Team:NCTU Formosa/project

Motivation

Serious Problem in The World ( Insect Damage )

From ancient times to the present, harmful insects always play roles in our life, and we don’t have any appropriate solution to these annoying pests. Every year, harmful insects cause a great amount of loss to our agriculture, which seriously disturbs our people livelihood and economy.

Take Brazil for example. We can find several crucial economic crops such as coconut, coffee, and sugarcane are damaged by harmful insects. The total loss of crops in Brazil is about 11 billion US dollars. In addition, we can also find that people spent nearly 1.4 billion controlling these annoying pests. What's more? They have used a great number of insecticides containing some toxic substances. However, this method will make human beings exposed to horrible situation.

fig.1-1-1 Losses caused by pests in Brazil. This chart demonstrates the losses of various crops caused by pests.

Also, there is a data about the IPM (Integrated pest management) of USA. IPM means the ability to control pests. The IPM is reality number in global, and the lower of the number is, the more efficiently we control. In the data, we can see that lots of areas in USA are used to plant crops. There are several kinds of plants which are still under attack by harmful insects. We can only control the insect damage fairly well in a small limited production area. Therefore, as the production areas expand to an extent, the effect of control declines to a point. Even though the production areas become larger, the total production is lower.

Therefore, what we need is invent a brand new method to solve this problem.

fig.1-1-2 This chart demonstrates the area we plant of crops IPM of various crops in USA.

Common Methods of Solving Insect Damage Problem

Chemical Control Method

Because of the hazard of insects, human beings have come up with lots of ideas to kill these harmful insects. In the 15^th century, people used heavy metals such as Arsenic, Mercury and Plumbum to kill harmful insects, which caused a catastrophe to the environment. Pesticides became more powerful along with the technology, in the 20^th century, the agriculture developed rapidly just because of the evolution of pesticides. But the pesticides are not only fatal to the insects but also harmful to the human beings. People found this problem after several decades. The toxin of the pesticides will be kept in creatures by the food chain, and finally, goes into human's body.

fig.1-1-3 Human health and environmental cost from pesticides in the United States is estimated at $9.6 billion.

Damage to Human Body

Pesticides includes substances that kill weeds (herbicides), insects (insecticides), fungus (fungicides), rodents (rodenticides), and others. The use of toxic pesticides to manage pest problems has become a common practice around the world. Pesticides are used almost everywhere, causing human health hazards, ranging from short-term impacts such as headaches and nausea to chronic impacts like cancer and reproductive harm. There is also mounting evidence that exposure to pesticides disrupts the endocrine system, wreaking havoc with the complex regulation of hormones, the reproductive system, and embryonic development.

Damage to The Environment

Pesticides wreak havoc on the environment, threatening biodiversity and weakening the natural systems upon which human survival depends. Pesticides have led to abnormal mass mortality of America's honeybees. The populations of bees have dropped by 29% to 36% each year since 2006. To our astonishment, fully 1/3 of the food we eat depends on bees for pollination!

Secondly, some scientists believe that amphibians and bats have become more susceptible to deadly disease because their immune systems are weakened by pesticides. What's more, one kind of pesticides called herbicide, which can give a male frog a sex change. Genetically, the frogs are still males, but morphologically they are completely female and they can even mate successfully with other males and lay viable eggs. Pesticides have also contaminated waterways and endangered fish and birds, causing ecological damage.

By the way, pesticide resistance makes insecticides ineffective. Other factors in the speed with which a species evolves resistance are generation time and fecundity, that is, causing shorter generations and more offspring lead to resistance more quickly.

Biological Control Method

Since most chemical control methods are poisonous to the environment, humans began to look for other alternative ways. One of the most well known example is bacillus thuringiensis (Bt), which can efficiently kill insects with its crystal proteins. However, recent researches have shown that more and more insects have been resistant to this kind of protein.

Chemical control method have caused fetal environmental damge and more and more harmful insects can be resistant to it. Also, biological bacillus thuringiensis (Bt) control method can be resisted by more and more insects. However, it's not too late to improve this situation. We can create an evolution of agriculture by a new method, and that is what we do!

fig.1-1-4 Comparison of both solutions.

Pheromone trap is the novelist method to solve the insect damage problem nowadays. What's more, pheromone is a pollution-free substance and a substance of insect's itself. Thus, pheromone doesn't have any insect's resistance problem. Thus, we initially took biological synthesis of pheromone in E.coli into consideration.

Novel and Efficient Ways to Solve Insect Damage Problem - Pheromone Trap

Introduction of Pheromones

A pheromone is a secreted or excreted chemical factor that triggers a social response in members of the same species. Pheromones are chemicals capable of acting outside the body of the secreting individual to impact the behavior of the receiving individual.

There are many kinds of pheromones such as alarm pheromones, food trail pheromones, sex pheromones, and many others that affect behavior or physiology.

Sex pheromones are an important factor in finding a mating partner. When a female releases chemicals, the mate search is initiated, and the male moths begin their upwind motion toward their potential partner. Sex pheromones in particular are associated with long-range chemical communication of sex substances used in signaling a mating partner. Mate finding in moths involve sex pheromones that have the ability to propel long-distances and are emitted by the females abdominal glands in most cases.

How to Make Pheromones？

There are 2 main ways to make pheromones. One is chemical synthesis, the other one is biosynthesis approach.

Biosynthesis Pathway in Insects

Biosynthesis approach needs lots of enzymes to catalyzed, which is too complicated for E.coli to carry out. In addition, E.coli is lack of Glycoproteins that can do posttranslational modification. Therefore, it is nearly impossible for E.coli to produce pheromones in a normal way.

fig.1-2-2 If we want to produce the pheromone of Gypsy moth, Lymantria dispar. We have to pass at least four complicated pathways. That will be high loading for E.coli,and it may also cause low efficiency of producing pheromone.

Chemical Synthesis Approach Used in Factory to Mass Produce Pheromone

It is difficult to make pheromone by chemical synthesis approach. This approach needs to use many kinds of chemical compound which may cause some pollution to the environment. Furthermore, chemical approach needs to control the condition of reaction that may consume a lot of energy. An example of chemical synthesis approach: leafroller moth, Bonagota cranaodes.

fig.1-2-1 If we want to produce the pheromone of leafroller moth and Bonagota cranaodes by Chemical synthesis approach ,we have to buy some expensive equipments to control the conditions.And it may produce some byproduct which is toxic.

Difficulty and Disadvantage of Pheromone Production

As above mentioned, because pheromone is a very complicated substance, it absolutely has many problems to produce it. Speaking of chemistry production, though pheromone can be mass produced to save the cost, chemistry production still has many derived problems, like high producing cost ( difficult and very complex technique needed to control many reaction conditions ) and most importantly, serious environmental pollution ( using many chemical reagents to purify and produce pheromone ). Thus, it is no mentioned to produce pheromone in biosynthesis method with E.coli. Therefore, we decided to find a more easy and better way to reach our goal.

Idealist Method of Solving Insect Damage Problem - PBAN

Though, it is impossible to biosynthesize pheromone with E.coli, we still tried our best to find another more feasible way. As above mentioned, our method should be pollution-free, efficient, insects' resistance-free and cheap. Conclude all these advantages elements, and we found PBAN, which can help us let target harmful insects produce pheromone for us. Moreover, we even can use biosynthesis method to make our project multifunctional. We hope we can use PBAN to solve the target harmful insects below. ( Check photos )

Reference

PBAN ( Pheromone Biosynthesis Activating Neuropeptide )

PBAN (Pheromone Biosynthesis Activating Neuropeptide) is one kind of peptides that can activate biosynthesis of pheromones of insects we target. Once a PBAN binds with the G-protein coupled receptor on an insect’s pheromone gland, the signal sent by the G-protein coupled receptor activates the kinase and phosphatase, and then kinase and phosphatase can activate enzymes that participate in the biosynthesis of insect sex pheromone, which will be emitted.

In nature, female insects such as moths release PBAN during mating to stimulate the synthesis of pheromones in order to attract their male counterparts. PBAN can also facilitate the release of non-sex pheromones such as trail pheromones for ants.

fig.1-2-3 Working mechanism of PBAN

Features of PBAN

1. Species-Specific：PBAN is species-specific just like pheromones, that means every kinds of insects which can produce pheromone have it's specific PBAN, which can only bind with it's specific receptor and only stimulate the biosynthesis of a specific pheromone.

2. Small Simple Peptide：The coding sequence for a PBAN is usually around 100 base pairs. Therefore, it is easy for E.coli to express. Thus, all we have to do is to cultivate the E.coli to express our PBAN so our cost is very low. In addition, we can even combine several different kinds of PBAN sequences into one BioBrick assembly or even make different regulations for different PBANs without DNA length problem. Last but not least, PBAN is a peptide unlike pesticide so it dosen't cause any damage to the environment.　

3. Natural Substance of Insects' Itself： Because PBAN is a natural substance of insects' itself, insects have no possibility to resist it. Thus, our PBAN unlike pesticide and bacillus thuringiensis (Bt) can cause target insects to continuously produce pheromone for us if it can come in contact to the pheromone gland.

Effect Testing of PBAN from Reference

Because we regard PBAN as a leading character in our project, we have to realize what kind of problem we would meet with if we want to make use of PBAN. First, we had to confirm that whether PBAN can work when getting in the moth’s body from outside, we got a paper titled “Pheromonotropic Activity of Naturally Occurring Pyrokinin Insect Neuropeptides (FXPRLamide) in Helicoverpa zea” from ELSEVIER, experiments of the research were conducted with Helicoverpa zea (corn earworm) in this research, researchers injected different amount (from 0.05 pmol to 200 pmol) of PBAN of Helicoverpa zea (named Hez-PBAN) into their bodies directly, and then measured the amount of pheromone produced, the result (fig.1-2-4) showed that PBAN has function as long as it gets in moth’s body, and the 2 pmol dose of Hez-PBAN was the minimum dose required to stimulate production of the maximum amount of pheromone, that revealed we are able to stimulate the production of pheromone with only little amount of PBAN which gets in moth’s body, whether we fed or injected PBAN.

fig.1-2-4 Effect of dose of Hez-PBAN on stimulation of in vivo pheromonotropic activity in vitrugin females of H. zea (bars indicate SEM. n=8).

Difficulty of Natural PBAN about Peptidase Degradation

But we still had the second problem, we had known that peptidase in the insect’s body can degrade peptides including PBAN, so the effect of PBAN can’t continue for a long time. In order to solve this problem, we search some former papers and we found another paper from ELSEVIER, it was titled “Enhanced oral availability/pheromonotropic activity of peptidase-resistanttopical amphiphilic analogs of pyrokinin/PBAN insect neuropeptides”, Heliothis virescens was used in this research, researchers produced amphiphilic analogs of pyrokinin/PBAN with chemical synthesis method, they used hydroxyproline (Hyp) and octahydroindole-2-carboxyl (Oic) to replace the Proline (Pro) of the PBAN C-terminal penpapeptides (Phe-Thr-Pro-Arg-Leu-NH2), that made PBAN be able to resist peptidase, they compared the stability of the natural pyrokinin/PBAN analog LPK, and the peptidase-resistant pyrokinin/PBAN analogs (5 nmol was used), we can see that natural PBAN will be reduced over time in the moth’s body (fig.1-2-5).

fig.1-2-5 Stability of the natural pyrokinin/PBAN analog LPK (_), and the peptidase-resistant pyrokinin/PBAN analogs Hex-Phe-Thr-Hyp-Trp-Gly-NH2 (901)(_) and Hex-Phe-Thr-Oic-Trp-Gly-NH2 (904) (_) to hydrolysis by peptidases bound to corn earworm (H. zea) Malpighian tubule tissue. Measurement of the amount of remaining peptide was made by HPLC at 30, 60, and 120 min. The data points represent the means of at least three replicates

Our New Idea of Using PBAN from Oral Testing in Refernece

This results above sounded not good for us, but there was other experiment interested us in the same research, researchers also conducted oral test with their PBAN (fig.1-2-6), in this experiment, they fed moths with sugar solution which contained their PBAN analogs, and measure the amount of pheromone produced over time, that gave us inspiration, although natural PBAN can’t be maintained for a long time in moth’s body, we could solve this problem by feeding moths with PBAN continually, after combining this thought with the method of the oral experiment, we figured out an idea, instead of killing all of the moths.

It is very possible that the PBAN in the food will penetrate into moths’ body from the digestive system. Thus, if female moths suck the food, PBAN in the moths’ body will be replenished, which solves the problem that PBAN can not maintain for a long time in the moths’body. As to how we produce PBAN and apply our concept to capture wanted harmful insects, we will explain in the following.

fig.1-2-6 Amount of pheromone, relative to injected PBAN, produced by Hyp-analog 901 and Oic-analog 904, 1.5, 3, 4, and 6 h following oral administration.Dotted line at 100% denotes maximal production of injected PBAN (positive control).

How We Are Going to Use PBAN?

fig.1-2-7 Our Project Overview

In our project, we will biologically synthesize PBAN with our E.coli. We store the PBAN inside a trapping device (check this out at our Device page). In the device, there will be appropriate lighting and nutrient sources that will attract insects.

Once an insect is attracted into our device and ingests the nutrient sources we provide, it will also inevitably come in contact with our PBAN. As the PBAN works its magic and activates the pheromone synthesis of the attracted insect, more of this species of insect’s counterparts will be attracted and later captured.

Owing to the first feature mentioned above, PBAN are species-specific, which means that it doesn't matter if other kind of insect fly into our device and eat PBAN, because the insects we don't want to catch will not be stimulated by PBANs to produce pheromone; our PBAN are only for what we want to catch, and we are sure that our method won't affect other kinds of insects.

Biobrick Design

Basic Biobrick Design to Use Our PBAN

fig.1-3-1 This is our basic design of biobrick.

We searched the DNA sequences of the PBANs from many kinds of insects on NCBI, then contrasted to the amino sequences from papers so that we can select the DNA fragments which directly correspond to gland-stimulating function. By ligating the constitutive promoter (J23101), ribosome binding site (B0034) and PBAN DNA sequence with a terminator (J61048) at the last (we delimit this sequence as basic part), we were able to make E.coli directly produce these PBANs continusely instead of the original complex process of PBAN biosynthesis in insects.

Best Potential of Our PBAN Biobrick - Multifunctional

fig.1-3-2 Best Potential of Our PBAN Biobrick - Multifunctional.

We can assemble these basic parts together because the base pairs of these basic parts are small. Therefore, we can assemble different basic parts which contain different PBAN DNA sequences to resolve different pest problems. Therefore, our biobrick design can be customized to different pest problems. For instance, there is a farm damaged by 3 kinds of moths : Lymantria dispar, Spodoptera litura and Mamestra brassicae. What we only have to do is ligate the PBAN DNA sequence of 3 basic parts into one plasmid and let the E.coli to express these PBAN for us. After these three kinds of moth ingest these three kinds of PBANs. Subsequently, these 3 species of moths will produce their own pheromones to attract the same species. Brief to say, our PBAN basic part can assemble together with any combination method in infinite possibility.

For much more creative ideas, each PBAN basic part can use different promoter, RBS and terminator to make many different regulation. Thus, our PBAN basic part can not only assemble together but also make different regulations ( use different promoters or RBS ) on each PBAN gene. Thus, our PBAN biobrick really have infinite potential！- Multifunction

Reference

Device

fig1-4-1 Our blue light pyramidal device

Introduction

Since insects behave widely different to the gravity force, we design a device which could catch specific kind of insect species that we want. For example, Agrotis ypsilon Rottemberg and Spodoptera litura falls into the kind of moth that have negative geotaxis. So we made a trap with accessible pathway in the bottom. Once an insect enters the device, it could only goes up and be trapped inside the pyramid. However, after field investigation, we found some insects still escape from the device. Then we came out with a new version trap with doubled layers, inner shell and outer shell.

Mechanism

Device Design

More detail information about how we design our pyramidal device can download the file below.

Device_Design_Download

Process to Assemble Our Device & Main Idea of Our Device Design


Outer Shell：



fig.1-4-3 Outer Shell Real Product


fig.1-4-4 Outer Shell Schematic Diagram

　We shaped the device into a pyramid. Its special layout also enriches the device with mysterious colors.

1. Outer shell is composed of 4 triangular acrylic planes which has a trapezoid entrance to let bugs in and release the smell of pheromone produced by the insects which eat our PBAN.


Inner Shell：



fig.1-4-5 Inner Shell Real Product


fig.1-4-6 Inner Shell Schematic Diagram

　Similar to the outer one, inner shell are also composed of 4 trapezoid planes and removable from the base. The only different part is that its top is not sealed in order to contain the captured insects.

1. There would be a PBAN solution placed on the bottom.

2. A blue LED light bulb will be installed around the top of the inner shell plane to attract the first female insect.


Latch：



fig.1-4-7 Latch Real Product


fig.1-4-8 Latch Schematic Diagram

A part to stabilize the pyramid.

1. Stabilize 4 corners of the bottom.

2. To make sure the outer shell can combine with the inner one tightly.








Assembling Process

We can assemble the outer shell、inner shell and tenon together to get our completed pyramid device.

　　　　　　　　






Advantages


1.We successfully apply the geotaxis of targeted female moth to trap them in our device, forcing them releasing sex pheromone by our PBAN to attract more same-kind insects.
2.The hook attached to the purse-string bag can seal it simultaneously when we want to remove the outer shell. By doing so, no insects would flee from the bag and safety problems can also be solved.
3.Inner shell is removable so it’s easier to add new PBAN solution.
4.Compared to conventional light bulb, LED bulb is much brighter and conserves more energy. It could powered by battery so it’s also easier to establish.
5.Purse-string bag is cheap and easy to switch.
6.The PBAN system can run day and night. Its function won’t be affected by sunlight.
7.Pyramid is good at looking and can enriches the entire device with a technological feeling.