Difference between revisions of "Team:NYMU-Taipei/Notebook-Lab Book-fruit flies hemolymph"

Latest revision as of 20:13, 19 October 2016

Overview

This section of our wiki contains the protocols for our insect experiments, including cultivation, infection rate tests, and hemolymph extraction.

Related experiment schedule & Lab note

Experiments

Experiments related to B. dorsalis	Number	Date
Diameter of the tunnel that allows passage to one B. dorsalis	1	8/30～9/1
Infecting B. dorsalis with non-designed M. anisopliae	2-1	9/30~10/7
infecting female B. dorsalis by copulation with infected male B. dorsalis	2-2	10/10~10/17

Experiments related to B. dorsalis	Number	Date
Diameter of the tunnel that allows passage to one B. dorsalis	1	8/30～9/1
Infecting B. dorsalis with non-designed M. anisopliae	2-1	9/30~10/7
infecting female B. dorsalis by copulation with infected male B. dorsalis	2-2	10/10~10/17

Experiments about hemolymph extraction	Number	Date
Blatta lateralis	1	9/20~9/30
Bactrocera dorsalis	2	9/29~10/5
Bombyx mori	3	10/3~10/

Experiments about hemolymph extraction	Number	Date
Blatta lateralis	1	9/20~9/30
Bactrocera dorsalis	2	9/29~10/5
Bombyx mori	3	10/3~10/

Routine works

Routine tasks of feeding Bactrocera dorsalis	Date
Produce & change the tube with the specific agar	8/24~9/20
Produce & change water gel and the specific ratio fodder	9/22~10/

Routine tasks of feeding Bactrocera dorsalis	Date
Produce & change the tube with the specific agar	8/24~9/20
Produce & change water gel and the specific ratio fodder	9/22~10/

Routine tasks of hemolymph extraction	Date
Collect the mulberry leaves to feed the silkworm	10/3~10/
Sterilize the related equipments	9/20~10/

Routine tasks of hemolymph extraction	Date
Collect the mulberry leaves to feed the silkworm	10/3~10/
Sterilize the related equipments	9/20~10/

[Collapse all] | [Expand all]

8/23

Oriental fruit flies sent to lab and cultivated by large transparent box to avoid oriental fruit flies flying out.

[Collapse]

8/24

We used both of centrifuge tube and feeding box to feed oriental fruit flies for comparing and ensuring the status of oriental fruit flies that established the independent space for observing oriental fruit flies.

[Collapse]

8/25

B. dorsalis started to break through the cocoon. We conducted to put B. dorsalis to centrifuge tubes, avoiding the feeding box cannot load all oriental fruit flies. We poured the special agar to centrifuge tubes, and put centrifuges into entrance to catch oriental fruit flies. Then we sealed the centrifuge tubes by cotton.

(The team members put agar into centrifuge tubes which would be their feed.)

(The left picture is that oriental fruit flies broke through cocoon; the right picture is the feeding box & centrifuge tubes where fed oriental fruit flies.)

[Collapse]

8/30

First time to Conducting Experiment 1

[Collapse]

9/3

Changing the centrifuge tubes where oriental fruit flies lived. (Avoid the environment being mess.) /p>

[Collapse]

9/5

Changing the centrifuge tubes where oriental fruit flies lived.

[Collapse]

9/6

The oriental fruit flies in centrifuge tubes are dead quickly, so we conducted trouble shooting and sent the mail to professors who research oriental fruit flies.

We took the trap to Beitou orchard to try to catch oriental fruit flies.

[Collapse]

9/7

Second time to Conducting Experiment 1.

Conducting trouble shooting.

[Collapse]

9/10

Searching the store selling the silkworm for extracting hemolymph which is used to induce pMcl1.

[Collapse]

9/13

Visiting two farmers working in the orchard to ask the information about oriental fruit flies in the nature environment, and installing the trap of catching oriental fruit flies.

(Left picture: the team member install the trap; Right picture: the farmer and our team member took a picture)

Sent the mail to researchers to ask for making public the content of the mail.

[Collapse]

9/15

Communicating with Chang Ming ecological education sericulture, asking the information of buying silkworm.

[Collapse]

9/16

Picking up the oriental fruit fly larval from rotten guavas and feeding them.

Left picture: the team members were picking up the oriental fruit fly larval from rotten guavas; Right picture: the larval have picked up and put into another box. Expected becoming pupae after six days.

[Collapse]

9/22

For the situation that our fruit flies in the centrifuge tubes were dead quickly , so we visited Dr. Yu-Bing Huang in Taiwan Agricultural Research Institute, Applied Zoology Division, and asking the problems about the right way for feeding oriental fruit flies. We change our way of feeding oriental fruit flies.

[Collapse]

9/23

Producing new feeding box which is larger and ventilation, and design the experimental boxes for Experiment 2: Infecting B. dorsalis with non-designed M. anisopliae.

(Left picture: experimental boxes; Right picture: the internal space of feeding box)

[Collapse]

9/30

The new oriental fruit flies broke through cocoon in the new feeding box

the new way for feeding oriental fruit flies is using gauze to be the windows that could be ventilation, and we can put watered agar and artificial feed on the top of gauze, then there were no water and feed in the box which lead to the environment being mess, and they also could have enough water and feed.

Infecting B. dorsalis with non-designed M. anisopliae started to conduct.

Photos shows three concentration condition and negative control environment of experiment "Infecting B. dorsalis with non-designed M. anisopliae"

[Collapse]

10/1

Keeping on Experiment 2-1 (9/30~10/7)

[Collapse]

10/7

Experiment 2-1 finished.

[Collapse]

10/10

Experiment 2-2 "infecting female B. dorsalis by copulation with infected male B. dorsalis" started to conduct.

[Collapse]

Q&A SectionHow to CULTURE oriental fruit flies?
	The oriental fruit flies can grow in a transparent large container and centrifuge tubes with a piece of cotton placed at the opening to prevent them from escaping while still allowing air exchange.
	Feeding on sucrose-peptone agar (sited by ATCC) when you choose the centrifuge tubes as their living environment; a ratio of composition below is the way to feed the fruit flies in a larger container. The larval need the other formula to grow up whether living in the centrifuge tubes or the larger transparent container. The adult feeding formula without peptone can also alive the oriental fruit flies but not breeding.
                 Larva formula                                            Adult formula ratio
	Yeast extraction	35g                       Sucrose: yeast extraction: peptone
	Sucrose			60g                       3:1:1
	Oat		                120g
	Sodium benzoate	1g
	Hydrochloric acid	4.5ml, 37%
	Water			450ml
	The advantage of cultivating in large container is avoiding the tight space that oriental fruit flies can almost only walk and then causing the growth medium spoil easily. On the other hand, cultivating the fruit flies in the centrifuge tubes can easily distinguish the situation of the oriental fruit flies, for example, gender, health, quantity to take the correct actions.
How to prevent COMTAMINATION?
Adding preservatives, for instance, sodium benzoate, and decreasing environmental humidity can decease the growth of the mold on agar.
Sterilize cotton according common lab procedure or just buy pre-sterilized one.

How do you KEEP the oriental fruit flies in place during experiments?
Using carbon dioxide gas to induce comatose and try your best to contain the gas within the tube and the oriental fruit flies should be unconscious after 10-20 seconds.
Induce coma under low temperature.

How about the methyl eugenol? Are there any suggestions on attracting the male oriental fruit flies or the actual situation that farmer using it to prevent the oriental fruit fly pest?
It is best to complete experiments using methyl eugenol before 2:00 PM.
Several time experiments may cause the male oriental fruit flies became desensitized to methyl eugenol, so it's important to change the sample you test on the experiments routinely to avoid the factor to interfere the result.
The attraction traps putting in the orchard need to add pure and undiluted methyl eugenol to capture the oriental fruit flies. It also needs some cottons or gauzes to separate the oriental fruit flies and methyl eugenol to prevent the chain of fruit flies and eventually causing the death.
It's not a good idea to set the trap when (a) it's a rainy day (b) it's not the harvest season of the guava or tangerine, or the number of the fruit flies captured will not high.

AcknowledgementsBeitou orchard
S. J. Chen 	Grower
Beitou orchard
Z. L. Liao		Grower
Bioresource Collection and Research Center
G. Y. Liou		Fellow
Chiayi Agricultural Experiment Branch
P. H. Chen	Research assistant
Kaohsiung District Agricultural Research and Extension Station, COA, EY
M. N. Tseng	Research director
National Chung Hsing University, the Department of Entomology
S. Y. Hwang	Professor
National Chung Hsing University, the Department of Entomology
Y. Y. Tseng		Research assistant
Taiwan Agricultural Research Institute, Applied Zoology Division
M. Y. Chiang	Research assistant
Taiwan Agricultural Research Institute, Applied Zoology Division
Y. B. Huang	Professor
/p>

Overview

This section of our wiki contains the protocols for our insect experiments, including cultivation, infection rate tests, and hemolymph extraction.

Related experiment schedule & Lab note

Experiments

Experiments related to B. dorsalis	Number	Date
Diameter of the tunnel that allows passage to one B. dorsalis	1	8/30～9/1
Infecting B. dorsalis with non-designed M. anisopliae	2-1	9/30~10/7
infecting female B. dorsalis by copulation with infected male B. dorsalis	2-2	10/10~10/17

Experiments about hemolymph extraction	Number	Date
Blatta lateralis	1	9/20~9/30
Bactrocera dorsalis	2	9/29~10/5
Bombyx mori	3	10/3~10/

Routine works

Routine tasks of feeding Bactrocera dorsalis	Date
Produce & change the tube with the specific agar	8/24~9/20
Produce & change water gel and the specific ratio fodder	9/22~10/

Routine tasks of hemolymph extraction	Date
Collect the mulberry leaves to feed the silkworm	10/3~10/
Sterilize the related equipments	9/20~10/

[Collapse all] | [Expand all]

8/23

Oriental fruit flies sent to lab and cultivated by large transparent box to avoid oriental fruit flies flying out.

[Collapse]

8/24

We used both of centrifuge tube and feeding box to feed oriental fruit flies for comparing and ensuring the status of oriental fruit flies that established the independent space for observing oriental fruit flies.

[Collapse]

8/25

B. dorsalis started to break through the cocoon. We conducted to put B. dorsalis to centrifuge tubes, avoiding the feeding box cannot load all oriental fruit flies. We poured the special agar to centrifuge tubes, and put centrifuges into entrance to catch oriental fruit flies. Then we sealed the centrifuge tubes by cotton.

(The team members put agar into centrifuge tubes which would be their feed.)

(The left picture is that oriental fruit flies broke through cocoon; the right picture is the feeding box & centrifuge tubes where fed oriental fruit flies.)

[Collapse]

8/30

First time to Conducting Experiment 1

[Collapse]

9/3

Changing the centrifuge tubes where oriental fruit flies lived. (Avoid the environment being mess.) /p>

[Collapse]

9/5

Changing the centrifuge tubes where oriental fruit flies lived.

[Collapse]

9/6

The oriental fruit flies in centrifuge tubes are dead quickly, so we conducted trouble shooting and sent the mail to professors who research oriental fruit flies.

We took the trap to Beitou orchard to try to catch oriental fruit flies.

[Collapse]

9/7

Second time to Conducting Experiment 1.

Conducting trouble shooting.

[Collapse]

9/10

Searching the store selling the silkworm for extracting hemolymph which is used to induce pMcl1.

[Collapse]

9/13

Visiting two farmers working in the orchard to ask the information about oriental fruit flies in the nature environment, and installing the trap of catching oriental fruit flies.

(Left picture: the team member install the trap; Right picture: the farmer and our team member took a picture)

Sent the mail to researchers to ask for making public the content of the mail.

[Collapse]

9/15

Communicating with Chang Ming ecological education sericulture, asking the information of buying silkworm.

[Collapse]

9/16

Picking up the oriental fruit fly larval from rotten guavas and feeding them.

Left picture: the team members were picking up the oriental fruit fly larval from rotten guavas; Right picture: the larval have picked up and put into another box. Expected becoming pupae after six days.

[Collapse]

9/22

For the situation that our fruit flies in the centrifuge tubes were dead quickly , so we visited Dr. Yu-Bing Huang in Taiwan Agricultural Research Institute, Applied Zoology Division, and asking the problems about the right way for feeding oriental fruit flies. We change our way of feeding oriental fruit flies.

[Collapse]

9/23

Producing new feeding box which is larger and ventilation, and design the experimental boxes for Experiment 2: Infecting B. dorsalis with non-designed M. anisopliae.

(Left picture: experimental boxes; Right picture: the internal space of feeding box)

[Collapse]

9/30

The new oriental fruit flies broke through cocoon in the new feeding box

the new way for feeding oriental fruit flies is using gauze to be the windows that could be ventilation, and we can put watered agar and artificial feed on the top of gauze, then there were no water and feed in the box which lead to the environment being mess, and they also could have enough water and feed.

Infecting B. dorsalis with non-designed M. anisopliae started to conduct.

Photos shows three concentration condition and negative control environment of experiment "Infecting B. dorsalis with non-designed M. anisopliae"

[Collapse]

10/1

Keeping on Experiment 2-1 (9/30~10/7)

[Collapse]

10/7

Experiment 2-1 finished.

[Collapse]

10/10

Experiment 2-2 "infecting female B. dorsalis by copulation with infected male B. dorsalis" started to conduct.

[Collapse]

Q&A SectionHow to CULTURE oriental fruit flies?
	The oriental fruit flies can grow in a transparent large container and centrifuge tubes with a piece of cotton placed at the opening to prevent them from escaping while still allowing air exchange.
	Feeding on sucrose-peptone agar (sited by ATCC) when you choose the centrifuge tubes as their living environment; a ratio of composition below is the way to feed the fruit flies in a larger container. The larval need the other formula to grow up whether living in the centrifuge tubes or the larger transparent container. The adult feeding formula without peptone can also alive the oriental fruit flies but not breeding.
Larva formula                                            
Yeast extraction	35g
Sucrose			60g
Oat		                120g
Sodium benzoate	1g
Hydrochloric acid	4.5ml, 37%
Water			450ml

Adult formula ratio
Sucrose: yeast extraction: peptone
3:1:1
	The advantage of cultivating in large container is avoiding the tight space that oriental fruit flies can almost only walk and then causing the growth medium spoil easily. On the other hand, cultivating the fruit flies in the centrifuge tubes can easily distinguish the situation of the oriental fruit flies, for example, gender, health, quantity to take the correct actions.
How to prevent COMTAMINATION?
Adding preservatives, for instance, sodium benzoate, and decreasing environmental humidity can decease the growth of the mold on agar.
Sterilize cotton according common lab procedure or just buy pre-sterilized one.

How do you KEEP the oriental fruit flies in place during experiments?
Using carbon dioxide gas to induce comatose and try your best to contain the gas within the tube and the oriental fruit flies should be unconscious after 10-20 seconds.
Induce coma under low temperature.

How about the methyl eugenol? Are there any suggestions on attracting the male oriental fruit flies or the actual situation that farmer using it to prevent the oriental fruit fly pest?
It is best to complete experiments using methyl eugenol before 2:00 PM.
Several time experiments may cause the male oriental fruit flies became desensitized to methyl eugenol, so it's important to change the sample you test on the experiments routinely to avoid the factor to interfere the result.
The attraction traps putting in the orchard need to add pure and undiluted methyl eugenol to capture the oriental fruit flies. It also needs some cottons or gauzes to separate the oriental fruit flies and methyl eugenol to prevent the chain of fruit flies and eventually causing the death.
It's not a good idea to set the trap when (a) it's a rainy day (b) it's not the harvest season of the guava or tangerine, or the number of the fruit flies captured will not high.

Is the common characteristic among the hemolymph of Bactrocera dorsalis and other insects sufficiently confirmed the accuracy of the experiment result?

Can the designed M. anisopliae actually invade B. dorsalis?

AcknowledgementsBeitou orchard
S. J. Chen 	Grower
Beitou orchard
Z. L. Liao		Grower
Bioresource Collection and Research Center
G. Y. Liou		Fellow
Chiayi Agricultural Experiment Branch
P. H. Chen	Research assistant
Kaohsiung District Agricultural Research and Extension Station, COA, EY
M. N. Tseng	Research director
National Chung Hsing University, the Department of Entomology
S. Y. Hwang	Professor
National Chung Hsing University, the Department of Entomology
Y. Y. Tseng		Research assistant
Taiwan Agricultural Research Institute, Applied Zoology Division
M. Y. Chiang	Research assistant
Taiwan Agricultural Research Institute, Applied Zoology Division
Y. B. Huang	Professor
/p>

@@ Line 12: / Line 12: @@
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+  margin-left: auto;
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+  list-style-type: disc;
+}
+div.thelanguage img {
+   width: 100%;
+}
+.openlanguage{ /*class added to contents of 2nd demo when they are open*/
+color: #666666;
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@@ Line 61: / Line 115: @@
    font-size: 18px !important;
    text-decoration:none
+}
 }
@@ Line 109: / Line 164: @@
 <body>
-<div id="wrap">
+   <div class="prototypeprototypesp">
+        <div class="imageimage00">
+            <img src="https://static.igem.org/mediawiki/2016/1/17/%E5%88%86%E9%A0%81_labnote_%E5%8A%A0%E5%AD%97%E7%89%88_labnote.jpg" width="100%" height="100%" />
+        </div>
+    </div>
-<div class="imageimage1">
+    <div class="prototypeprototype">
-<div class="wordword1">
+        <div class="imageimage00">
+            <img src="https://static.igem.org/mediawiki/2016/1/17/%E5%88%86%E9%A0%81_labnote_%E5%8A%A0%E5%AD%97%E7%89%88_labnote.jpg" width="100%" height="100%" />
-<div class="satisfy">
+        </div>
-<h1>Lab note</h1></div>
+    </div>
-<div class="satisfysp">
-<h1>Lab note</h1></div>
-<hr />
-</div></div><br />
+<div class="prototypeprototype">
+<div id="wrap">
 <div class="fund">
 <h2 style="margin-top:30px; margin-bottom:10px;">Overview</h2><hr>
@@ Line 194: / Line 250: @@
 　</tr>
 　<tr>
-　    <td style="width:600px;">Bactro</td>
+　    <td style="width:600px;">Bactrocera dorsalis</td>
 　    <td style="width:150px;">2</td>
 　    <td style="width:150px;">9/29~10/5</td>
@@ Line 218: / Line 274: @@
 　</tr>
 　<tr>
-　    <td style="width:600px;">Bactro</td>
+　    <td style="width:600px;">Bactrocera dorsalis</td>
 　    <td style="width:150px;">2</td>
 　    <td style="width:150px;">9/29~10/5</td>
@@ Line 308: / Line 364: @@
 <div class="technology">8/23</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Oriental fruit flies sent to lab and cultivated by large transparent box to avoid oriental fruit flies flying out.</p></li>
+<li><p style="font-size: 16px;">Oriental fruit flies sent to lab and cultivated by large transparent box to avoid oriental fruit flies flying out.</p></li>
 <div class="imageimage">
@@ Line 320: / Line 376: @@
 <div class="technology">8/24</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">We used both of centrifuge tube and feeding box to feed oriental fruit flies for comparing and ensuring the status of oriental fruit flies that established the independent space for observing oriental fruit flies.</p></li>
+<li><p style="font-size: 16px;">We used both of centrifuge tube and feeding box to feed oriental fruit flies for comparing and ensuring the status of oriental fruit flies that established the independent space for observing oriental fruit flies.</p></li>
 <div class="imageimage">
@@ Line 339: / Line 395: @@
 <div class="technology">8/25</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">B. dorsalis started to break through the cocoon. We conducted to put B. dorsalis to centrifuge tubes, avoiding the feeding box cannot load all oriental fruit flies. We poured the special agar to centrifuge tubes, and put centrifuges into entrance to catch oriental fruit flies. Then we sealed the centrifuge tubes by cotton. </li></p>
+<li><p style="font-size: 16px;">B. dorsalis started to break through the cocoon. We conducted to put B. dorsalis to centrifuge tubes, avoiding the feeding box cannot load all oriental fruit flies. We poured the special agar to centrifuge tubes, and put centrifuges into entrance to catch oriental fruit flies. Then we sealed the centrifuge tubes by cotton. </li></p>
 <div class="imageimage">
@@ Line 346: / Line 402: @@
 </div>
-<p style="font-size: 18px;">(The team members put agar into centrifuge tubes which would be their feed.)</p>
+<p style="font-size: 16px;">(The team members put agar into centrifuge tubes which would be their feed.)</p>
 <div class="imageimage">
@@ Line 355: / Line 411: @@
 </div>
-<p style="font-size: 18px;">(The left picture is that oriental fruit flies broke through cocoon; the right picture is the feeding box & centrifuge tubes where fed oriental fruit flies.)</p>
+<p style="font-size: 16px;">(The left picture is that oriental fruit flies broke through cocoon; the right picture is the feeding box & centrifuge tubes where fed oriental fruit flies.)</p>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 2); return false">[Collapse]</a></p>
@@ Line 362: / Line 418: @@
 <div class="technology">8/30</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">First time to Conducting Experiment 1</p></li>
+<li><p style="font-size: 16px;">First time to Conducting Experiment 1</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 3); return false">[Collapse]</a></p>
@@ Line 369: / Line 425: @@
 <div class="technology">9/3</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Changing the centrifuge tubes where oriental fruit flies lived. (Avoid the environment being mess.) /p></li>
+<li><p style="font-size: 16px;">Changing the centrifuge tubes where oriental fruit flies lived. (Avoid the environment being mess.) /p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 4); return false">[Collapse]</a></p>
@@ Line 376: / Line 432: @@
 <div class="technology">9/5</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Changing the centrifuge tubes where oriental fruit flies lived.</p></li>
+<li><p style="font-size: 16px;">Changing the centrifuge tubes where oriental fruit flies lived.</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 5); return false">[Collapse]</a></p>
@@ Line 383: / Line 439: @@
 <div class="technology">9/6</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">The oriental fruit flies in centrifuge tubes are dead quickly, so we conducted trouble shooting and sent the mail to professors who research oriental fruit flies.</p></li>
+<li><p style="font-size: 16px;">The oriental fruit flies in centrifuge tubes are dead quickly, so we conducted trouble shooting and sent the mail to professors who research oriental fruit flies.</p></li>
-<li><p style="font-size: 18px;">We took the trap to Beitou orchard to try to catch oriental fruit flies. </p></li>
+<li><p style="font-size: 16px;">We took the trap to Beitou orchard to try to catch oriental fruit flies. </p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 6); return false">[Collapse]</a></p>
@@ Line 391: / Line 447: @@
 <div class="technology">9/7</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Second time to Conducting Experiment 1.</p></li>
+<li><p style="font-size: 16px;">Second time to Conducting Experiment 1.</p></li>
-<li><p style="font-size: 18px;">Conducting trouble shooting.</p></li>
+<li><p style="font-size: 16px;">Conducting trouble shooting.</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 7); return false">[Collapse]</a></p>
@@ Line 399: / Line 455: @@
 <div class="technology">9/10</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Searching the store selling the silkworm for extracting hemolymph which is used to induce pMcl1.</p></li>
+<li><p style="font-size: 16px;">Searching the store selling the silkworm for extracting hemolymph which is used to induce pMcl1.</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 8); return false">[Collapse]</a></p>
@@ Line 406: / Line 462: @@
 <div class="technology">9/13</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Visiting two farmers working in the orchard to ask the information about oriental fruit flies in the nature environment, and installing the trap of catching oriental fruit flies.</p></li>
+<li><p style="font-size: 16px;">Visiting two farmers working in the orchard to ask the information about oriental fruit flies in the nature environment, and installing the trap of catching oriental fruit flies.</p></li>
-<p style="font-size: 18px;">(Left picture: the team member install the trap; Right picture: the farmer and our team member took a picture)</p>
+<p style="font-size: 16px;">(Left picture: the team member install the trap; Right picture: the farmer and our team member took a picture)</p>
 <div class="imageimage">
 <div style="position:relative; width:50%; margin-right:10%;">
-<img src="https://static.igem.org/mediawiki/2016/2/2b/Vejvlkevblkemblemb.jpg" width="100%" /></div>
+<img src="https://static.igem.org/mediawiki/2016/d/d5/T--NYMU-Taipei--photo-OFF-related-14740977_120300000687733839_596021223_n.jpg" width="100%" /></div>
 <div style="position:relative; width:90%; margin-top:10%;">
 <img src="https://static.igem.org/mediawiki/2016/7/74/T--NYMU-Taipei--photo-OFF-related-14741635_120300000673924526_1894584987_n.jpg" width="100%" /></div>
 </div>
-<li><p style="font-size: 18px;">Sent the mail to researchers to ask for making public the content of the mail.</p></li>
+<li><p style="font-size: 16px;">Sent the mail to researchers to ask for making public the content of the mail.</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 9); return false">[Collapse]</a></p>
@@ Line 424: / Line 480: @@
 <div class="technology">9/15</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Communicating with Chang Ming ecological education sericulture, asking the information of buying silkworm.</p></li>
+<li><p style="font-size: 16px;">Communicating with Chang Ming ecological education sericulture, asking the information of buying silkworm.</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 10); return false">[Collapse]</a></p>
@@ Line 431: / Line 487: @@
 <div class="technology">9/16</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">Picking up the oriental fruit fly larval from rotten guavas and feeding them.</p></li>
+<li><p style="font-size: 16px;">Picking up the oriental fruit fly larval from rotten guavas and feeding them.</p></li>
-<li><p style="font-size: 18px;">Left picture: the team members were picking up the oriental fruit fly larval from rotten guavas; Right picture: the larval have picked up and put into another box.   Expected becoming pupae after six days. </p></li>
+<li><p style="font-size: 16px;">Left picture: the team members were picking up the oriental fruit fly larval from rotten guavas; Right picture: the larval have picked up and put into another box.   Expected becoming pupae after six days. </p></li>
 <div class="imageimage">
-<div style="position:relative; width:98%;">
+<div style="position:relative; width:48%;">
 <img src="https://static.igem.org/mediawiki/2016/f/f9/T--NYMU-Taipei--photo-OFF-related-14699910_120300000674856561_1048345374_n.jpg" width="100%" /></div>
-<div style="position:relative; width:98%;">
+<div style="position:relative; width:48%;">
 <img src="https://static.igem.org/mediawiki/2016/e/e4/T--NYMU-Taipei--photo-OFF-related-14627744_120300000676522237_1703903936_n.jpg" width="100%" /></div>
 </div>
@@ Line 444: / Line 500: @@
 </div>
-<div class="technology">Week13</div>
+<div class="technology">9/22</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">9/11: Standard part PgpdA amplification PCR with KOD polymerase</p></li>
+<li><p style="font-size: 16px;">For the situation that our fruit flies in the centrifuge tubes were dead quickly , so we visited Dr. Yu-Bing Huang in Taiwan Agricultural Research Institute, Applied Zoology Division, and asking the problems about the right way for feeding oriental fruit flies. We change our way of feeding oriental fruit flies. </p></li>
-<li><p style="font-size: 18px;">9/11: pBARGPE1 digested for pBAR_PgpdA_EL222_TtrpC construction</p></li>
-<li><p style="font-size: 18px;">9/11: TtrpC KOD PCR amplification</p></li>
-<li><p style="font-size: 18px;">9/11: TtrpC KOD PCR amplification</p></li>
-<li><p style="font-size: 18px;">9/11: pBAR_PgpdA_EL222_TtrpC containing culture used in 3 in 1</p></li>
-<li><p style="font-size: 18px;">9/11: pBAR_KR transformed into competent cell</p></li>
-<li><p style="font-size: 18px;">9/11: pBAR_KR transformed into competent cell</p></li>
-<li><p style="font-size: 18px;">9/12: pBAR_KR performed plasmid extraction and plasmid PCR (successful)</p></li>
-<li><p style="font-size: 18px;">9/12: pBAR_PgpdA_EL222_TtrpC checked with restriction digestion</p></li>
-<li><p style="font-size: 18px;">9/12: pBAR_PgpdA_EL222_TtrpC amplified with plasmid PCR</p></li>
-<li><p style="font-size: 18px;">9/12: Successfully constructed the plasmid pBAR_PgpdA_EL222_TtrpC</p></li>
-<li><p style="font-size: 18px;">9/12: Standard part PgpdA mutation PCR (mutated PstI)</p></li>
-<li><p style="font-size: 18px;">9/12: Standard part PMCL1 amplification PCR with KOD polymerase</p></li>
-<li><p style="font-size: 18px;">9/13: Standard Part PMCL1 mutation PCR (mutated SpeI) </p></li>
-<li><p style="font-size: 18px;">9/13: Standard part TtrpC amplification PCR with KOD polymerase</p></li>
-<li><p style="font-size: 18px;">9/13: pBAR _RFP_TtrpC transformed into competent cell</p></li>
-<li><p style="font-size: 18px;">9/13: RFP_TtrpC PCR to amplify the target sequence</p></li>
-<li><p style="font-size: 18px;">9/13: pBAR _RFP_TtrpC did 3 in 1</p></li>
-<li><p style="font-size: 18px;">9/13: pBAR _RFP_TtrpC digestion check</p></li>
-<li><p style="font-size: 18px;">9/14: pBAR _RFP_TtrpC plasmid extraction</p></li>
-<li><p style="font-size: 18px;">9/14: RFP_TtrpC digested</p></li>
-<li><p style="font-size: 18px;">9/14: K118400 and TtrpC used PCR to amplify the target sequence</p></li>
-<li><p style="font-size: 18px;">9/14: KR_TtrpC used PCR to amplify the target sequence</p></li>
-<li><p style="font-size: 18px;">9/15: KR_TtrpC digested with restriction enzymes</p></li>
-<li><p style="font-size: 18px;">9/15: Standard part TtrpC mutation PCR (mutated XbaI)</p></li>
-<li><p style="font-size: 18px;">9/15: pBARGPE1 digested for pBAR_KR_TtrpC construction</p></li>
-<li><p style="font-size: 18px;">9/16: pBAR_KR_TtrpC transformed into competent cell</p></li>
-<li><p style="font-size: 18px;">9/16: Transformed pBAR_KR_TtrpC into competent cell</p></li>
-<li><p style="font-size: 18px;">9/16: Digested pBAR_KR for transforming into Metarhizium anisopliae</p></li>
-<li><p style="font-size: 18px;">9/16: Extracted plasmid BBa_K118400</p></li>
-<li><p style="font-size: 18px;">9/16: pBAR_EL222_TtrpC did plasmid PCR</p></li>
-<li><p style="font-size: 18px;">9/16: pBAR_EL222_TtrpC did plasmid PCR (confirmed successfully)</p></li>
-<li><p style="font-size: 18px;">9/16: pBAR _RFP_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/17: pBAR_RFP_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/17: pBAR_EL222_TtrpC plasmid stored and transformed cultures cryopreserved</p></li>
-<li><p style="font-size: 18px;">9/17: pBAR_KR_TtrpC did 3 in 1</p></li>
-<li><p style="font-size: 18px;">9/17: Extracted plasmid pBAR_KR_TtrpC</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 12); return false">[Collapse]</a></p>
 </div>
-<div class="technology">Week14</div>
+<div class="technology">9/23</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">9/18: Transformed pBAR_RFP_TtrpC into competent cell</p></li>
+<li><p style="font-size: 16px;">Producing new feeding box which is larger and ventilation, and design the experimental boxes for Experiment 2: Infecting B. dorsalis with non-designed M. anisopliae.</p></li>
-<li><p style="font-size: 18px;">9/18: pBAR_RFP_TtrpC did 3 in 1</p></li>
-<li><p style="font-size: 18px;">9/18: pBAR_KR_TtrpC did plasmid PCR and digestion check</p></li>
+<div class="imageimage">
-<li><p style="font-size: 18px;">9/18: BBa_E0040 did plasmid PCR</p></li>
+<div style="position:relative; width:48%;">
-<li><p style="font-size: 18px;">9/19: Amplified GFP from plasmid BBa_E0040</p></li>
+<img src="https://static.igem.org/mediawiki/2016/9/91/T--NYMU-Taipei--photo-OFF-related-14697029_120300000683154255_1538186769_n.jpg" width="100%" /></div>
-<li><p style="font-size: 18px;">9/19: Extracted plasmid BBa_E0040 </p></li>
+<div style="position:relative; width:48%;">
-<li><p style="font-size: 18px;">9/20: KR_TtrpC digestion</p></li>
+<img src="https://static.igem.org/mediawiki/2016/3/30/T--NYMU-Taipei--photo-OFF-related-14699699_120300000675475754_434414381_n.jpg" width="100%" /></div>
-<li><p style="font-size: 18px;">9/20: Transformed pBAR_KR_TtrpC into competent cell</p></li>
+</div>
-<li><p style="font-size: 18px;">9/20: pBAR_KR_TtrpC did 3 in 1</p></li>
-<li><p style="font-size: 18px;">9/20: Extracted plasmid pBAR_KR_TtrpC </p></li>
+<p style="font-size: 16px;">(Left picture: experimental boxes; Right picture: the internal space of feeding box)</p>
-<li><p style="font-size: 18px;">9/20: pBAR_KR_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/20: Extracted plasmid pBAR_RFP_TtrpC </p></li>
-<li><p style="font-size: 18px;">9/20: pBAR_RFP_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/20: RFP_TtrpC digestion</p></li>
-<li><p style="font-size: 18px;">9/21: pBAR_KR_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/21: pBAR_RFP_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/21: pBAR_RFP_TtrpC did 2 in 1</p></li>
-<li><p style="font-size: 18px;">9/21: pBARGPE1 and RFP_TtrpC digestion</p></li>
-<li><p style="font-size: 18px;">9/22: Amplified RFP_TtrpC with KOD polymerase</p></li>
-<li><p style="font-size: 18px;">9/22: Extracted plasmid pBAR_KR_TtrpC </p></li>
-<li><p style="font-size: 18px;">9/22: pBAR_KR_TtrpC did plasmid PCR</p></li>
-<li><p style="font-size: 18px;">9/22: pMCL1 and pBAR_KR_TtrpC digestion (pMCL1 was not digested successfully)</p></li>
-<li><p style="font-size: 18px;">9/22: pBAR_PgpdA_EL222_TtrpC digested for transforming into Metarhizium anisopliae </p></li>
-<li><p style="font-size: 18px;">9/23: Amplified pMCL1 with KOD polymerase</p></li>
-<li><p style="font-size: 18px;">9/24: pBAR_RFP_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/24: Extracted plasmid pBAR_RFP_TtrpC (from 9/5 plate)</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 13); return false">[Collapse]</a></p>
 </div>
-<div class="technology">Week15</div>
+<div class="technology">9/30</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">9/25: Transformed pBAR_RFP_TtrpC into competent cell</p></li>
+<li><p style="font-size: 16px;">The new oriental fruit flies broke through cocoon in the new feeding box</p></li>
-<li><p style="font-size: 18px;">9/25: RFP_TtrpC digestion</p></li>
-<li><p style="font-size: 18px;">9/25: Amplified RFP-TtrpC with KOD polymerase </p></li>
+<p style="font-size: 18px;">the new way for feeding oriental fruit flies is using gauze to be the windows that could be ventilation, and we can put watered agar and artificial feed on the top of gauze, then there were no water and feed in the box which lead to the environment being mess, and they also could have enough water and feed.</p>
-<li><p style="font-size: 18px;">9/25: Amplified RFP_TtrpC with KOD polymerase</p></li>
+<br />
-<li><p style="font-size: 18px;">9/25: pMCL1 and pBAR_KR_TtrpC digestion</p></li>
+<li><p style="font-size: 16px;">Infecting B. dorsalis with non-designed M. anisopliae started to conduct.</p></li>
-<li><p style="font-size: 18px;">9/25: Transformed pBAR_pMCL1_KR_TtrpC into competent cell</p></li>
-<li><p style="font-size: 18px;">9/25: Transformed pBAR_pMCL1_KR_TtrpC into competent cell</p></li>
+<div class="imageimage">
-<li><p style="font-size: 18px;">9/25: pBAR_pMCL1_KR_TtrpC did 3 in 1</p></li>
+<div style="position:relative; width:48%;">
-<li><p style="font-size: 18px;">9/26: Extracted plasmid pBAR_pMCL1_KR_TtrpC</p></li>
+<img src="https://static.igem.org/mediawiki/2016/c/c5/T--NYMU-Taipei--photo-OFF-related-14696875_120300000677699020_204079543_n.jpg" width="100%" /></div>
-<li><p style="font-size: 18px;">9/26: pBAR_RFP_TtrpC did 2 in 1</p></li>
+<div style="position:relative; width:48%;">
-<li><p style="font-size: 18px;">9/26: Extracted plasmid pBAR_RFP_TtrpC </p></li>
+<img src="https://static.igem.org/mediawiki/2016/4/46/T--NYMU-Taipei--photo-OFF-related-14696862_120300000674829370_219600743_n.jpg" width="100%" /></div>
-<li><p style="font-size: 18px;">9/26: pBAR_RFP_TtrpC did digestion check</p></li>
+</div>
-<li><p style="font-size: 18px;">9/26: pBAR_RFP_TtrpC did digestion check</p></li>
+<br />
-<li><p style="font-size: 18px;">9/26: Amplified pMCL1 with KOD polymerase</p></li>
+<div class="imageimage">
-<li><p style="font-size: 18px;">9/27: Amplified pMCL1 with KOD polymerase </p></li>
+<div style="position:relative; width:48%;">
-<li><p style="font-size: 18px;">9/28: pBAR_pMCL1_KR_TtrpC did digestion check</p></li>
+<img src="https://static.igem.org/mediawiki/2016/b/b9/T--NYMU-Taipei--photo-OFF-related-14686253_120300000675039507_1525612271_n.jpg" width="100%" /></div>
-<li><p style="font-size: 18px;">9/28: pBAR_RFP_TtrpC did plasmid PCR</p></li>
+<div style="position:relative; width:48%;">
-<li><p style="font-size: 18px;">9/28: Successfully constructed the plasmid pBAR_RFP_TtrpC</p></li>
+<img src="https://static.igem.org/mediawiki/2016/c/c7/T--NYMU-Taipei--photo-OFF-related-14627961_120300000679852339_2089167734_n.jpg" width="100%" /></div>
-<li><p style="font-size: 18px;">9/29: Amplified pMCL1 with KOD polymerase</p></li>
+</div>
-<li><p style="font-size: 18px;">9/30: pBAR_pMCL1_KR_TtrpC did digestion check</p></li>
-<li><p style="font-size: 18px;">9/30: Received the junction primer of GFP-TtrpC</p></li>
+<p style="font-size: 16px;">Photos shows three concentration condition and negative control environment of experiment "Infecting B. dorsalis with non-designed M. anisopliae"</p>
-<li><p style="font-size: 18px;">9/30: GFP_TtrpC ran ligation PCR</p></li>
-<li><p style="font-size: 18px;">10/1: GFP_TtrpC ran ligation PCR</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 14); return false">[Collapse]</a></p>
 </div>
-<div class="technology">Week16</div>
+<div class="technology">10/1</div>
 <div class="thelanguage">
-<li><p style="font-size: 18px;">10/2: GFP_TtrpC digested for ligation with pBARGPE1 </p></li>
+<li><p style="font-size: 16px;">Keeping on Experiment 2-1 (9/30~10/7)</p></li>
-<li><p style="font-size: 18px;">10/2: Transformed pBAR_GFP_TtrpC into competent cells</p></li>
-<li><p style="font-size: 18px;">10/2: pBARGPE1 digested for pBAR_GFP_TtrpC construction</p></li>
-<li><p style="font-size: 18px;">10/2: pBARGPE1 resuspended (from cryopreservation)</p></li>
-<li><p style="font-size: 18px;">10/3: Extracted plasmid pBARGPE1</p></li>
-<li><p style="font-size: 18px;">10/3: pBAR_GFP_TtrpC containing cultures used in 2 in 1</p></li>
-<li><p style="font-size: 18px;">10/3: Extracted plasmid pBAR_GFP_TtrpC</p></li>
-<li><p style="font-size: 18px;">10/3: pBAR_GFP_TtrpC digestion check(failure)</p></li>
-<li><p style="font-size: 18px;">10/3: pBAR_GFP_TtrpC PCR check(failure)</p></li>
-<li><p style="font-size: 18px;">10/3: Transformed pBAR_GFP_TtrpC into competent cells</p></li>
-<li><p style="font-size: 18px;">10/3: pBARGPE1 checked with restriction digestion (correct)</p></li>
-<li><p style="font-size: 18px;">10/4: pBARGPE1 plasmid stored</p></li>
-<li><p style="font-size: 18px;">10/4: pBAR_GFP_TtrpC containing cultures used in 2 in 1</p></li>
-<li><p style="font-size: 18px;">10/4: pBAR_GFP_TtrpC plasmid extraction</p></li>
-<li><p style="font-size: 18px;">10/4: pBAR_GFP_TtrpC digestion check</p></li>
 <p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 15); return false">[Collapse]</a></p>
 </div>
-<script>if (window.runOnloadHook) runOnloadHook();</script>
+<div class="technology">10/7</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Experiment 2-1 finished.</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 16); return false">[Collapse]</a></p>
+</div>
+<div class="technology">10/10</div>
+<div class="thelanguage">
+<p style="font-size: 16px;">Experiment 2-2 "infecting female B. dorsalis by copulation with infected male B. dorsalis" started to conduct.</p>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 17); return false">[Collapse]</a></p>
+</div>
+<div id="wrap">
+<div class="fund" style="white-space: pre-wrap;">
+<h2 style="margin-top:30px; margin-bottom:10px;">Q&A Section</h2><hr />
+<p style="font-size:16px;">How to CULTURE oriental fruit flies?</p>
+<p style="font-size:16px;">	The oriental fruit flies can grow in a transparent large container and centrifuge tubes with a piece of cotton placed at the opening to prevent them from escaping while still allowing air exchange.</p>
+<p style="font-size:16px;">	Feeding on sucrose-peptone agar (sited by ATCC) when you choose the centrifuge tubes as their living environment; a ratio of composition below is the way to feed the fruit flies in a larger container. The larval need the other formula to grow up whether living in the centrifuge tubes or the larger transparent container. The adult feeding formula without peptone can also alive the oriental fruit flies but not breeding.</p>
+<p style="font-size:16px;">                 Larva formula                                            Adult formula ratio
+	Yeast extraction	35g                       Sucrose: yeast extraction: peptone
+	Sucrose			60g                       3:1:1
+	Oat		                120g
+	Sodium benzoate	1g
+	Hydrochloric acid	4.5ml, 37%
+	Water			450ml
+</p>
+<p style="font-size:16px;">	The advantage of cultivating in large container is avoiding the tight space that oriental fruit flies can almost only walk and then causing the growth medium spoil easily. On the other hand, cultivating the fruit flies in the centrifuge tubes can easily distinguish the situation of the oriental fruit flies, for example, gender, health, quantity to take the correct actions.</p>
+<p style="font-size:16px;">How to prevent COMTAMINATION?</p>
+<li><p style="font-size:16px;"></p>Adding preservatives, for instance, sodium benzoate, and decreasing environmental humidity can decease the growth of the mold on agar.</li>
+<li><p style="font-size:16px;"></p>Sterilize cotton according common lab procedure or just buy pre-sterilized one.</li><br />
+<p style="font-size:16px;">How do you KEEP the oriental fruit flies in place during experiments?</p>
+<li><p style="font-size:16px;"></p>Using carbon dioxide gas to induce comatose and try your best to contain the gas within the tube and the oriental fruit flies should be unconscious after 10-20 seconds.</li>
+<li><p style="font-size:16px;"></p>Induce coma under low temperature.</li><br />
+<p style="font-size:16px;">How about the methyl eugenol? Are there any suggestions on attracting the male oriental fruit flies or the actual situation that farmer using it to prevent the oriental fruit fly pest?</p>
+<li><p style="font-size:16px;">It is best to complete experiments using methyl eugenol before 2:00 PM.</p></li>
+<li><p style="font-size:16px;">Several time experiments may cause the male oriental fruit flies became desensitized to methyl eugenol, so it's important to change the sample you test on the experiments routinely to avoid the factor to interfere the result.</p></li>
+<li><p style="font-size:16px;">The attraction traps putting in the orchard need to add pure and undiluted methyl eugenol to capture the oriental fruit flies. It also needs some cottons or gauzes to separate the oriental fruit flies and methyl eugenol to prevent the chain of fruit flies and eventually causing the death.</p></li>
+<li><p style="font-size:16px;">It's not a good idea to set the trap when (a) it's a rainy day (b) it's not the harvest season of the guava or tangerine, or the number of the fruit flies captured will not high.</p></li><br />
+</div>
+<div class="fund" style="white-space: pre;">
+<h2 style="margin-top:30px; margin-bottom:10px;">Acknowledgements</h2><hr />
+<p style="font-size:16px;">Beitou orchard
+S. J. Chen 	Grower
+</p>
+<p style="font-size:16px;">Beitou orchard
+Z. L. Liao		Grower
+</p>
+<p style="font-size:16px;">Bioresource Collection and Research Center
+G. Y. Liou		Fellow
+</p>
+<p style="font-size:16px;">Chiayi Agricultural Experiment Branch
+P. H. Chen	Research assistant
+</p>
+<p style="font-size:16px;">Kaohsiung District Agricultural Research and Extension Station, COA, EY
+M. N. Tseng	Research director
+</p>
+<p style="font-size:16px;">National Chung Hsing University, the Department of Entomology
+S. Y. Hwang	Professor
+</p>
+<p style="font-size:16px;">National Chung Hsing University, the Department of Entomology
+Y. Y. Tseng		Research assistant
+</p>
+<p style="font-size:16px;">Taiwan Agricultural Research Institute, Applied Zoology Division
+M. Y. Chiang	Research assistant
+</p>
+<p style="font-size:16px;">Taiwan Agricultural Research Institute, Applied Zoology Division
+Y. B. Huang	Professor
+/p>
+</div>
+</div>
+<script>if (window.runOnloadHook) runOnloadHook();</script></div>
+<div class="prototypeprototypesp">
+<div id="wrap">
+<div class="fund">
+<h2 style="margin-top:10px; margin-bottom:10px;">Overview</h2><hr>
+<p style="font-size:16px;">
+This section of our wiki contains the protocols for our insect experiments, including cultivation, infection rate tests, and hemolymph extraction.</p>
+</div>
+<div class="fund">
+<h2 style="margin-top:30px; margin-bottom:10px; height:50px; line-height: 24px;">Related experiment schedule & Lab note</h2><hr>
+<h4 style="margin-top:30px; margin-bottom:10px;">Experiments</h4>
+<div class="tablesatisfysp" style="overflow-x:auto; margin-top:-50px;">
+<table border="1" >
+　<tr>
+　    <td style="width:600px;">Experiments related to B. dorsalis</td>
+　    <td style="width:150px;">Number</td>
+　    <td style="width:150px;">Date</td>
+　</tr>
+　<tr>
+　    <td style="width:600px;">Diameter of the tunnel that allows passage to one B. dorsalis</td>
+　    <td style="width:150px;">1</td>
+　    <td style="width:150px;">8/30～9/1</td>
+　</tr>
+　<tr>
+　    <td style="width:600px;">Infecting B. dorsalis with non-designed M. anisopliae</td>
+　    <td style="width:150px;">2-1</td>
+　    <td style="width:150px;">9/30~10/7</td>
+　</tr>
+　<tr>
+　    <td style="width:600px;">infecting female B. dorsalis by copulation with infected male B. dorsalis</td>
+　    <td style="width:150px;">2-2</td>
+　    <td style="width:150px;">10/10~10/17</td>
+　</tr>
+</table>
+</div>
+<div class="tablesatisfysp" style="overflow-x:auto; margin-top:-50px;">
+<table border="1">
+　<tr>
+　    <td style="width:600px;">Experiments about hemolymph extraction</td>
+　    <td style="width:150px;">Number</td>
+　    <td style="width:150px;">Date</td>
+　</tr>
+　<tr>
+　    <td style="width:600px;">Blatta lateralis</td>
+　    <td style="width:150px;">1</td>
+　    <td style="width:150px;">9/20~9/30</td>
+　</tr>
+　<tr>
+　    <td style="width:600px;">Bactrocera dorsalis</td>
+　    <td style="width:150px;">2</td>
+　    <td style="width:150px;">9/29~10/5</td>
+　</tr>
+　<tr>
+　    <td style="width:600px;">Bombyx mori</td>
+　    <td style="width:150px;">3</td>
+　    <td style="width:150px;">10/3~10/</td>
+　</tr>
+</table>
+</div>
+<br /><h4 style="margin-top:30px; margin-bottom:10px;"">Routine works</h4>
+<div class="tablesatisfysp" style="overflow-x:auto; margin-top:-50px;">
+<table border="1">
+　<tr>
+　    <td style="width:750px;">Routine tasks of feeding Bactrocera dorsalis</td>
+　    <td style="width:150px;">Date</td>
+　</tr>
+　<tr>
+　    <td style="width:750px;">Produce & change the tube with the specific agar</td>
+　    <td style="width:150px;">8/24~9/20</td>
+　</tr>
+　<tr>
+　    <td style="width:750px;">Produce & change water gel and the specific ratio fodder</td>
+　    <td style="width:150px;">9/22~10/</td>
+　</tr>
+</table>
+</div>
+<div class="tablesatisfysp" style="overflow-x:auto; margin-top:-50px;">
+<table border="1">
+　<tr>
+　    <td style="width:750px;">Routine tasks of hemolymph extraction</td>
+　    <td style="width:150px;">Date</td>
+　</tr>
+　<tr>
+　    <td style="width:750px;">Collect the mulberry leaves to feed the silkworm</td>
+　    <td style="width:150px;">10/3~10/</td>
+　</tr>
+　<tr>
+　    <td style="width:750px;">Sterilize the related equipments</td>
+　    <td style="width:150px;">9/20~10/</td>
+　</tr>
+</table>
+</div>
+</div>
+</div><br /><br />
+<div class="expand">
+<center><p style="font-size:24px; color: #666666 !important; text-shadow: 0px 0px 1px #000000 !important; margin-bottom:100px;">
+<a href="#" onclick="ddaccordion.collapseall('technology'); return false"
+style="color: #666666 !important; text-shadow: 0px 0px 1px #000000 !important;  /*margin: 12px !important;*/  font-size: 18px !important;  text-decoration: none;">[Collapse all]</a>  |
+<a href="#" onclick="ddaccordion.expandall('technology'); return false"
+style="color: #666666 !important; text-shadow: 0px 0px 1px #000000 !important;  /*margin: 12px !important;*/  font-size: 18px !important;  text-decoration: none;">[Expand all]</a></p></center></div>
+<div class="technology">8/23</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Oriental fruit flies sent to lab and cultivated by large transparent box to avoid oriental fruit flies flying out.</p></li>
+<div class="imageimage">
+<div style="position:relative; width:100%;">
+<img src="https://static.igem.org/mediawiki/2016/2/2a/T--NYMU-Taipei--photo-OFF-related-14724253_120300000672924380_2135741640_o.jpg" width="100%" /></div>
+</div>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 0); return false">[Collapse]</a></p>
+</div>
+<div class="technology">8/24</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">We used both of centrifuge tube and feeding box to feed oriental fruit flies for comparing and ensuring the status of oriental fruit flies that established the independent space for observing oriental fruit flies.</p></li>
+<div class="imageimage">
+<div style="position:relative; width:100%;">
+<img src="https://static.igem.org/mediawiki/2016/4/42/T--NYMU-Taipei--photo-OFF-related-%E6%8B%8D%E7%85%A7.jpg" width="100%" /></div>
+</div>
+<br />
+<div class="imageimage">
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/3/3d/T--NYMU-Taipei--photo-OFF-related-14658400_120300000674554374_884455077_n.jpg" width="100%" /></div>
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/7/7d/T--NYMU-Taipei--photo-OFF-related-14686491_120300000678083870_2056369023_n.jpg" width="100%" /></div>
+</div>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 1); return false">[Collapse]</a></p>
+</div>
+<div class="technology">8/25</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">B. dorsalis started to break through the cocoon. We conducted to put B. dorsalis to centrifuge tubes, avoiding the feeding box cannot load all oriental fruit flies. We poured the special agar to centrifuge tubes, and put centrifuges into entrance to catch oriental fruit flies. Then we sealed the centrifuge tubes by cotton. </li></p>
+<div class="imageimage">
+<div style="position:relative; width:100%;">
+<img src="https://static.igem.org/mediawiki/2016/5/5c/T--NYMU-Taipei--photo-OFF-related-14697277_120300000677127274_438898518_o.jpg" width="100%" /></div>
+</div>
+<p style="font-size: 16px;">(The team members put agar into centrifuge tubes which would be their feed.)</p>
+<div class="imageimage">
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/e/ea/T--NYMU-Taipei--photo-OFF-related-14741005_120300000677094868_1093963562_n.jpg" width="100%" /></div>
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/4/40/T--NYMU-Taipei--photo-OFF-related-14696947_120300000676485877_378050777_n.jpg" width="100%" /></div>
+</div>
+<p style="font-size: 16px;">(The left picture is that oriental fruit flies broke through cocoon; the right picture is the feeding box & centrifuge tubes where fed oriental fruit flies.)</p>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 2); return false">[Collapse]</a></p>
+</div>
+<div class="technology">8/30</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">First time to Conducting Experiment 1</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 3); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/3</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Changing the centrifuge tubes where oriental fruit flies lived. (Avoid the environment being mess.) /p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 4); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/5</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Changing the centrifuge tubes where oriental fruit flies lived.</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 5); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/6</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">The oriental fruit flies in centrifuge tubes are dead quickly, so we conducted trouble shooting and sent the mail to professors who research oriental fruit flies.</p></li>
+<li><p style="font-size: 16px;">We took the trap to Beitou orchard to try to catch oriental fruit flies. </p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 6); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/7</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Second time to Conducting Experiment 1.</p></li>
+<li><p style="font-size: 16px;">Conducting trouble shooting.</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 7); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/10</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Searching the store selling the silkworm for extracting hemolymph which is used to induce pMcl1.</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 8); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/13</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Visiting two farmers working in the orchard to ask the information about oriental fruit flies in the nature environment, and installing the trap of catching oriental fruit flies.</p></li>
+<p style="font-size: 16px;">(Left picture: the team member install the trap; Right picture: the farmer and our team member took a picture)</p>
+<div class="imageimage">
+<div style="position:relative; width:50%; margin-right:10%;">
+<img src="https://static.igem.org/mediawiki/2016/d/d5/T--NYMU-Taipei--photo-OFF-related-14740977_120300000687733839_596021223_n.jpg" width="100%" /></div>
+<div style="position:relative; width:90%; margin-top:10%;">
+<img src="https://static.igem.org/mediawiki/2016/7/74/T--NYMU-Taipei--photo-OFF-related-14741635_120300000673924526_1894584987_n.jpg" width="100%" /></div>
+</div>
+<li><p style="font-size: 16px;">Sent the mail to researchers to ask for making public the content of the mail.</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 9); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/15</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Communicating with Chang Ming ecological education sericulture, asking the information of buying silkworm.</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 10); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/16</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Picking up the oriental fruit fly larval from rotten guavas and feeding them.</p></li>
+<li><p style="font-size: 16px;">Left picture: the team members were picking up the oriental fruit fly larval from rotten guavas; Right picture: the larval have picked up and put into another box.   Expected becoming pupae after six days. </p></li>
+<div class="imageimage">
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/f/f9/T--NYMU-Taipei--photo-OFF-related-14699910_120300000674856561_1048345374_n.jpg" width="100%" /></div>
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/e/e4/T--NYMU-Taipei--photo-OFF-related-14627744_120300000676522237_1703903936_n.jpg" width="100%" /></div>
+</div>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 11); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/22</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">For the situation that our fruit flies in the centrifuge tubes were dead quickly , so we visited Dr. Yu-Bing Huang in Taiwan Agricultural Research Institute, Applied Zoology Division, and asking the problems about the right way for feeding oriental fruit flies. We change our way of feeding oriental fruit flies. </p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 12); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/23</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Producing new feeding box which is larger and ventilation, and design the experimental boxes for Experiment 2: Infecting B. dorsalis with non-designed M. anisopliae.</p></li>
+<div class="imageimage">
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/9/91/T--NYMU-Taipei--photo-OFF-related-14697029_120300000683154255_1538186769_n.jpg" width="100%" /></div>
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/3/30/T--NYMU-Taipei--photo-OFF-related-14699699_120300000675475754_434414381_n.jpg" width="100%" /></div>
+</div>
+<p style="font-size: 16px;">(Left picture: experimental boxes; Right picture: the internal space of feeding box)</p>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 13); return false">[Collapse]</a></p>
+</div>
+<div class="technology">9/30</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">The new oriental fruit flies broke through cocoon in the new feeding box</p></li>
+<p style="font-size: 18px;">the new way for feeding oriental fruit flies is using gauze to be the windows that could be ventilation, and we can put watered agar and artificial feed on the top of gauze, then there were no water and feed in the box which lead to the environment being mess, and they also could have enough water and feed.</p>
+<br />
+<li><p style="font-size: 16px;">Infecting B. dorsalis with non-designed M. anisopliae started to conduct.</p></li>
+<div class="imageimage">
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/c/c5/T--NYMU-Taipei--photo-OFF-related-14696875_120300000677699020_204079543_n.jpg" width="100%" /></div>
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/4/46/T--NYMU-Taipei--photo-OFF-related-14696862_120300000674829370_219600743_n.jpg" width="100%" /></div>
+</div>
+<br />
+<div class="imageimage">
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/b/b9/T--NYMU-Taipei--photo-OFF-related-14686253_120300000675039507_1525612271_n.jpg" width="100%" /></div>
+<div style="position:relative; width:48%;">
+<img src="https://static.igem.org/mediawiki/2016/c/c7/T--NYMU-Taipei--photo-OFF-related-14627961_120300000679852339_2089167734_n.jpg" width="100%" /></div>
+</div>
+<p style="font-size: 16px;">Photos shows three concentration condition and negative control environment of experiment "Infecting B. dorsalis with non-designed M. anisopliae"</p>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 14); return false">[Collapse]</a></p>
+</div>
+<div class="technology">10/1</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Keeping on Experiment 2-1 (9/30~10/7)</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 15); return false">[Collapse]</a></p>
+</div>
+<div class="technology">10/7</div>
+<div class="thelanguage">
+<li><p style="font-size: 16px;">Experiment 2-1 finished.</p></li>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 16); return false">[Collapse]</a></p>
+</div>
+<div class="technology">10/10</div>
+<div class="thelanguage">
+<p style="font-size: 16px;">Experiment 2-2 "infecting female B. dorsalis by copulation with infected male B. dorsalis" started to conduct.</p>
+<p style="text-align:right;font-size:1.3em;"><a href="#" class="collapseLink" onClick="ddaccordion.collapseone('technology', 17); return false">[Collapse]</a></p>
+</div>
+<div id="wrap">
+<div class="fund" style="white-space: pre-wrap;">
+<h2 style="margin-top:30px; margin-bottom:10px;">Q&A Section</h2><hr />
+<p style="font-size:16px;">How to CULTURE oriental fruit flies?</p>
+<p style="font-size:16px;">	The oriental fruit flies can grow in a transparent large container and centrifuge tubes with a piece of cotton placed at the opening to prevent them from escaping while still allowing air exchange.</p>
+<p style="font-size:16px;">	Feeding on sucrose-peptone agar (sited by ATCC) when you choose the centrifuge tubes as their living environment; a ratio of composition below is the way to feed the fruit flies in a larger container. The larval need the other formula to grow up whether living in the centrifuge tubes or the larger transparent container. The adult feeding formula without peptone can also alive the oriental fruit flies but not breeding.</p>
+<p style="font-size:16px;">Larva formula
+Yeast extraction	35g
+Sucrose			60g
+Oat		                120g
+Sodium benzoate	1g
+Hydrochloric acid	4.5ml, 37%
+Water			450ml
+Adult formula ratio
+Sucrose: yeast extraction: peptone
+:1:1
+</p>
+<p style="font-size:16px;">	The advantage of cultivating in large container is avoiding the tight space that oriental fruit flies can almost only walk and then causing the growth medium spoil easily. On the other hand, cultivating the fruit flies in the centrifuge tubes can easily distinguish the situation of the oriental fruit flies, for example, gender, health, quantity to take the correct actions.</p>
+<p style="font-size:16px;">How to prevent COMTAMINATION?</p>
+<li><p style="font-size:16px;"></p>Adding preservatives, for instance, sodium benzoate, and decreasing environmental humidity can decease the growth of the mold on agar.</li>
+<li><p style="font-size:16px;"></p>Sterilize cotton according common lab procedure or just buy pre-sterilized one.</li><br />
+<p style="font-size:16px;">How do you KEEP the oriental fruit flies in place during experiments?</p>
+<li><p style="font-size:16px;"></p>Using carbon dioxide gas to induce comatose and try your best to contain the gas within the tube and the oriental fruit flies should be unconscious after 10-20 seconds.</li>
+<li><p style="font-size:16px;"></p>Induce coma under low temperature.</li><br />
+<p style="font-size:16px;">How about the methyl eugenol? Are there any suggestions on attracting the male oriental fruit flies or the actual situation that farmer using it to prevent the oriental fruit fly pest?</p>
+<li><p style="font-size:16px;">It is best to complete experiments using methyl eugenol before 2:00 PM.</p></li>
+<li><p style="font-size:16px;">Several time experiments may cause the male oriental fruit flies became desensitized to methyl eugenol, so it's important to change the sample you test on the experiments routinely to avoid the factor to interfere the result.</p></li>
+<li><p style="font-size:16px;">The attraction traps putting in the orchard need to add pure and undiluted methyl eugenol to capture the oriental fruit flies. It also needs some cottons or gauzes to separate the oriental fruit flies and methyl eugenol to prevent the chain of fruit flies and eventually causing the death.</p></li>
+<li><p style="font-size:16px;">It's not a good idea to set the trap when (a) it's a rainy day (b) it's not the harvest season of the guava or tangerine, or the number of the fruit flies captured will not high.</p></li><br />
+<p style="font-size:16px;">Is the common characteristic among the hemolymph of Bactrocera dorsalis and other insects sufficiently confirmed the accuracy of the experiment result?</p><br />
+<p style="font-size:16px;">Can the designed M. anisopliae actually invade B. dorsalis?</p>
+</div>
+<div class="fund" style="white-space: pre-wrap;">
+<h2 style="margin-top:30px; margin-bottom:10px;">Acknowledgements</h2><hr />
+<p style="font-size:16px;">Beitou orchard
+S. J. Chen 	Grower
+</p>
+<p style="font-size:16px;">Beitou orchard
+Z. L. Liao		Grower
+</p>
+<p style="font-size:16px;">Bioresource Collection and Research Center
+G. Y. Liou		Fellow
+</p>
+<p style="font-size:16px;">Chiayi Agricultural Experiment Branch
+P. H. Chen	Research assistant
+</p>
+<p style="font-size:16px;">Kaohsiung District Agricultural Research and Extension Station, COA, EY
+M. N. Tseng	Research director
+</p>
+<p style="font-size:16px;">National Chung Hsing University, the Department of Entomology
+S. Y. Hwang	Professor
+</p>
+<p style="font-size:16px;">National Chung Hsing University, the Department of Entomology
+Y. Y. Tseng		Research assistant
+</p>
+<p style="font-size:16px;">Taiwan Agricultural Research Institute, Applied Zoology Division
+M. Y. Chiang	Research assistant
+</p>
+<p style="font-size:16px;">Taiwan Agricultural Research Institute, Applied Zoology Division
+Y. B. Huang	Professor
+/p>
+</div>
+</div>
+<script>if (window.runOnloadHook) runOnloadHook();</script></div>
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