCAGATGAAGCTTGCTTCTTTGCTGACGAG TGGCGGACGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACG TCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGG CTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGG AGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTA AAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACT CCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCAC GCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTA GAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGAC GAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGA CTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAA AACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGT CTTGACATCCACGGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCG TGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGA GACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTAC AATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCA ACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCG CCCGTCACACCATGGGAGTGGGTTGCAAAAGTAGTAGAGTGGGTTAGACTCTTCGCTAGAGCTCATTATACAGCTTCGATCAC CCCGCCG >AB548582_Escherichia_coli ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGTAACAGGAAGCAGCTTGCTTCTTTGCTGACGAGTGGCGGA CGGGTGAGTAATGTCTGGGAAACTGCCTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAG ACCAAAGAGGGGGACCTTCGGGCCTCTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCT AGGCGACGATCCCTAGCTGGTCTGAGAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGC AGTGGGGAATATTGCACAATGGGCGCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACT TTCAGCGGGGAGGAAGGGAGTAAAGTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCC

AGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAG ATGTGAAATCCCCGGGCTCAACCTGGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGG TGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGC GAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAG GCGTGGCTTCCGGAGCTAACGCGTTAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGG GCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTC AGAGATGAGAATGTGCCTTCGGGAACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAA GTCCCGCAACGAGCGCAACCCTTATCCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGG AGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGA AGCGACCTCGCGAGAGCAAGCGGACCTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGG AATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGT GGGTTGCAAAAGAAGTAGGTAGCTTAACCTTCGGGAGGGCGCTTACCACTTTGTGATTCATGACTGGGGTGAAGTCGTAACA AGGTAACCGTAGGGGAACCTGC >EU423304_Ramlibacter_sp. GGCTCAGATTGAACGCTGGCGGAATGCTTTACACATGCAAGTCGAACGGCAGCACGGGGGCAACCCTGGTGGCGAGTGGCGA ACGGGTGAGTAATACATCGGAACGTGCCCAGTCGTGGGGGATAACGTAGCGAAAGCTACGCTAATACCGCATACGAACTCTG GTTGAAAGCGGGGGATCGCAAGACCTCGCGCGATTGGAGCGGCCGATGGCAGATTAGGTAGTTGGTGGGGTAAAGGCTCACC AAGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCA GCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATTCCGCGTGCAGGATGAAGGCCCTCGGGTTGTAAACTG CTTTTGGACGGAACGAAAAGGCCTTTTCTAATACAGAAGGCTCATGACGGTACCGTCAGAATAAGCACCGGCTAACTACGTG CCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTGATGTAAGAC AGGTGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGTGACTGCATCGCTGGAGTGCGGCAGAGGGGGATGGAATTCCG CGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGGCCTGCACTGACGCTCATGCA CGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACTGGTTGTTGGGTCTTCACT GACTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTGAAACTCAAAGGAATTGACGGG GACCCGCACAAGCGGTGGATGATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCTACCCTTGACATGTCTGGAATTGTG CAGAGATGTGCAAGTGCCCGAAAGGGAGCCAGAACACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGT TAAGTCCCGCAACGAGCGCAACCCTTGTCATTAGTTGCTACGAAAGGGCACTCTAATGAGACTGCCGGTGACAAACCGGAGG AAGGTGGGGATGACGTCAAGTCCTCATGGCCCTTATGGGTAGGGCTACACACGTCATACAATGGCTGGTACAGAGGGTTGCC AACCCGCGAGGGGGAGCTAATCCCATAAAACCAGTCGTAGTCCGGATCGTAGTCTGCAACTCGACTGCGTGAAGTCGGAATC GCTAGTAATCGCGGATCAGCATGTCGCGGTGAATACGTTCCCGGGTCTTGTACACACCGCCCGTCACACCATGGGAGCGGGTT CTGCCAGAAGTAGTTAGCCTAACCGCAAGGAGGGCGATTACCACGGCAGGGTTCGTGACTGGGGTGAAGTC >AJ551148_Acinetobacter_sp. AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGGGAAATGTAGCTTGCTACAT TACCTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCG CATACGCCCTACGGGGGAAAGCAGGGGATCTTCGGACCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGG GTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTC CTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGGAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTTT GGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACCTAGATTAATACTCTGGGATAGTGGACGTTACTCGCAGAATAAGCACC GGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGTGCGTAGGT GGCCAATTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTGGCTAGAGTATGGGAGAGGA TGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGGATACCGATGGCGAAGGCAGCCATCTGGCCTAATAC TGACACTGAGGTACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCG TTGGGGTCTTTGAGACTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAA ATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACA TAGTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTG AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTGCCAGCGGGTTAAGCCGGGAACTTTAAGGATACTGC CAGTGACAAACTGGAGGAAGGCGGGGACGACGTCAAGTCATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGT CGGTACAAAGGGTTGCTACCTCGCGAGAGGATGCTAATCTCAAAAAGCCGATCGTAGTCCGGATTGGAGTCTGCAACTCGAC TCCATGAAGTCGGAATCGCTAGTAATCGCGGATCAGAATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCA CACCATGGGAGTTTGTTGCACCAGAAGTAGGTAGTCTAACCGCAAGGAGGACGCTTACCACGGTGTGGCCGATGACTGGGGT GAAGTCGTAACAAGGTAACC >NR041500_Flavisolibacter_ginsengisoli TGGCTCAGGATGAACGCTAGCGGCAGGCTTAATACATGCAAGTCGAGGGGCAGCAGGACTGTAGCAATACAGTTGCTGGCGA CCGGCAAACGGGTGCGGAACACGTACGCAACCTACCCAAAACTGGGGAATAGCCCGGGGAAACCCGGATTAATACCTCGTA ACCTATTGGAGTGGCATCACTTTAATAGTATAGCTCCGGCGGTTTTGGATGGGCGTGCGCCTGATTAGGTAGTTGGTGAGGGT AACGGCCCACCAAGCCTGCGATCAGTAACTGGTGTGAGAGCACGACCAGTCACACGGGCACTGAGACACGGGCCCGACTCCT ACGGGAGGCAGCAGTAAGGAATATTGGTCAATGGACGCAAGTCTGAACCAGCCATGCCGCGTGAAGGATGAAGGTCCTCTG GATTGTAAACTTCTTTTATATGGGACGAAACCCGGGAATTCTTTCCCGATTGACGGTACCATARGAATAAGCACCGGCTAACT CCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTATCCGGATTCACTGGGTTTAAAGGGTGCGTAGGAGGGTAGGT AAGTCAGTGGTGAAATCTTCGAGCTTAACTCGGAAACTGCCGTTGATACTATCTATCTTGAATACCGTGGAGGTGAGCGGAAT ATGTCATGTAGCGGTGAAATGCTTAGATATGACATAGAACACCAATTGCGAAGGCAGCTCGCTACACGAATATTGACTCTGA GGCACGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGGATACTCGACATACGCGATA CACAGTGTGTGTCTGAGCGAAAGCATTAAGTATCCCACCTGGGAAGTACGACCGCAAGGTTGAAACTCAAAGGAATTGGCGG GGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCTGGGCTAGAATGTAGTCTGACCG TGGGTGAAAGCTCATTTTGTAGCAATACACAGATTATAAGGTGCTGCATGGCTGTCGTCAGCTCGTGCCGTGAGGTGTTGGGT TAAGTCCCGCAACGAGCGCAACCCCCATCACTAGTTGCCATCAGGTAACGCTGGGAACTCTAGTGAAACTGCCGTCGTAAGA CGCGAGGAAGGAGGGGATGATGTCAAGTCATCATGGCCTTTATGCCCAGGGCTACACACGTGCTACAATGGGGCGTACAAAG GGCTGCCACTTAGCGATAAGGAGCCAATCCCAAAAAACGCCTCTCAGTTCAGATTGGAGTCTGCAACTCGACTCCATGAAGC TGGAATCGCTAGTAATCGTATATCAGCAACGATACGGTGAATACGTTCCCGGACCTTGCACACACCGCCCGTCAAGCCATGG AAGCTGGGTGTACCTAAAGTCGGTAACCGCAAGGAGCCGCCTAGGGTAAAACTAGTAACTGGGGCTAAGTCGTAACAAGGT >AY922995_Halomonas_phoceae ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCACGGGGAGCTTGCTCCCTGGTGGCGAGCGGCGGAC GGGTGAGTAATGCATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGG GGAAAGCGGGGGACCTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAA GGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC

AGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACT TTCAGTGAGGAAGAAGGCCTGCGGGTTAATAGCCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGC CAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCG GTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCC GGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGT GCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCG AGCACTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGG GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTG CCGGAGACGGCGGAGTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTT AAGTCCCGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACC GGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGG GTTGCAAGACGGCGACGTGGAGCTAATCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTC GGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGA GTGGACTGCACCAGAAGTGGTTAGCTTAACCTTCGGGAGAGCGATCACCACGGTGTGGTTCATGACTGGGGTG >FN997635_Shewanella_haliotis GCTACACATGCAGTCGAGCGGTAACATTTCAAAAGCTTGCTTTTGAAGATGACGAGCGGCGGACGGGTGAGTAATGCCTGGG AATTTGCCCATTTGTGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTC GGGCCTTGCGCTGATGGATAAGCCCAGGTGGGATTAGCTAGTAGGTGAGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTG GTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACA ATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGG GTGTAAGTTAATACCTTACATCTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACG GAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCT CAACCTGGGAACCGCATTTCGAACTGGCAAACTAGAGTCTTGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGT AGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAGGCACGAAAGCGTGGGGAGCAAA CAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTCGGAGTTTGGTGTCTTGAACACTGGGCTCTCAAGCTAA CGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGA GCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCACAGAATTTGGTAGAGATACCTCAGTGCCT TCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAA CCCCTATCCTTACTTGCCAGCGGGTAATGCCGGGAACTTTAGGGAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGAC GTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCTACAATGGTCAGTACAGAGGGTTGCGAAGCCGCGAGGTGG AGCTAATCCCATAAAGCTGGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGA TCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGA TAGCTTAACCTTCGGGAGGGCGTT >AB604659_Wolbachia_of_Curculio_okumai GGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTA ATGTATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATT TATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGAGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGA GAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGC GAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGT ACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGG CGTAAAGGGCGCATAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAAC CTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGG CGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGT AAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGC AAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCT TACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGGTTTCACACAGGTGTTGCATGGCTG TCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGG GGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTA CACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATT GTACTCTGCAACTCGAGTGCATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCACGCCACGGTGAATACGTTCTCGGGTCTT GTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAACGACCTAACCGCAAGGAGAGAGTTATTTAAAGTG GGATTGGTGACTGGGGTG >Clone1_egg ATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCAGCACGGGGAGCTTGCTCCCTGGTGGCGAGCGGCGGAC GGGTGAGTAATGCATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGG GGAAAGCGGGGGACCTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAA GGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGC AGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACT TTCAGTGAGGAAGAAGGCCTGCGGGTCAATAACCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGC CAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCG GTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCC GGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGT GCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCG AGCACTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGG GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTG CCGGAGACGGCGGAGTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTT AAGTCCCGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACC GGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGG GTTGCAAGACGGCGACGTGGAGCTAATCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTC GGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGA GTGGACTGCACCAGAAGTGGTTAGCTTAACCTTCGGGAGAGCGATCACCACGGTGTGGTTCATGACTGGGGTG >Clone2_egg GGTCACGGGGAAGCTTGCTCCCTGGTGGCGAGCGGCGGAGGGGGGGGGGGCATAGGAATCTGCCCGGTAGTGGGGGATAAC CTGGGGAAAACCAGGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGACCTTCGGGCCTCGCGCTATCGGATGAGCCT ATGTCGAATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACT GGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCATTCCAT

GCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTGAGGAAGAAGGCCTGCGGGTCAATAACCGGCAGGAAG GACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATT ACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACT GTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGG CGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCA CGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGA GTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACG CGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGAGACGGCGGAGTGCCTTCGGGAACCGAGTGACAGGTGCTG CATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATT CGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGG TAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAAGACGGCGACGTGGAGCTATTCCCATAAAGCCTGCCTCA GTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTC CCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAAGT >Clone4_egg CAGCCTGATCATAGCAGTCGAGCGGCGCACGGGGAGCTTGCTCCCTGGTGGCGAGCGGCGGACGGGTTAGTAATGCATAGGA ATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGACCTTCG GGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCGTAGCTGG TCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGACCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAA TGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTGAGGAAGAAGGCC TGCGGGTCAATAACCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACG GAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAGCCCCGGGCT CAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAATGCG TAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAGCAA ACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTTAGTGGCGCAGTTA ACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGG AGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGAGACGGCGGAGTGCC TTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAA CCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACG TCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAAGACGGCGACGTGGA GCTAATCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGAAT CAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAAGTGGTT GCTTAACTCTCGGAGAGCG >Clone6_egg_1492r TGGGGAATATTGCCCAATGGGGGCCACCCCTGATCCCAGCCAAGACGCGTGTCGTGAAGAAGGCCCTTCGGGTTGGTAAAGC ACTTTCCAGTGAGGAAGAAGGCCTTGCGGGTTAATAGCCGGCCAGGAAGGACATCACTCACAGAAGAAGCACCCGCTAAATT CCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGAT AAGCCGGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGA ATTCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACT GAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTG CCTCGAGCACTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATT GACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGG AATCTGCCGGAGACGGCGGAGTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGT TGGGTTAAGTCCCGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGAC AAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTAC AAAGGGTTGCAAGACGGCGACGTGGAGCTATAGAGTTAGATCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTG AAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCAT GGGAGTGGACTGCACCAGATTGCTTAGTTTGCTTCTCGGCTGCTAGTTT >Clone7_egg TTGGCGGCGCCGCGGAGCAGCCAGAACAACCAGCAAGTCGACGGAGTTTATTGAAGCTTGCTATGGTATAACTTAGTGGCAG ACGGGTGAGTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACG GGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTAT AGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTG GACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGA TAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGG AATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAA AACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAG TGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGT CCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGAC TACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGC GAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTT GCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGG TAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGG AGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTC AGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTT CTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTATTGACTTAACCCCAAGGATGGGAG >Clone8_egg AGAGCAGCCTGATTATAGCAGTCGAGACGGCGCACAGTGAGCTTGCTCCCTGGTGGCGAGCGGCGGCCCCTTTAAGGGGCAT AGGAATCTGCCCGGTAGTGGGGGATAACCCCTTTTAAACCCAGGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGAC CTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCGTAG CTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGG ACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTGAGGAAGAA GGCCTGCGGGTTAATAGCCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAAT ACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAGCCCCGG GCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAAT GCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAG CAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTTAGTGGCGCA

GTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGAGACGGCGGA GTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAG CGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGA CGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAAGACGGCGAC GTGGAGCTATTCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCG TGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAAG TGGTAGATTAACTTCGGGGTAGCTTCGTCGA >Clone10_egg AGACCTAGGATAATAGCAAGTCGAGCGGCAGCACGGGGAAGCTTGCTCCCTGGTGGCGAGCGGCGGACGGGTGAGTAATGC ATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGG ACCTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCGT AGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATT GGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTGAGGAAG AAGGCCTGCGGGTTAATAGCCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGT AATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAGCCC CGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGA AATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGG TAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTTAGTGGC GCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAA GCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGAGACGGC GGAGTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAAC GAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGG GGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAAGACGGC GACGTGGAGCTAATCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTA ATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCA GAAGTGGTAGCTTAACTTCGGAGAGCGTATTCGTCGTCGTGTGTTTTCTC >Clone11_egg GCTCAGTCGAATCATAGCAAGTCGAGCGGCGCACGGGGAGCTTGCTCCCTGGTGGCGAGCGGCGGACGGGTGAGTAATGCAT AGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGGGGAAAGCGGGGGAC CTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGATCCGTAG CTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGG ACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTGAGGAAGAA GGCCTGCGGGTTAATAGCCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAAT ACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAGCCCCGG GCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGCGGTGAAAT GCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAG CAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTTAGTGGCGCA GTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCG GTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGAGACGGCGGA GTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAG CGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGA CGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAAGACGGCGAC GTGGAGCTATTCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCG TGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAAG TGTTAGCTTAACTTCGGAGAGCGTATCTAGCTGCGTGCTGTGT >Clone12_egg CGAGAGCAGCTCAGACATCTAGCAGTCGAGCGGCAGCACGGGGAGCTTGCTCCCTGGTGGCGAGCGGCGGACGGGTGAGTA ATGCATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGGGGAAAGCG GGGGACCTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACGA TCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGGA ATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTGA GGAAGAAGGCCTGCGGGTTAATAGCCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCC GCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAA AGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAGC GGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGC GTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTTA GTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGC ACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGAGA CGGCGGAGTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCG TAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGGGGAAG GTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAAGA CGGCGACGTGGAGCTAATCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCT AGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGC ACCAGAAGTGGTTAGCTTAACCTTCGGGATAGCGTATCTAGCTGCGTGTCTGGTGTGCTGCGGGCGCGAC >Clone13_egg AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGTAACATTTCAAAAGCTTGCTT TTGAAGATGACGAGCGGCGGACGGGTGAGTAATGCCTGGGAATTTGCCCATTTGTGGGGGATAACAGTTGGAAACGACTGCT AATACCGCATACGCCCTACGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCTGATGGATAAGCCCAGGTGGGATTAGCTAGT AGGTGAGGTAAAGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCC CAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAA GGCCTTCGGGTTGTAAAGCACTTTCAGCGAGGAGGAAAGGGTGTAAGTTAATACCTTACATCTGTGACGTTACTCGCAGAAG AAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTG CGCAGGCGGTTTGTTAAGCGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACCGCATTTCGAACTGGCAAACTAGAGTCTTG TAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGG ACAAAGACTGACGCTCAGGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCT

ACTCGGAGTTTGGTGTCTTGAACACTGGGCTCTCAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTA AAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTAC TCTTGACATCCACAGAATCTGGTAGAGATACCTCAGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTC GTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCTATCCTTACTTGCCAGCGGGTAATGCCGGGAACTTTAGG GAGACTGCCGGTGATAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGAGTAGGGCTACACACGTGCT ACAATGGTCAGTACAGAGGGTTGCGAAGCCGCGAGGTGGAGCTAATCCCATAAAGCTGGTCGTAGTCCGGATTGGAGTCTGC AACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCACGGTGAATACGTTCCCGGGCCTTGTACACACC GCCCGTCACACCATGGGAGTGGGCTGCACCAGAAGTAGATAGCTTAACCTTCGGGAGGGCGTTTACCACGGTGTGGTTCATG ACTGGGGTGAAGTCGTAACAAGGTAACC >Clone15_egg_1492r GGATTAGCTGGTTGGTGGAGTAACGGCTTAACCAAGGCGGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTTGG ACTGAGACACGGCCCCAGACTCCTACGGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCCAGCCAT GCCGCGTGTGTGAAGAAGGCCTTTCGGGTTGTAAAGCACTTTCAGTGAGGAAGAAGGCCTGCGGGTCAATAACCGGCCAGGA AGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGA ATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGA ACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCCGGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCA GTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAG TCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTG GGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGC AACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGAGACGGCGGAGTGCCTTCGGGAACCGAGTGACAGGT GCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGC GATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTAC GGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAAGACGGCGACGTGGAGCTAATCCCATAAAGCCTGCC TCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCGCTAGTAATCGTGAATCAGAATGTCACGGTGAATAC GTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTGCACCAGAA >Clone16_egg_27f GCCACGTGTTCACCGAGGAGAAGCTGATCATAGCAGTCGAGCGGGACATTTCAAAAGCTTGCTTTTGAAGATGACGAGCGGC GGACGGGTGAGTAATGCCTGGGAATTTGCCCATTTGTGGGGGATAACAGTTGGAAACGACTGCTAATACCGCATACGCCCTA CGGGGGAAAGCAGGGGACCTTCGGGCCTTGCGCTGATGGATAAGCCCAGGTGGGATTAGCTAGTAGGTGAGGTAAAGGCTC ACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGG CAGCAGTGGGGAATATTGCACAATGGGGGAAACCCTGATGCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAG CACTTTCAGCGAGGAGGAAAGGGTGTAAGTTAATACCTTACATCTGTGACGTTACTCGCAGAAGAAGCACCGGCTAACTCCG TGCCAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGCAGGCGGTTTGTTAAG CGAGATGTGAAAGCCCCGGGCTCAACCTGGGAACCGCATTTCGAACTGGCAAACTAGAGTCTTGTAGAGGGGGGTAGAATTC CAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACAAAGACTGACGCTCAG GCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCTACTCGGAGTTTGGTGTCT TGAACACTGGGCTCTCAAGCTAACGCATTAAGTAGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGAC GGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACTCTTGACATCCACAGAAT CTGGTAGAGATACCTCAGTGCCTTCGGGAACTGTGAGACAGGTGCTGCATGGCTGTCTTCAGCTCCTG >Clone20_egg GCTAGAGTCGCAGACGAGTACTAGCAAGTCGAAGGAGTTTAAGGAAACTTGCTATGGTATAACTTAGTGGCAGACGGGTGAG TAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAA TTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCT GAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGG GCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACG GTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTG GGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTA ACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAA GGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCT GTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTC GCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAA CCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTTGGCCGGATTTCACACAGGTGTTGCATGG CTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGC TGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGG CTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCG GATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGG TCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGTTATTGA >Clone21_egg TCACGAGGTCACGCCTAGATACAGCAAGTCGAGCGGCGCACGGGAAGCTTGCTCCCTGGTGGCGAGCGGCGGACGGGTGAGT AATGCATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGGGGAAAGC GGGGGACCTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCGACG ATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGGGG AATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAGTG AGGAAGAAGGCCTGCGGGTTAATAGCCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGC CGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGTGA AAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCGGTGTAG CGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGAAAG CGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCACTT AGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCC GCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGGA GACGGCGGAGTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCC CGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAGGA AGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGCAA GACGGCGACGTGGAGCTAATCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAATCG CTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGACTG CACCAGAAGTGTTAGATTAACTTCGGGGAGCGTATCTAGCTCGTTGCTGTGTTGCTCGGCTGGCGACC

>Clone22_egg TTACGCCCCCGGGGCAGCGACAACCTAGCAGTCGAGCGGCGCACCGTAAGCTTGCTCCCTGGTGGCGAGCGGCGGACGGGTG AGTAATGCATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGGGGGAA AGCGGGGGACCTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCAAGGCG ACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTGG GGAATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCACTTTCAG TGAGGAAGAAGGCCTGCGGGTCAATAACCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGCCAGCA GCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCGGTTGT GAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCCCGGTG TAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGTGCGA AAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCGAGCA CTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGC CCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTGCCGG AGACGGCGGAGTGCCTTCGGGAACCGAGTGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGT CCCGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACCGGAG GAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGGGTTGC AAGACGGCGACGTGGAGCTATTCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTCGGAAT CGCTAGTAATCGTGAATCAGAATGTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGA CTGCACCAGAAGTG >Clone28_egg CTACGGCGCGGCGGAGCCACCAGACACTCGAGGCAGTCGAGCGGCGCACGGGGAGCTTGCTCCCTGGTGGCGAGCGGCGGA CGGGTGAGTAATGCATAGGAATCTGCCCGGTAGTGGGGGATAACCTGGGGAAACCCAGGCTAATACCGCATACGCCCTACGG GGGAAAGCGGGGGACCTTCGGGCCTCGCGCTATCGGATGAGCCTATGTCGGATTAGCTGGTTGGTGAGGTAACGGCTCACCA AGGCGACGATCCGTAGCTGGTCTGAGAGGATGATCAGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAG CAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTCGGGTTGTAAAGCAC TTTCAGTGAGGAAGAAGGCCTGCGGGTTAATAGCCGGCAGGAAGGACATCACTCACAGAAGAAGCACCGGCTAACTCCGTGC CAGCAGCCGCGGTAATACGGAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTGGCTTGATAAGCCG GTTGTGAAAGCCCCGGGCTCAACCTGGGAACGGCATCCGGAACTGTCAGGCTAGAGTGCAGGAGAGGAAGGTAGAATTCCC GGTGTAGCGGTGAAATGCGTAGAGATCGGGAGGAATACCAGTGGCGAAGGCGGCCTTCTGGACTGACACTGACACTGAGGT GCGAAAGCGTGGGTAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTAGCCGTTGGGTGCCTCG AGCACTTAGTGGCGCAGTTAACGCGATAAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGG GGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTACCCTTGACATCCTCGGAATCTG CCGGAGACGGCGGAGTGCCTTCGGGAACCGAGTGACACGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTT AAGTCCCGTAACGAGCGCAACCCCTGTCCCTATTTGCCAGCGATTCGGTCGGGAACTCTAGGGAGACTGCCGGTGACAAACC GGAGGAAGGTGGGGACGACGTCAAGTCATCATGGCCCTTACGGGTAGGGCTACACACGTGCTACAATGGCAGGTACAAAGG GTTGCAAGACGGCGACGTGGAGCTAATCCCATAAAGCCTGCCTCAGTCCGGATCGGAGTCTGCAACTCGACTCCGTGAAGTC GGAATCGCTAGTAATCGTGAATCAGTTTTCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCCCCATGGGAGTGG ACTGCACCAGAAGTGGTAGCT >Clone2_midgut_plasmid AGCTACACATGCAGTCGAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGA ATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTA GATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATC AGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGA TCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAA GAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGC GCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGA AAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGT TCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATG TTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACT CAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTG ACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCG >Clone3_midgut AGCTACACATGCAGTCGAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGA ATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTA GATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATC AGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGA TCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAA GAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGC GCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGA AAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGT TCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATG TTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACT CAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTG ACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTT >Clone3_midgut_plasmid AGCTACACATGCAGTCGAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGA ATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTA GATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATC AGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGA TCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAA GAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGC GCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGA AAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGT TCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATG TTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACT

CAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTG ACATGGAAATTATACCTATTCGAA >Clone7_midgut CATGGCGCGGCCTACATGACAGTCGACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATAT ATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATT GCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGG ATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAA GCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTC ACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTA AAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAG AGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTC TATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAAC GATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGA TTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACC ACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGT CAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGAC TTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACA CGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTAC TCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTAC ACACTGCCCGTAACGGCCATGGGAATTGGTTTCACTCGAAGCTAATGACCTAACCGCAAGGAGGGAGTCTGCTGTGTGTTGCC GCCGCTC >Clone8_midgut CCCTTAGAGTTTGATCCTGGCTCAGGATGAACGCTAGCGGCAGGCTTAATACATGCAAGTCGAGGGGCAGCAGGGGTGTAGC AATACACTCGCTGGCGACCGGCAAACGGGTGCGGAACACGTACGCAATCTACCCAAAACTGATGAATAGCCCTCCGAAAGGA GGATTAATACATCGTAACATATTAGAGTGGCATCACTTTATTATTATAGCTCCGGCGGTTTTGGATGAGCGTGCGCCTGATTA GGTAGTTGGCGGGGTAACGGCCCACCAAGCCTTCGATCAGTAACTGGTGTGAGAGCACGACCAGTCACACGGGCACTGAGAC ACGGGCCCGACTCCTACGGGAGGCAGCAGTAAGGAATATTGGTCAATGGACGCAAGTCTGAACCAGCCATGCCGCGTGAAG GATGAAGGTCCTCTGGATTGTAAACTTCTTTTATATGGGACGAAACCCCCGAATTCTTTCGGGATTGACGGTACCATAAGAAT AAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAAGCGTTATCCGGATTCACTGGGTTTAAAGGGTG CGTAGGAGGGCAGGTAAGTCAGTGGTGAAATCTCCGAGCTTAACTTGGAAACTGCCGTTGATACTATCTGTCTTGAATATTGT GGAGGTGAGCGGAATATGTCATGTAGCGGTGAAATGCTTAGATATGACATAGAACACCAATTGCGAAGGCAGCTCGCTACAC ATATATTGACTCTGAGGCACGAAAGCGTGGGGATCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGGATAC TCGACATACGCGATACACAGTGTGTGTCTGAGCGAAAGCATTAAGTATCCCACCTGGGAAGTACGACCGCAAGGTTGAAACT CAAAGGAATTGGCGGGGGTCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGATACGCGAGGAACCTTACCTGGGCTAG AATGCTGGTGGATCGTGGGTGAAAGCTCACTTTGTAGCAATACACCGCCAGTAAGGTGCTGCATGGCTGTCGTCAGCTCGTGC CGTGAGGTGTTGGGTTAAGTCCCGCAACGAGCGCAACCCCCATCACTAGTTGCCATCAGGTAACGCTGGGAACTCTAGTGAA ACTGCCGTCGTAAGACGCGAGGAAGGAGGGGATGATGTCAAGTCATCATGGCCTTTATGCCCAGGGCTACACACGTGCTACA ATGGGGCGTACAAAGGGCTGCCACTTAGCGATAAGGAGCCAATCCCAAAAAACGCCTCTCAGTTCAGATTGGAGTCTGCAAC TCGACTCCATGAAGCTGGAATCGCTAGTAATCGTATATCAGCAATGATACGGTGAATACGTTCCCGGACCTTGCACACACCGC CCGTCAAGCCATGGAAGCTGGGTGTACCTAAAGTCGGTAACCGCAAGGAGCCGCCTAGGGTAAAACTAGTAACTGGGGCTAA GTCGTAACAAGGTA >Clone9_midgut GACAGTCGACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGT AGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTA TATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTG GAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATG CCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGG CTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTG GTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATA GAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTG ACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATG GGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAA TTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAA ATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAG ATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCA GTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGG CTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTAC ATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGC CATGGGAATTGGTTTCACTCGAAGCTATTGACCTAACCGCAAGGGAGGGAGTCTGCGTGTTGTGTTCCGCGCCCCGAC >Clone10_midgut_27f AGTCGACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAGT ACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATT AGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAAC TGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCA TGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAAC TCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAG TAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGA ATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAAGCGTCTATCTGGTTCAAATCTGACGCT GAAGCGCGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTTAACGATGAATGTTAAATATGGGGA AGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGA CGGGGACCCGCACAAGCGGTGGAGCATGTGGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATT ATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATG TTGGGGTTAAGTCCCCCAACGAAGCGAACCCTCATCCTTAGTTACCATCAGGGAATGCTGGGGGACTTTAAGGAACCTGCCA GCGATAAACTGGAGGAAGGTGGGGGATG >Clone16a_midgut

AGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGCAGGCTTAACACATGCAAGTCGAGCGGGGAAAAGTAGCTTGCTACTT TACCTAGCGGCGGACGGGTGAGTAATGCTTAGGAATCTGCCTATTAGTGGGGGACAACATTTCGAAAGGAATGCTAATACCG CATACGTCCTACGGGAGAAAGCAGGGGACCTTCGGGCCTTGCGCTAATAGATGAGCCTAAGTCGGATTAGCTAGTTGGTGGG GTAAAGGCCTACCAAGGCGACGATCTGTAGCGGGTCTGAGAGGATGATCCGCCACACTGGGACTGAGACACGGCCCAGACTC CTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGGGCAACCCTGATCCAGCCATGCCGCGTGTGTGAAGAAGGCCTTTT GGTTGTAAAGCACTTTAAGCGAGGAGGAGGCTACTTAGATTAATACTCTGAGATAGTGGACGTTACTCGCAGAATAAGCACC GGCTAACTCTGTGCCAGCAGCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGATTTACTGGGCGTAAAGCGTGCGTAGGT GGCCAATTAAGTCAAATGTGAAATCCCCGAGCTTAACTTGGGAATTGCATTCGATACTGGTTGGCTAGAGTATGGGAGAGGA TGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGGAGGAATACCGATGGCGAAGGCAGCCATCTGGCCTAATAC TGACACTGAGGTACGAAAGCATGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCATGCCGTAAACGATGTCTACTAGCCG TTGGGGTCTTTGAGACTTTAGTGGCGCAGCTAACGCGATAAGTAGACCGCCTGGGGAGTACGGTCGCAAGACTAAAACTCAA ATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACA TAGTAAGAACTTTCCAGAGATGGATTGGTGCCTTCGGGAACTTACATACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTG AGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTTTCCTTATTTGCCAGCGGGTTAAGCCGGGAACTTTAAGG >Clone25_midgut GTCGAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGAGGGAGGGAATATATAGGAATCTACCTAGTAGTA CGGAATAATTGTTGGAAAAGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTA GATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACT GAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCAT GAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACT CCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGT AAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAA TTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGACCACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGA GGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTT TACTTTCTGTATAACAGCTAACGCGTAAAACATCCCGCCTGGGGACTCCGGTCGCAAGATAAAAACTCAAAGGAATGGACGG GCCCCCGCACAAGCGGTGGAGCATGTGTTTTATTTCGAGCCACCGAGAAAACCCTTACCACTCCTTGACATGGAAATTATACC TATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGG TTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAA CTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATG GGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTT GGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAA TTGGTTTCACTCGAAGCT >Clone26a_midgut TAGAGTTTGATCCTGGCTCAGATTGAACGCTGGCGGAATGCTTTACACATGCAAGTCGAACGGCAGCACGGGGGCAACCCTG GTGGCGAGTGGCGAACGGGTGAGTAACACATCGGAACGTGCCCAGTCGTGGGGGATAACGTAGCGAAAGCTACGCTAATAC CGCATACGAACTCTGGTTGAAAGCGGGGGACTCGCAAGGGCCTCGCGCGATTGGAGCGGCCGATGGCAGATTAGGTAGTTGG TGGGGTAAAGGCTCACCAAGCCGACGATCTGTAGCTGGTCTGAGAGGACGACCAGCCACACTGGGACTGAGACACGGCCCA GACTCCTACGGGAGGCAGCAGTGGGGAATTTTGGACAATGGGCGCAAGCCTGATCCAGCCATTCCGCGTGCAGGATGAAGGC CCTCGGGTTGTAAACTGCTTTTGGACGGAACGAAAAGGCTTTTCCTAATACGGAAAGCTCATGACGGTACCGTCAGAATAAG CACCGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGGTGCGAGCGTTAATCGGAATTACTGGGCGTAAAGCGTGCGC AGGCGGTGATGTAAGACAGGTGTGAAATCCCCGGGCTTAACCTGGGAACTGCATTTGTGACTGCATCGCTGGAGTGCGGCAG AGGGGGATGGAATTCCGCGTGTAGCAGTGAAATGCGTAGATATGCGGAGGAACACCGATGGCGAAGGCAATCCCCTGGGCC TGCACTGACGCTCATGCACGAAAGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCCCTAAACGATGTCAACT GGTTGTTGGGTCTTCACTGACTCAGTAACGAAGCTAACGCGTGAAGTTGACCGCCTGGGG >Clone34_midgut CTTGCTATGGTATAACTTAGTGGCAGAGGGGGAGGAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGG CAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTA ATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTA CGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGG TTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTA ATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCG AGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAA TTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAG CAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTA ACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGA GCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCG GTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGC AACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATG ATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGC TAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTG GATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTA ATCGACCTAACCGCAAGGAGGGAGTATTTAAAGTGG >Clone39_midgut AGTCGAACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAG TACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATAT TAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAA CTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGC ATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAA CTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTA GTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGG AATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGC TGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGA AGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGA CGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTA TACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGA

TAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTAC AATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGA AGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATG GGAATTGGTTTCACTCGAAGCTAATGACCTAACCGCAAGGAGGGAGTCTGCA >Clone40_midgut CGGCGCCGGGACGCAGACACCTAGCAAGTCGACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGA GTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAA ATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATC TGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATG GGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGAC GGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATT GGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCT AACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGA AGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGC TGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGT CGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAA ACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATG GCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATG CTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGG GCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTC GGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGG GTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTATCGACTTAACCGCAAGGAGGGAG >Clone41_midgut ACACATGCAAGTCGACGGTAACGGAACAGCTTGCTTCTTTGCTGACGAGTGGCGGACGGGTGAGTAATGTCTGGGAAACTGC CTGATGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCT CTTGCCATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGA GAGGATGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGC GCAAGCCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAA GTTAATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGT GCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCT GGGAACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGAT CTGGAGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGAT TAGATACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTT AAGTCGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGA ACCGTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTAT CCTTTGTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTC ATCATGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGAC CTCATAAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAAT GCCACGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGAAGTAGGTAGCTTA ACCTTCGGGAGGGCGCT >Clone43_midgut AGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGCTTGCTAT GGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAA TACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCT ACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGG CAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAG CTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAG AGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAA CCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAA TATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTT AAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGG CCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCA AGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCT AATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAG CATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAACGACC TAACCGCAAGGAGGGAGTTATTTAAAGTGGGATCGGTGACTGGGGTGAAGTCGTAACAAGGTAACCGTA >Clone45_midgut CAGTCGTACGGGGCAACGCAGATAAGACTTGCTGCTTTGCTGACGAGTGGCGGAGGGGAGGAATGGCTGGGAAACTGCCTGA TGGAGGGGGATAACTACTGGAAACGGTAGCTAATACCGCATAACGTCGCAAGACCAAAGAGGGGGACCTTCGGGCCTCTTGC CATCGGATGTGCCCAGATGGGATTAGCTAGTAGGTGGGGTAACGGCTCACCTAGGCGACGATCCCTAGCTGGTCTGAGAGGA TGACCAGCCACACTGGAACTGAGACACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGCACAATGGGCGCAAG CCTGATGCAGCCATGCCGCGTGTATGAAGAAGGCCTTCGGGTTGTTAAAGTACTTTCAGCGGGGAGGAAGGGAGTAAAGTTA ATACCTTTGCTCATTGACGTTACCCGCAGAAGAAGCACCGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGGGTGCAA GCGTTAATCGGAATTACTGGGCGTAAAGCGCACGCAGGCGGTTTGTTAAGTCAGATGTGAAATCCCCGGGCTCAACCTGGGA ACTGCATCTGATACTGGCAAGCTTGAGTCTCGTAGAGGGGGGTAGAATTCCAGGTGTAGCGGTGAAATGCGTAGAGATCTGG AGGAATACCGGTGGCGAAGGCGGCCCCCTGGACGAAGACTGACGCTCAGGTGCGAAAGCGTGGGGAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCCGTAAACGATGTCGACTTGGAGGTTGTGCCCTTGAGGCGTGGCTTCCGGAGCTAACGCGTTAAGT CGACCGCCTGGGGAGTACGGCCGCAAGGTTAAAACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTT AATTCGATGCAACGCGAAGAACCTTACCTGGTCTTGACATCCACGGAAGTTTTCAGAGATGAGAATGTGCCTTCGGGAACCGT GAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTTGTGAAATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTATCCTTT GTTGCCAGCGGTCCGGCCGGGAACTCAAAGGAGACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGACGTCAAGTCATCA TGGCCCTTACGACCAGGGCTACACACGTGCTACAATGGCGCATACAAAGAGAAGCGACCTCGCGAGAGCAAGCGGACCTCAT

AAAGTGCGTCGTAGTCCGGATTGGAGTCTGCAACTCGACTCCATGAAGTCGGAATCGCTAGTAATCGTGGATCAGAATGCCA CGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGAGTGGGTTGCAAAAGA >Clone46_midgut_1492r TAACTGACGCTGAGGGACGAAAGCGTGGGAGCGAACAGGATTAGATACGCTGGTAGTCCACGCCGTAGACGATGCTAACTCG TATGTGGGGCGCAAGCTTCAGAGACCAAGCGAAAGTGATAAGTTAGCCACGTGGGGAGTACGTTCGCAAGAATGAAACTCA AAGGAATTGACGGGGGCCCGCACAAGCGGTGGATTATGTGGTCAAATTCGATGACACGCGAGGAACCTTACCAAGGCTTAAA TGGGAATTGACAGGTTTAGAAATAGACTCTCCTTCGGGCAATTTTCAAGGTGCTGCATGGTTGTCGTCAGCTCGTGCCGTGAG GTGTTAGGTTAAGTCCTGCAACGAGCGCAACCCCTGTCACTAGTTGCTAACATTAAGTTGAGGACTCTAGTGAGACTGCCTAC GCAAGTAGAGAGGAAGGTGGGGATGACGTCAAATCATCACGGCCCTTACGCCTTGGGCCACACACGTAATACAATGGCCGGT ACAGAGGGCAGCTACACAGCGATGTGATGCAAATCTCGAAAGCCGGTCTCAGTTCGGATTGGAGTCT >Clone47_midgut TTCGCCCTTAGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTA GCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAAC GGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGG TAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCC TACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTG GGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGG TAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCC CGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGA AATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGG AGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGGAAGTTTACTTTCTGTATTACAG CTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGT GGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGG GTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGA GCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGG GATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCG AGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAAT CGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA GCTAACGACCTAACCGCAAGGAGGGAGTTATTTAAAGTGGGATCGGTGACTGGGGTGAAGTCGTAACAAGGTAACCGTAAA GGGCGAAT >Clone49_midgut TCGCCCTTAGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAG CTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACG GCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGT AATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCT ACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGG GTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGT AATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCC GAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAA ATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGA GCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCT AACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGG AGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTC GGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCG CAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGAT GATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGG CTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGT GGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCT AACGACCTAACCGCAAGGAGGGAGTTATTTAAAGTGGGATCGGTGACTGGGGTGAAGTCGTAACAAGGTAACCGTAAAGGG CGAATTCGT >Clone50_midgut AGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGCTTGCTAT GGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAA TACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCT ACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGG CAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAG CTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAG AGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAA CCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAA TATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTT AAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGG CCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCA AGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCT AATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAG CATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAACGACC TAACCGCAAGGAGGGAGTTATTTAAAGTGGGATCGGTGACTGGGGTGAAGTCGTAACAAGGTAACCGTA >Clone52_midgut AGTCGAACGGAGTTATATTTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTA GTACGGAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTAT ATTAGATTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGG AACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCC GCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCT AACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGT

TAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGA GGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGAC GCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGG AAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTG ACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATT ATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATG TTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTG ATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTA CAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATG AAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCAT GGGAATTGGTTTCACTCGAAGCTAATGACCTAACCGCAAGGAGGGAG >Clone53_midgut_1492r TTAGCTAGTTGTGGGGTAATACCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGA TACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGT GAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGT GCCAGCAGCCGCGGTAATACGGAGAGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGT TAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCC TAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGC GCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTAC TTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGA CCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTAT TCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTA AGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACT GGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGG CTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGG AATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATT GGTTTCACTCGAA >Clone54_midgut_1492r GGGAATATTGGACAATGGGCGAAAGCCTGATCCAAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTT AGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCT AGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGG AACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAG GAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAG ATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACAT TCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAA TTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATT TCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTT AGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATC ATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCT TAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCA CGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAA >Clone55_midgut TCGACGGAGTTATATTGTAGCTTGCTATGGTATAACTTAGTGGCAGACGGGGAGGAATATATAGGAATCTACCTAGTAGTACG GAATAATTGTTGGAAACGGCAACTAATACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGA TTAGCTAGTTGGTGGGGTAATAGCCTACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGA GATACGGTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGA GTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAWTTTT CTTTGCCAGCAGCCGCGGTAATACGGAGAGGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAG TAAGTTAAAAAGTGAAATCCCGAGGCTTAACCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGG AATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGC TGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGA AGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGA CGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTA TACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGT TGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGA TAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTAC AATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGA AGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCGCCCATGG GAATTGGTTTCACTCGAAGCTAATAGACCTAACCGCAAGGAGG >Clone56_midgut AGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGCTTGCTAT GGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAA TACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCT ACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGG CAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAG CTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAG AGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAA CCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAA TATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTT AAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGG CCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCA AGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCT AATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAG

CATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAACGACC TAACCGCAAGGAGGGAGTTATTTAAAGTGGGATCGGTGACTGGGGTGAAGTCGTAACAAGGTA >Clone57_midgut AGAGTTTGATCCTGGCTCAGAATGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAACGGAGTTATATTGTAGCTTGCTAT GGTATAACTTAGTGGCAGACGGGTGAGTAATATATAGGAATCTACCTAGTAGTACGGAATAATTGTTGGAAACGGCAACTAA TACCGTATACGCCCTACGGGGGAAAAATTTATTGCTATTAGATGAGCCTATATTAGATTAGCTAGTTGGTGGGGTAATAGCCT ACCAAGGCAATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGG CAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAG CTCTTTTAGTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAG AGGGCTAGCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAA CCTTGGAACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAA TATTAGGAGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAG GATTAGATACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTT AAACATTCCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGT GGTTTAATTCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGG CCGGATTTCACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCT CATCCTTAGTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCA AGTCATCATGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCT AATCCCTTAAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAG CATGCCACGGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTAACGACC TAACCGCAAGGAGGGAGTTATTTAAAGTGGGATCGGTGACTGGGGTGAAGTCGTAACAAGGTAACCGTA >Clone59_midgut_1492r AATGATCTATAGCTGATCTGAGAGGATGATCAGCCACACTGGAACTGAGATACGGTCCAGACTCCTACGGGAGGCAGCAGTG GGGAATATTGGACAATGGGCGAAAGCCTGATCCAGCCATGCCGCATGAGTGAAGAAGGCCTTTGGGTTGTAAAGCTCTTTTA GTGAGGAAGATAATGACGGTACTCACAGAAGAAGTCCTGGCTAACTCCGTGCCAGCAGCCGCGGTAATACGGAGAGGGCTA GCGTTATTCGGAATTATTGGGCGTAAAGGGCGCGTAGGCTGGTTAGTAAGTTAAAAGTGAAATCCCGAGGCTTAACCTTGGA ACTGCTTTTAAAACTGCTAACCTAGAGATTGAAAGAGGATAGAGGAATTCCTAGTGTAGAGGTGAAATTCGTAAATATTAGG AGGAACACCAGTGGCGAAGGCGTCTATCTGGTTCAAATCTGACGCTGAGGCGCGAAGGCGTGGGGAGCAAACAGGATTAGA TACCCTGGTAGTCCACGCTGTAAACGATGAATGTTAAATATGGGAAGTTTACTTTCTGTATTACAGCTAACGCGTTAAACATT CCGCCTGGGGACTACGGTCGCAAGATTAAAACTCAAAGGAATTGACGGGGACCCGCACAAGCGGTGGAGCATGTGGTTTAAT TCGATGCAACGCGAAAAACCTTACCACTCCTTGACATGGAAATTATACCTATTCGAAGGGATAGGGTCGGTTCGGCCGGATTT CACACAGGTGTTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTCATCCTTA GTTACCATCAGGTAATGCTGGGGACTTTAAGGAAACTGCCAGTGATAAACTGGAGGAAGGTGGGGATGATGTCAAGTCATCA TGGCCCTTATGGAGTGGGCTACACACGTGCTACAATGGTGGCTACAATGGGCTGCAAAGTCGCGAGGCTAAGCTAATCCCTT AAAAGCCATCTCAGTTCGGATTGTACTCTGCAACTCGAGTACATGAAGTTGGAATCGCTAGTAATCGTGGATCAGCATGCCAC GGTGAATACGTTCTCGGGTCTTGTACACACTGCCCGTCACGCCATGGGAATTGGTTTCACTCGAAGCTATGACTTAACCCCAA GGAGGG