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's livelihood and economy.

Take Brazil for example. We can find several crucial economic crops such as coconut, coffee, and sugarcane which 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 situations.

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 the number is, the more efficient we are able to 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 efficiently for a small, limited area. Therefore, as the production areas expand to an extent, the effect of control declines exponentially. Even though the production areas become larger, the total production is might be lower.

Therefore, what we need is 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 idea 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 were 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 eventually, enters 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 on. Pesticides have led to abnormal massive mortality of America's honeybees. The populations of bees have dropped by 29% to 36% each year since 2006. To our astonishment, approximately 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.

As a matter of fact, 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 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 damage and insects have grown resistant to both the chemicals substances and Bt. It is, however, not too late to improve this situation. We can create an agricultural evolution with our new method!

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

Pheromone trap is currently the most novel method to solve the insect damage problem. What's more, pheromone is a pollution-free substance that is produced by the insects, meaning that insects cannot grow resistant to it. This is why 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 among members of the same species. Pheromones are capable of acting outside the body of the secreting individual to impact the behavior of the receiving individuals.

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 mating search is initiated, and the male moths begin their upwind motion toward their potential partner. 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 requires the cooperation of different enzymes, which is too complicated for E.coli to carry out. In addition, E.coli lacks Glycoproteins that can do posttranslational modification. Therefore, it is nearly impossible for E.coli to produce pheromones biosynthetically.

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, the control of reacting condition 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 toxic byproduct.

Difficulty and Disadvantage of Pheromone Production

As mentioned above, pheromone is too complicated to be produced either artificially or biosynthetically. Although the cost of artificial chemically synthesis can be lowered through massive production, the environmental damage it causes is fatal. With that said, we decided to find an easier and better way to reach our goal.

Idealist Method of Solving Insect Damage Problem - PBAN

To solve the impossibility of biosynthesizing pheromone with E.coli, we still managed to find another feasible way. As mentioned above, our method should be pollution-free, efficient, insect resistance-free, and cheap. Combining all these advantageous elements, we introduce you PBAN. ( Check photos )

Reference

PBAN ( Pheromone Biosynthesis Activating Neuropeptide )

PBAN (Pheromone Biosynthesis Activating Neuropeptide) is a kind of peptide that can activate biosynthesis of pheromones of the targeted insects. Once a PBAN binds with the G-protein coupled receptor located at an insect’s pheromone gland, the signal sent by the G-protein coupled receptor activates kinase and phosphatase, which in turns activate other enzymes that participate in the biosynthesis of insect sex pheromones. These pheromones are eventually emitted.

In nature, female insects such as moths release PBAN during mating to stimulate the synthesis of pheromones in order to attract male moths. 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, meaning that every kind of insect produces specific PBAN that only binds with it's targeted receptor, resulting in the production of a particular pheromone.

2. Small Simple Peptide：The coding sequence for a PBAN is only around 100 base pairs. To E.coli 100 base pairs is totally within its working capacity. and therefore, E.coli can be our low-cost PBAN factory. By synthesizing the DNA sequences for different PBAN into our factory, we can even produce a verity of PBANs. In addition, this factory is totally environmental friendly, unlike any pesticidewe have seen.　

3. Insects' own secretion： Because PBAN is a insect's own secretion, insects could not form resistance it. In addition, it can easily trigger pheromone production by coming in contact with its receptor.

Our Main Idea

We plan to produce PBANs and make them come in contact with the target insect. The target insect will then start producing pheromones and attract more of the target insects for us.

PBAN Can Indeed Lead to Pheromone Production

Before we could employ PBAN to accomplish our goals, we need to solve few problems. First, we had to confirm that whether an insect can take in vitro PBAN and allow it to function. From “Pheromonotropic Activity of Naturally Occurring Pyrokinin Insect Neuropeptides (FXPRLamide) in Helicoverpa zea” of ELSEVIER, this method has been proven possible. In this experiment, 0.05 pmol to 200 pmol of PBAN had been injected in to corn earworm and the amount of pheromone produced had been measured. The result (fig.1-2-4) showed that PBAN can effectively increase the production of pheromone. The maximized amount of pheromone production was reached with only 2 pmol of PBAN. This experiment also showed that, as long as PBAN enters the worm’s body, it can function, whether it is injected or fed.

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).

Problem of being degraded by peptidase

Our second problem is that PBAN would be degraded quickly in a insect’s body. In order to solve this problem, we search some former papers and we found another paper from ELSEVIER, “Enhanced oral availability/pheromonotropic activity of peptidase-resistanttopical amphiphilic analogs of pyrokinin/PBAN insect neuropeptides.” In this experiment, Heliothis virescens was used and amphiphilic analogs of pyrokinin/PBAN was used instead of natural PBAN. This way, the analog can resist peptidase, as shown in the graph below. The amount natural PBAN decreases quickly while the analogs degrades slowly. (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

Solution ( Inspired by Paper, Led to Our Design )

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.

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.

Reference

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！

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

First, we divide our design chart into two parts－exterior and interior. The exterior is just like the appearance of pyramid, and the interior is used to equip PBAN and bag for pests. When the harmful insects we want to catch eat our PBAN, they will release pheromone, and attract the same species. We can use blue light or the smell of pheromone to attract insect at first. After they go into our device, the design of our device will take advantages of their characteristic that insects always fly high to escape and make them stuck in our device. When we take away the outer shield, the hock on the outer shield will close the bag, and the insects will be caught. In addition, the four tenons at the corner can firm up our device.

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.