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 fig 7, COI gene evolutionary tree, the bar labeled with 10.00 at the bottom is branch length unit, it is a measure unit of all branch length. It means “10 substitutions per 100 residues”.

There are 7 major clades and two independent sequences, which are mt-bud37 and mz-bud17.

These insects which make same types of galls are grouped together.

The first clade consists of 3 out-group species, Asphondylia sphaera, Asphondylia gennadii, and Asphondylis itoi. Their sequence divergences are 12.94%, 14.13% and 16.02%.

The second clade consists of all blister morphospecies, which are mj-blister56, mt-blister36, mo-blister76. Their sequence divergences are 22.50%, 19.51%, and 27.95%.

The third clade includes all spindle morphospecies, and it can be divided into two small clades. All sequence divergences in this clade range from 0.25% to 11.86%. The first small clade consist of mp-spindle68, mt-spindle38, and their sequence divergence is 0.25%. The second small clade consists of mm-spindle68, mz-spindle28, and mj-spindle58. Their sequence divergences are 0.51% and 2.05%.

The fourth clade consists of all bulb morphospecies, which are mjk-bulb45, mj-bulb55, mm-bulb25. The sequence divergences are 2.83%, 9.43%, 9.73%. The fifth clade consists of all bullet morphospecies, and it can be divided into three small clades. Sequence divergences among all sequences range from 0.51% to 6.65%. The first small clade consist of mz-bullet19, mm-bullet29, and their sequence divergence is 0.76%. The second small clade consists of mo-bullet79, mjk-bullet49, mp-bullet69, and their sequence divergences are 0.76%, 0.76%, and 0.51%. The third small clade consists of mt-bullet39, mj-bullet59, and their sequence divergence is 6.60%.

The sixth clade consists of all sequences of mice morphospecies, which are mj-mice51, mz-mice11, mjk-mice41, mt-mice31, and mm-mice21. Their sequence divergences are 0.25%, 2.58% and 2.84%. The seventh clade consists of three different gall-making midges, which are mt-bell32, mm-bell22, mjk-bell42, mz-bell12, mt-club34, mo-club74, mk-bird83,

mp-club64, mj-club54, mjk-club44. The first three sequences are totally the same, and all sequence divergences range from 0.51% to 2.84%.

3.1.4 Gall midge 12s gene evolutionary tree

Same with 3.1.3 COI evolutionary tree, we used the “Evolution” function in SeqWeb to get our result. The bar labeled with 10.00 at the bottom is branch length unit, it is a measure unit of all branch length. It means “10 substitutions per 100 residues”. In fig 8, 12s gene evolutionary tree, there are 28 sample sequences and 3 out-group sequences, witch constitute seven major clades.

The first clade is made of mt-bud37, mz-bud 17, and their sequence divergence is 0.61%. The second clade has 4 spindle sequences, mz-spindle18, mm-spindle28, mj-spindle58, mp-spindle68, the sequence divergence ranges form 0.31% to 8.12%. It can be divided into two small clades. The first clade consists of mz-spindl18, mm-spindle28, and the sequence divergence is 0.31%. The second clade consists of mj-spindle58, mp-spindle68, and the sequence divergence is 5.76%.

The third clade consisted of 3 out-group sequences, Asphondylia sphaera, Asphondylia gennadii, and Asphondylis itoi. Their sequence divergences are 14.88%, 15.89%, and 18.33%.

The fourth group consists of three bulb morphospecies, they are mm-bulb25, mj-bulb55, and mz-bulb15. Their sequence divergences are 5.83%, 12.19%, and 16.68%. The fifth clade is bullet morphospecies, which includes mt-bullet39, mj-bullet59, mp-bullet69, mjk-bullet49, mo-bulle79, mz-bullet19, and mm-bullet29. They can be divided into three small clades, and all the sequence divergence ranges from 1.92% to 13.63%. The first small clade consists of mt-bullet49, mj-bullet59, and the sequence divergence is 2.61%. The second small clade consists of mp-bullet69, mjk-bullet49, mo-bllet79, and their sequence divergences are 0.95%, 1.92%, and 2.25%.

The sixth clade consists of 5 mice morphospecies, mjk-mice41, mt-mice31, mj-mice51,

mz-mice11, and mm-mice21. Their sequence divergence ranges from 0.95% to 13.63%. It can also be divided into three small clades. The first small clade consists of mjk-mice41, mt-mice31, and the sequence divergence is 0.64%. The second small clade consists of mj-mice51, mz-mice11, and sequence divergence is 3.96%. The last clade is mm-mice21, the average sequence divergence between it among others is about 10%.

The last clade consists of bell- and club morphospecies, they are mt-bell32, mm-bell22, mjk-bell42, mo-club74, mt-club34, mj-club54, and mz-bell12. This clade can be divided further into two small clades, one consists of mt-bell32, mm-bell22, mjk-bell42, and sequence divergences range from 0.63% to 1.59%. The other group consists of mo-club74, mt-club34, mj-club54, and mz-bell12, and sequence divergences range from 2.89% to 4.87%.

3.1.5 Gall midge evolutionary relationships

Besides midge taxonomy, we also want to know the evolutionary relationships between each midge tribe, genus and species. Therefore, we use UPGMA method in evolutionary analysis and get an ultrametric tree. There are differences between NJ-tree and UPGMA-tree.

First, ultrametric trees are rooted trees while NJ-trees are unrooted. Second, ultrametric trees are rooted trees in which all the end nodes are equidistant from the root of the tree. The branch length in NJ-tree indicates the genetic change and the relationships between taxa, but the branch length of an ultrametric tree is proportional to the divergent time. Therefore, we use UPGMA-tree to analyse the phylogeny and evolutionary relationship between gall midges.

In figure 9, COI gene evolutionary tree-UPGMA, we added another two Drosophila species as out-group besides the original 3 out-groups we used, in order to identify the root of all gall midges. Generally speaking, midges in the same clade make same type of galls. The root of gall midges is in the middle of blister morphospecies and other gall-making midges. In the compounded group of gall-making midges, the node divided into mt-bud37 and other

gall-making midges, which forms one big clade. Then the big clade divided into two second big clades. One contains spindle morphospecies, mz-bud17, and out-group insects; the other clade contains bulb, bullet, mice, bell, club, and bird morphospecies. Then the bulb morphospecies divided from other gall-making midges. In the remaining clade, mt-bullet39 and mj-bullet59 divided from other gall-making midges. In the remaining clade, there are three major groups: bullet morphospecies, mice morphospecies and a last group, which contains bell, club, and bird morphospecies.

In figure 10, 12S gene evolutionary tree-UPGMA, we also added two Drosophila species as additional out-groups in order to determine the root of gall midges. The outcome is as same as COI gene evolutionary tree-UPGMA, midges in the same clade make same type of galls. The root, which means the ancestral species, diversified into two clades. The clade at right contains bud and spindle-making midges; the clade at left contains other gall making midges and the original out-groups. In the left clade, out-group species diversified first, then diversified bulb morphospecies. In the remaining clade, which contains mice, bell, club, and bullet morphospecies, divided into two little clades. The little clade at left contains all bullet morphospecies. The little clade at right divided into another two little clades, one is mice morphospecies; the other contains bell and club gall making midges.

3.1.6 Gall midge larvae morphology anatomy and gene analysis

In Dr. Tung’s research in TFRI, the anatomical structures of all midge larvae can be classified into 4 types according to their spatula and anus. (Table 3, Table 4) In type I, there are two salient parts on larva’s spatula and no terminal papillae on anus. In type II, the button part of larva’s spatula is healed but the top of spatula is crotched. The shape of anus looks like a peach. In type III, the spatula shaped like chisel, and there are terminal papillae on anus. In type IV, the spatula is shorter than other type’s spatula, but the middle part of it is bigger. The anus forms two segments but there is only one segment has papillae. Each larva in each type

of galls can be classified into one type.

Among all four types of larvae, the type one larvae are the largest group. It contains all bulb-, bullet-, mice-, bell-, club-, and bird-gall-making-midges. Type two larvae only contains mt-bud37 midge. Type three larvae include mz-bud17, and all blister morphospecies. Type four larvae contain all spindle morphospecies.

We combine this morphological anatomy result with gene sequence analysis, (fig 5-8) and we also put sketches of gall types on this diagram.

From these diagrams, we can clearly see that midges with same anatomical structures were grouped together. In previous experiments, we know that midges which made same types of galls are closer species, or even same species. Therefore, the anatomical structures are the same among midges which made same types of galls.

3.2 Plant Proteomics

These 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.

In order to analyse the impacts on which gall-inducing insects cause to plants, we used 2-dimensional electrophoresis technique to study the protein changes and differences between gall tissues, galled leaves, and un-galled leaves. First, we compared galled and ungalled leaves to determine whether gall-inducing insect caused damage to health leaves or not.

Second, we compared the protein pattern differences between three Machilus leaves for excluding the original differences existed between different Machilus leaves at further comparisons. Third, we compared the protein pattern difference between two types of galls on single Machilus leaves to understand whether theses two types of galls caused different impacts or not. Then, we compared same type of galls but grow on different Machilus. At last, we compared two types of gall-tissues and Machilus leaf tissues on each Machilus in order to figure out the common protein differences between gall tissues and leaf tissues. After all the analyses of protein patterns, we combined the results of gall-insect taxonomy and discussed together.

3.2.1 Comparison of galled and ungalled leaves

In figure 12, (A) is 2D image of protein pattern of healthy leaf tissues and (B) is protein pattern of galled leaf tissues. Both samples are from Machilus zuihoensis var. mushaensis (MM). Healthy leaf tissue means that there are no galls or other damages on leaf surface.

Galled leaf means that there are galls on leaf surface, but we took the ungalled part of leaf tissue.

3.2.2 Protein pattern differences between three Machilus leaves

After comparing protein differences between galled and ungalled leaves, we compared the protein pattern differences among three Machilus leaves. Botanists regard these three Machilus as different species, and we want to know whether there are protein differences between them. Then we can exclude these difference proteins among gall tissues on different Machilus, because these proteins are original differences between leaves.

In figure 13, (A) is the 2D image of Machilus zuihoensis var. zuihoensis. (MZ) leaf tissues (B) is 2D image of Machilus zuihoensis var. mushaensis.(MM) leaf tissues (C) is leaf tissues of Machilus thunbergii.(MT) Labeled protein spots of the gels are those differentially expressed by at least ten-fold in comparison with each other. We classified these three Machilus leaves into three groups, (A) and (B), (B) and (C), (A) and (C), then we compared leaf protein patterns with each other in each group.

As figure 13 shows, in group (A) (B), the different proteins are protein No. 1. in MT-leaf tissue (A), and protein No. 2, 3, 4, 5 in MM-leaf tissues (B). There are total 5 different proteins in this group.

In group (A) (C), the different proteins are protein No. 6-11 in MT-leaf tissues (A) and protein No. 12-25 in MZ-leaf tissues (C). There are total 20 different proteins in this group.

In group (B) (C), the different proteins are protein No.2, 4, 5, 6-11 in MM-leaf tissues (B) and protein No. 1, 12-25 in MZ-leaf tissues (C). There are total 24 different proteins in this group.

In figure 14, we made a three-circle graph of original protein differences among three Machilus leaves. One circle represents one Machilus species, and in each circle are their own proteins. We can clearly see that the protein differences between MZ and MM leaves are smaller than the differences between MM-MT, and MZ-MT. The protein patterns of MZ and MM leaves are alike, there are 6 proteins in both MZ and MM leaves which don’t appear in MT leaves (protein 6-11). There are 14 proteins, which only appear in MT leaves (protein 12-25). Protein 2, 4, and 5 only appear in MM leaves. There are no distinctive proteins which

only appear in MZ leaves.

3.2.3 Protein pattern differences between two types of gall on single Machilus leaves In this experiment, we analyzed the protein pattern differences between two types of gall on single Machilus leaves. In figure 15, each graph are 2D images of our samples.

Samples were classified into three groups according to three Machilus species: (D) and (G), (E) and (H), (F) and (I). There are two types of galls in each group, bell gall and mice gall. (D) is bell-gall tissues of Machilus zuihoensis var. zuihoensis. (MZ-bell). (E) is bell-gall tissues of Machilus zuihoensis var. mushaensis. (MM-bell). (F) is bell-gall tissues of Machilus thunbergii. (MT-bell) (G) is mice tissues of Machilus zuihoensis var. zuihoensis. (MZ-mice).

(H) is mice-gall tissues of Machilus zuihoensis var. mushaensis. (MM-mice). (I) is mice-gall tissues of Machilus thunbergii. (MT-mice).The protein spots labeled are those who differentially expressed by at least ten-fold in comparison with each other.

In group (D) (G), bell and mice galls on MZ, the different proteins are protein No. 34, 104, 105 in MZ-bell (D) and protein No. 29, 70, 71 in MZ-mice (G). There are total 6 different proteins in this group.

In group (E) (H), bell and mice galls on MM, the different proteins are protein No. 5, 37, 70 in MM-bell (E) and protein No. 34, 46 in MM-mice (H). There are total 5 different proteins in this group.

In group (F) (I), bell and mice galls on MZ, the different proteins are protein No. 65, 95, 102 in MT-mice (I), and there are no special protein in MT-bell (F). There are total 3 different proteins in this group.

As the result shows, there are little protein pattern differences between bell and mice galls, which grow on same Machilus species.

3.2.4 Protein pattern differences between bell galls on three Machilus leaves

Next, we compared the protein pattern difference between bell galls on three Machilus leaves in order to see whether Machilus species affects gall tissues proteins. The bell-galls of three Machilus were classified into three groups, (D) and (E), (D) and (F), (E) and (F). In Fig.

16 and table 9, the bell-gall protein patterns were compared with each other. (D) is bell gall tissue of Machilus zuihoensis var. zuihoensis. (MZ-bell) (E) is bell gall tissues of Machilus zuihoensis var. mushaensis.(MM-bell). (F) is bell gall tissues of Machilus thunbergii.

(MT-bell). Labeled protein spots of the gels are those differentially expressed by at least ten-fold in comparison with each other.

In group (D) (E), bell galls on MZ and MM, the different proteins are protein No. 34, 104, 105 in MZ-bell (D), and protein No, 29, 70, 71 in MM-bell (E). There are total 6 different proteins between bell gall tissues on MZ and MM.

In group (D) (F), bell galls on MZ and MT, the different proteins are protein No. 1, 11, 31, 32, 34, 37, 42, 84, 104, 105 in MZ-bell (D) and protein No. 22, 70, 92, 94, 103, 106 in MT-bell. There are total 16 different proteins between bell gall tissues on MZ and MT.

In group (E) (F), bell galls on MM and MT, the different proteins are protein No. 1, 11, 32, 37, 42, 71, 84, 87 in MM-bell (E) and protein No. 2, 92, 94, 103, 106 in MT-bell (F).

There are total 13 different proteins between bell gall tissues on MM and MT.

There are total 13 different proteins between bell gall tissues on MM and MT.