Team:NYMU-Taipei/Notebook-Lab Book-fruit flies hemolymph
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/ |
Oriental fruit flies sent to lab and cultivated by large transparent box to avoid oriental fruit flies flying out.
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.
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.)
First time to Conducting Experiment 1
Changing the centrifuge tubes where oriental fruit flies lived. (Avoid the environment being mess.) /p>
Changing the centrifuge tubes where oriental fruit flies lived.
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.
Searching the store selling the silkworm for extracting hemolymph which is used to induce pMcl1.
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.
Communicating with Chang Ming ecological education sericulture, asking the information of buying silkworm.
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.
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.
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)
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"
Keeping on Experiment 2-1 (9/30~10/7)
Experiment 2-1 finished.
Experiment 2-2 "infecting female B. dorsalis by copulation with infected male B. dorsalis" started to conduct.
Q&A Section
How 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?
How do you KEEP the oriental fruit flies in place during experiments?
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.
Acknowledgements
Beitou 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/ |
Oriental fruit flies sent to lab and cultivated by large transparent box to avoid oriental fruit flies flying out.
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.
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.)
First time to Conducting Experiment 1
Changing the centrifuge tubes where oriental fruit flies lived. (Avoid the environment being mess.) /p>
Changing the centrifuge tubes where oriental fruit flies lived.
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.
Searching the store selling the silkworm for extracting hemolymph which is used to induce pMcl1.
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.
Communicating with Chang Ming ecological education sericulture, asking the information of buying silkworm.
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.
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.
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)
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"
Keeping on Experiment 2-1 (9/30~10/7)
Experiment 2-1 finished.
Experiment 2-2 "infecting female B. dorsalis by copulation with infected male B. dorsalis" started to conduct.
Q&A Section
How 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?
How do you KEEP the oriental fruit flies in place during experiments?
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?
Acknowledgements
Beitou 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>
