There are several researches about gall and plant physiology. For example, some scientists have tested the nitrogen concentration in plant primary metabolites, because nitrogen is usually regarded as the index of whether host plant could provide sufficient nutrition or not. But the outcomes are different among different researches. Hartley (1998) discovered that the nitrogen concentration is higher in Dasineuravicia galls, but not in other Diptera insect galls (Rhopalomyia sp.). Other researches revealed that the nitrogen concentrations are even higher in plant tissues than in gall tissues made by gall wasp, gall fly (Hartly, 1998) and some gall midge (Brewer et al, 1987).
Some researches indicated that there are obvious high concentration of starch, soluble carbohydrate, lipid and proteins in gall tissues. (Shannon 1980; Bronner, 1992; De Bruyn et al, 1998) But there are other researches indicated the opposite situations. (Anderson and mizell, 1987; Conell, 1983, Hartley, 1992, 1998; Yang 1998)
Most of previous researches in Taiwan about galls, focus on the description and comparison of newly discovered galls on plants. (Yang, 1984; Tao 1991; Yang and Tung, 1998). Some researches described more detailed insect life history and related information (Yang, 1996; Tung, 1998, Su 2002). There are also some researches about gall forms and tissues (Su, 2002; Tung, 1997; Liang, 1999; Weng, 2003; Chen, 2004). Other research about plant physiology and ecology includes Yang (1998) discuss the photo pigment and protein complex; Liao 2003 discussed the nutrition adaptation of gall-insects.
There are more and more researches about galls in Taiwan. Scientists put more
attentions and interests on these abundant galls. In our research, we use 2D electrophoresis technique to determine the protein differences between plant and gall tissues, in order to understand the protein changes. And we can provide further information of gall physiology.
We also use DNA barcode to determine gall midge phylogeny, combined with plant gall proteomics and larvae anatomy, in order to contribute some information to Cecidology.
2 .Materials and Methods
2.1 Insect gene molecular evolution
2.1.1 Gall midge sample collection
These samples was collected by Dr. Tung, Mr. Hsu and I. Total 9 morphospecies of galls were collected together with leaves and stems from 8 species of Machilus in Taiwan October 2004 through January 2005. Among these 9 morphospecies of galls, 3 are stem galls and 6 are leaf galls. We gave each plant species and galls a number. Since all midge larvae inside these galls are unknown species, we named these larvae according to plant names, gall types, and serial numbers. Detailed information is shown in Fig. 1 and Table 1.
Galls along with leaves and stems were put in zipped bags in 4℃ refrigerator. Larvae or pupae were picked from galls in two days after collection and preserved in 99.5% alcohol in 4
℃ for DNA extraction. Every larva or pupae were picked over under dissecting binocular microscope. Larvae and pupae which were distinguishable or suspected parasitized were excluded in order to avoid contamination with internal or external parasitoid larvae in DNA analysis.
The way we treated parasitized larvae:
Cecidomyiidae midge larvae are easily be parasitized by bees or other organisms such as fungi, bacteria…etc. When parasites were inside the larvae, we abandoned these larvae.
When parasites were outside the larvae, we picked parasites out from midge larvae. Then we put these once-parasitized larvae into another container, separated from those un-parasitized larvae in order to avoid contamination. We would use these once-parasitized larvae only when we ran out of un-parasitized larvae. In our experiment, we didn’t use these once-parasitized larvae.
2.1.2 DNA extraction
Due to the various body sizes and weights of larvae, an average of total weight 5 mg individuals from respective types of galls and host plants were used for DNA analysis.
Detailed data for speciment are shown in Table 1 and Table 2.
Total DNA were extracted from the whole body with QIAamp DNA Mini Kit (Qiagen) according to the steps in manufacturer’s specification. A ca. 430 bp long fragment of the 12S small ribosomal subunit was PCR-amplified using the primers SR-J-14199 (50-TAC TAT GTT ACG ACT TAT-30) and SR-N-14594 (50-AAA CTA GGA TTA GAT ACC C-30) (Kambhampati and Smith, 1995). Another region of cytochrome oxidase subunit I (COI) gene of mitochondria was amplified by using the following primer pair : forward, 5’-GGA TCA CCT GAT ATA GCA TTC CC-3’ (COIS) and reverse, 5’-CCC GGT AAA ATT AAA ATA TAA ACT TC-3’ (COIA) ( Funk, 1995). All PCR mixes had a total volume of 100μl and contained 0.1mM dNTPs, 2μM of each primer, 5-10μl genomic DNA , one unit of TaqDNA polymerase (Protaq), 10μl PCR buffer comes with TaqDNA polymerase (Protaq), and add ddH2O to 100μl. The thermocycling profile consisted of initial step of 5 min at 92℃, followed by 30 cycles of 1 min at 92℃, 1 min at 52℃, and 1 min at 72
℃, with the final step of 5 min at 72℃. PCR products were electrophoresed in 2.0% TAE agarose gels along with 100bp DNA markers (violet), stained with ethidium bromide, and visualized under UV light. In some cases, the DNA band in agarose gel needs to be purified by GFX PCR DNA and gel band purification kit (Amersham Biosiciences) according to the manufacturer’s instruction. The purified DNA is amplified the same way aforementioned.
PCR products of each sample were sequenced then.
2.1.3 DNA analysis
All DNA sequence data were uploaded to SeqWeb along with 3 out-group sequences
choose from NCBI. These 3 out-group sequences are also in the order Dipteria, but in different families with our sample midges. SeqWeb is provided by NHRI and it’s also the internet surface of Winkinson Package.
Two analysis methods were used in our study, PileUp and Evolution.
Pileup function creates multiple alignments of several sequences. The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster can then be aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences can be aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments that include increasingly dissimilar sequences and clusters, until all sequences have been included in the final pairwise alignment.
Before alignment, the sequences are first clustered by similarity to produce a dendrogram, or tree representation of clustering relationships. It is this dendrogram that directs the order of the subsequent pairwise alignments.
In our analysis, the gap creation penalty is 5 and extension penalty is 1.
Evolution function investigates the relationships within a group of sequences. DNA sequences were analyzed by neighbor-joining method. (Saiton N, 1987 and UPGMA method.
The arithmetic average of evolutionary distance was computed by Kimura 2-parameter.
(Kimura, 1980). Several sequences which presumed to be in the same family were used as out groups from NCBI.
Neighbor-Joining
This method is designed to find an approximation to the minimum evolution tree for a set of aligned sequences, using less computer time than the full algorithm for determining a minimum evolution tree. It works best when the distances are additive.
The neighbor-joining method clusters the sequences in a pairwise fashion. However, instead of picking the next pair to cluster by looking for the smallest distance in the distance matrix, this method seeks to form pairs that minimize the sum of the branch lengths for the entire tree. Therefore at each round of clustering, all possible pairs of entries are considered one at a time and the sum of the branch lengths for the resulting tree is calculated. The pairing that results in the smallest sum is the one that will be used to form the new cluster. This new cluster replaces its two constituent entries in the distance matrix (reducing the dimension of the distance matrix by one), and distances are calculated between the new cluster and the remaining entries in the distance matrix. The process continues until only two entries remain.
The resulting tree is an unrooted tree. Because this method attempts to build an additive tree from the data, negative branch lengths may result if the distance data are not exactly additive
UPGMA
This method (Sneath and Sokal, Numerical Taxonomy, Freeman, San Francisco (1973)) can be used to estimate a species tree or gene tree when the expected rate of gene substitution is constant and the distance measure is linear with evolutionary time (for example, distance is measured as amino acid substitutions). The distances must be ultrametric to obtain a correct tree using this method.
The two sequences that have the smallest distance in the distance matrix are combined to form a cluster. That cluster replaces the original sequence pair as a single entry in the distance matrix (reducing the dimension of the matrix by one), and distances between the cluster and the other entries are calculated. The entries in the new matrix that have the smallest distance are combined to form a new cluster, and the process continues until only a single cluster remains.
The resulting tree is a rooted tree.
Instead of using a simple average, the UPGMA method calculates the distances between a new cluster and the other entries in the distance matrix based on the total number of sequences in the cluster. If the new cluster C was formed by combining two clusters a and b, cluster a representing N(a) total sequences and cluster b representing N(b) total sequences, the distance between the new cluster C and another entry k is:
distance(k,C) = [ distance(k,a) * N(a) + distance(k,b) * N(b) ] / (N(a) + N(b))
Kimura Two-Parameter Distance
This method applies only to nucleic acids and takes into consideration the fact that transition substitutions (purine-purine or pyrimidine-pyrimidine) often occur much more frequently than transversion substitutions (purine-pyrimidine). Gap positions and ambiguous symbols other than R (purine) and Y (pyrimidine) are not scored.
P = transitions / positions_scored Q = transversions / positions_scored
distance = -(1)/(2) ln[ (1 - 2P - Q) * sqrt(1 - 2Q) ] M. Kimura, J. Mol. Evol. 16; 111-120 (1980).
This method gives better distance estimates than the Jukes-Cantor method when the rates of transitional and transversional substitutions are different. However, when the substitution pattern is more complex than this, this method underestimates the true distance for distantly related sequences.
SeqWeb http://v8803.nhri.org.tw:8003/mgr.shtml NCBI http://www.ncbi.nlm.nih.gov/
2.2 Plant proteomics
The following plant proteomic experiments ware done by Hung-Pin Chen, my laboratory colleague. I collected and arranged his experimental data, and discuss these data together with my experimental data.
2.2.1 Sample collection:
Both plant galls and Machilus leaves are collected from Taiwan Fu-Shan Research Station (TFRI). Plant galls are commonly found on Machilus zuihoensis var. mushaensis, Machilus zuihoensis var. zuihoensis, Machilus thunbergii, Machilus japonica, Machilus japonica kusanoi, Machilus philippinense, and Hamamelidaceae in TFRI. There are also several different types of galls on each plant species as gall midge collection table shows [Table 1]. Among all types of galls and plant species, the most abundant galls are bell galls and mice galls on Machilus thunbergii, Machilus zuihoensis var. mushaensis, Machilus zuihoensis var. zuihoensis. Therefore, we choose these two types of galls on three plant species, which equal to six sample category as our material. [Fig. 4]
2.2.2 Plant Gall tissues processing
The freshly collected galls and plant stems including leaves are preserved in zipped bags and quickly sent to 4℃ refrigerator in laboratory. In no more than ten days after collection, we would cut these plant galls by dissection knife and pick out the larvae by needles. We also slice away some plant tissues which near the larvae, in order to reduce the chance of contamination. The processed plant gall tissues can be used in following experiment steps right away or preserved in -80℃ refrigerator for future use.
2.2.3 Plant Leaves processing
The healthy and qualified plant leaves including stems are preserved in zipped bags in
-80℃ refrigerator if not being used for 2-D electrophoresis right away. Preserve the stems alone with plant leaves is to maintain the freshness of leaves.
2.2.4 Plant tissue powder preparation
We adapted the method from Wang, 2003, Electrophoresis (Wei Wang, 2003). First we ground our sample tissue with liquid N2 in stainless steel mortar and pestle. Then put 0.2g dry tissue powder into 2.0ml microtubes, added 1-2ml cold acetone vortexing thoroughly for 30s.
The mixture was centrifuged at 10000x g for 3 min at 4℃. Pour out acetone and repeat the above-mentioned steps for 2-3 times. The pellet was moved into mortar and dried at room temperature. The dried tissue pellet was ground into finer powder by adding quartz sand and then transferred into new microtubes. The fine tissue powder was sequentially rinsed with 10% cold TCA/acetone 3-4 times or until the supernatant is colorless. The powder was following rinsed with 10% TCA/H2O twice and cold 80% acetone twice. The pallet was vortexed and centrifuged as above-mentioned, and dried at room temperature. The dried powder can be use at following protein extraction and be stored at -80℃ refrigerator for future use.
2.2.5 Protein extraction & assay (phenol extraction) :
The dry tissue powder was resuspended in new 2.0ml microtubes with 0.8ml phenol buffer (Tris-buffered, pH 8.0, Sigma) and 0.8ml dense SDS buffer (30% sucrose, 2% SDS, 0.1M Tris-HCl, pH 8.0, 5% 2-mecaptoethanol), it was vortexed thoroughly for 30s then centrifuged at 10000x g for 3 min. The separated upper layer phenol was removed by pipette into fresh new microtubes, and be sure not to disturb the white interface SDS complex if there appeared any. At least 5-folds volume of cold 0.1M ammonium acetate/methanol was added into the phenol phase and stored in -20℃ refrigerator for 30 min. The precipitated proteins were centrifuged at 10000x g for 5 min to recover, and were poured out the upper layer cold
ammonium/methanol. The pellet was sequentially washed twice each with cold ammonium/methanol and 80% acetone acetate. The protein precipitate was dried at room temperature and dissolve in 2-DE rehydration buffer. (8M urea, 2%CHAPS, 0.5% IPG buffer, 0.002% bromophenol blue stock solution). The concentration of protein extracts were estimated by RC DC protein assay kit (Bio Rad), following it’s manual under 750nm.
2.2.6 2D-electrophoresis
2-DE was performed by a commercially available Ettan IPGphor IEF system and Hoefer SE600 Ruby (gel size 13cm x 15cm) from Amersham. The protein extracts were separated using gel strips and formed and immobilized nonlinear pH gradient from 3-10 (Immobiline Drystrip, pH3-10NL, 13cm, Amersham). Analytical IPG strips were rehydrated for 12h at 20℃ 30V with 250μL of the rehydration buffer including 100μg of protein extracts. IEF was performed at 20℃ in the Ettan IPGphor system (Amersham) for 1h at 500 V, 1h at 1000 V, 1h at 4000 V, and 2h at 8000V. Prior to the second dimension, the strips were equilibrated for 2 x 15 min in equilibration solution containing 6M urea, 75 mM Tris-HCl (pH 8.8), 29.3% v/v glycerol, 2% SDS, 0.002% bromophenol blue. DTT (1% w/v) was added to the first equilibration solution and 2.5 w/v iodoacetamide was added to the second one. For the second dimension, the strips were transferred onto SDS polyacrylamide gels (12.5%) with a run of 50mA per gel for 4-5h at 4℃. The 2DE gels were made in triplicate and sample proteins were from two independent extractions.
2.2.7 Protein staining and analysis of 2-DE gels
After electrophoresis, proteins were visualized by a modified silver-staining kit (Yan, J.S., 2000). Digital images of the gels were obtained by using an ImageScanner and were analyzed using ImageMaster 2D v3.1 elite software (Amersham). The spots were detected and the background was subtracted (mode: average on boundary), and the 2-DE gels were aligned
and matched. A quantitative determination of the spot volumes was performed (mode: total spot volume normalization). Specific spots were described during different treatments when their volumes significantly differed (at least ten-fold in relative abundance). The interesting proteins were identified by ESI-Q-TOF-MS analysis.
2.2.8 Protein identification by MS
For MS analysis, protein spots were excised from the gel and digested with trypsin according to published procedures ( Shevchenko, 1996). 34 labeled protein spots were sent to professor Chao-Hsiung Lin’s laboratory in NYMU for Mass Spectrometric analysis. Proteins were identified by searching the protein databases NCBInr using MASCOT
(http://www.matrixscience.com). To denote a protein as unambiguously identified, the Mowse scoring algorithms were sued. Only proteins whose score exceeded the significance threshold are discussed.
3. Result
3.1 Insect gene molecular evolution
In past studies, scientists regarded cecidomyiidae midges as highly host specific species (Harris, 1994). Cecidomyiidae midges are very fragile small insects usually only 2-3 mm. in length and many are less than 1 mm long. Unlike some gall-inducing insects of other plant species, most of Cecidomyiidae midges are unknown species and it’s hard to classify them by their appearance. In order to solve this problem, we adopt the molecular classification method and use both COI and 12S mitochondria gene.
3.1.1 Gall Midge COI gene sequence alignment
In PileUp dendrogram, Distance along the vertical axis is proportional to the difference between sequences; distance along the horizontal axis has no significance at all. In fig 5, COI sequence alignment, we can see several major clusters. Since it’s PileUp function in SeqWeb, the final output dendrogram is unrooted, and branch length has no meaning. There are 35 sample sequences and 3 out-group sequences. We choose these 3 out-group sequences from NCBI, All insects, which make same type of galls were grouped together. The first is constituted by same out-group species as 12s, Asphondylia sphaera, Asphondylia gennadii, and Asphondylis itoi. Second cluster is 3 blister morphospecies, which includes mj-blister56, mo-blister76, and mt-blister36. The third cluster includes 3 bulb morphospecies, they are mjk-bulb45, mj-bulb55, and m-bulb25. The forth cluster concludes 7 bullet morphospecies, which formed 3 small groups, each are mt-bullet39, mj-bullet59, mz-bulet10, mm-bullet29, mo-bullet79, mjk-bullet49, and mp-bullet69. The fifth cluster is made of 5 mice morphospecies, they are mj-mice51, mjk-mice41, mt-mice31, mz-mice11, and mm-mice21.
The sixth cluster is mixed with 4 bell, 5 club, and 1 bird morphospecies. They are mt-bell32, mm-bell22, mjk-bell42, mt-club34, mo-club74, mp-club64, mk-bird83, mz-bell12, mj-club54,
mjk-club44. Although in total they form a big cluster, but each different gall-making midges also grouped together to form smaller clusters. The seventh cluster contains 5 spindle morphospecies, which are mp-spindle68, mt-spindle38, mm-spindle28, mz-spindle18, and mj-spindle58and. The eighth cluster is 2 bud species, mz-bud17 and mt-bud37.
3.1.2 Gall midge 12s gene sequence alignment
In fig 6, 12s sequence alignment, we can see almost all the insects which make same type of galls were also grouped together. There are 28 sample sequences, 3 out-group sequences. All the sequences were clearly divided into 7 big clusters. The first cluster is 2 bud morphospecies grouped together, mt-bud37 and mz-bud37. The second cluster includes 4 spindle morphospecies, mz-spindle28 grouped with mm-spindle28; mj-spindle58, and mp-spindle68. The third cluster is 3 out-groups downloaded from NCBI, and these are also midges in the Cecidomyiidae family. The fourth cluster includes 3 bulb morphospecies, mm-bulb25 grouped with mj-bulb55, and mz-bulb15. The fifth cluster is 5 mice morphospecies, mjk-mice41 grouped with mt-mice31; mj-mice51 grouped with mz-mice11, and mm-mice21. The only exception is group six, which contains both 3 club morphospecies and 4 bell morphospecies while other cluster only contains same midges which made same types of galls. Sequences included in the sixth cluster are mj-club54, which grouped with bell12; mt-bell32 grouped with mjk-bell42 and mm-bell22; mo-clu74 grouped with mt-club34.
The last cluster includes 7 bullet morphospecies, mt-bullet39 grouped with mj-bullet59;
mz-bullet19 grouped with mz-bullet29; mp-bullet 69 grouped with mjk-bullet49 and mo-bullet79.
3.1.3 Gall midge COI gene evolutionary tree
The “Evolution” function in Seqweb investigates the evolutionary relationships within a group of sequences. It aligns a group of sequences, create a table of pairwise distances based
on the aligned sequences, and create a tree graph representing the sequence relationships. In
on the aligned sequences, and create a tree graph representing the sequence relationships. In