Team:NCTU Formosa/project

Motivation

Serious Problem

From ancient times to the present day, harmful insects always greatly affect our daily life, and we don’t have any appropriate solution to these troubling insects. Every year, harmful insects bring significant economic loss to agriculture, which seriously disturb people's livelihood and economy.

Take Brazil as an example, where the agricultural loss can reach up to 11 billion US dollars because most economic crops here such as coconut, coffee, and sugarcane are under serious insects attack. To control these obnoxious insects, 1.4 billion has to be invested to purchase insecticides annually. Terrible as it may seem, Brazil is just one of the many cases in the world! The truth is that farmers worldwide are doing the same thing! To ensure good harvest, they use pesticides without much consideration about its side effects. In other words, a great amount of insecticides containing toxic substances is discharged to the environment every day. Obviously, it’s never a good solution.

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 harmful insects. The IPM is a number that evaluates reality globally. The lower the number is, the more efficient we are able to control the pests in the evaluated area. In the data, we can see that crops are planted in lots of areas in USA. There are several kinds of plants which are still under attack by harmful insects. We can only control the insect damage efficiently within 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 might be lower.

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

Fig.1-1-2 This chart demonstrates IPMs of the various areas of different crops planted in the USA.

Common Solutions

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 was a catastrophe to the environment. Pesticides became more powerful along with the technology. In the 20^th century, the agriculture developed rapidly due to the evolution of pesticides alone. 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 remain in creatures through the food chain, and enter the human body eventually.

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 herbicides, insecticides, fungicides, rodenticides, which will kill weeds, insects, fungus, rodents, and others. In our project, we refer to insecticides as pesticides. The use of toxic pesticides to manage insect problems has become a common practice around the world. However, pesticides cause serious human health hazards, ranging from short-term impacts such as headaches and nausea to chronic impacts like cancer and reproductive harm. There are also many researches revealing that exposure to pesticides will disrupt the endocrine system, the reproductive system, and embryonic development.

Damage to The Environment

Pesticides pollute the environment such as threatening biodiversity and weakening the natural systems. Pesticides have led to abnormally high mortality of America's honeybees. The population of bees has dropped by 29% to 36% each year since 2006. To our amazement, approximately 1/3 of the food we eat depends on bees for pollination!

Some scientists believe that amphibians and bats have become more susceptible to deadly diseases because their immune systems are weakened by pesticides. Pesticides also contaminate waterways and endangered fish and birds, causing ecological damage.

Biological Control Method

Since most chemical control methods are poisonous to the environment, humans began to look for alternative solutions. One of the best 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 methods 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 revolution with our new method!

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

Pheromone trap is currently the most novel method to resolve insect damage problem. What's more, pheromone is a pollution-free substance that is produced by the insects, meaning that insects cannot resist it. That's why we take biological synthesis of pheromone in E.coli to solve the problem.

The 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 through chemical synthesis, the other through biosynthesis approach.

Chemical Synthesis Approach Used in Factory to Mass Produce Pheromone

It is difficult to make pheromone by chemical synthesis approach. This approach requires 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.

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.

Difficulty and Disadvantage of Pheromone Production

As mentioned above, pheromone is too complicated to be produced either artificially or bio-synthetically. 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. Click the photos to learn more!

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, it will send a signal to activate kinase and phosphatase, which in turns activate other enzymes that participate in the biosynthesis of insect sex pheromones. These pheromones are eventually emitted.⁽³⁴⁾

In nature, female insects such as moths release PBAN to stimulate the synthesis of pheromones in order to attract male moths during mating. PBAN can also facilitate the release of non-sex pheromones such as trail pheromones for ants. Overall, this kind of natural substance, PBAN have many advantages in the following, which absolutely can complete our goals perfectly.

Fig.1-2-3 This is the biosynthesis pathway of pheromones of the targeted insects. Once PBAN comes in contact with the PBAN receptor, the receptor will send a signal to activate other enzymes that participate in the biosynthesis of insect pheromones.

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 variety of PBANs. In addition, this factory is totally environmental friendly, unlike any pesticide we 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 (FXPR-Lamide) in Helicoverpa zea” of ELSEVIER⁽³⁶⁾, 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 produced by insects itself can effectively increase the production of pheromone. The maximized amount of pheromone production was reached with only "10 pmol" of PBAN. This experiment means as long as PBAN enters the body of target insect, it is much more possible that PBAN can stimulate the production of pheromone！

Fig.1-2-4 Effect of dose of Hez-PBAN on stimulation in vivo pheromonotropic activity in vitrugin females of H. zea (bars indicate SEM. n=8). This figure shows PBAN can efficiently stimulate pheromone production in just few amount (10 pmol).

Problem of Being Degraded by Peptidase

The only problem if we want to use PBAN 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-resistant topical amphiphilic analogs of pyrokinin/PBAN insect neuropeptides.”⁽³⁵⁾ In this experiment, Heliothis virescens was the experimental insect and amphiphilic analogs of pyrokinin/PBAN were 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). However, we found the amphiphilic analogs of pyrokinin/PBAN peptide have many special artificial modification, which means E.coli. absolutely can not produce this artificial PBAN which can easily resist peptidase. Thus, we have to make more brain storming to think how to use PBAN even if it can be degraded by peptidase.

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 and Hex-Phe-Thr-Oic-Trp-Gly-NH2 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 )

These results above sounded not good for us, but there was other experiment interested us in the same research, researchers also conducted oral test with artificial PBAN/pyrokinin analogs (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. (Blank：natural PBAN in low concentration) This result provoked our inspiration, although natural PBAN can’t be maintained for a long time in moth’s body, we could solve this problem by simply feeding moths with high concentration PBAN continually.

Fig.1-2-6 Amount of pheromone, relative to the maximum pheromone amount induced by injected PBAN, produced by Hyp-analog and Oic-analog, 1.5, 3, 4, and 6 h following oral administration.Dotted line at 100% denotes maximal pheromone production of injected PBAN (positive control). This figure shows a very important information that blank (natural PBAN in low concentration：50 pmol, the blank was measured when it was 2 hr after feeding with nature PBAN) have few effects of stimulating pheromone production. Thus, we decide to try to feed the female target insects with high concentration PBAN continuously as our project main idea.

Thus, as long as female moths suck the high concentration PBAN solution continuously. PBAN in the moths’ body will be replenished continually even if peptidase degradation. Then, PBAN will have high possibility of being absorbed into moths’ body from the digestive system and succeed in stimulating pheromone gland. Thus, if we feed the target female insects with high concentration PBAN solution continuously, the female insects will produce pheromone for us, which theoretically solves the problem that PBAN can not maintain for a long time in the insects’ body. As to how we produce PBAN and apply our concept to capture wanted harmful insects, we will explain in the following.

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 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 (BBa_J23101), ribosome binding site (BBa_B0034) and PBAN DNA sequence with a terminator (BBa_J61048) at 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. 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 ligating 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

Fig.1-4-1 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 at the bottom. Once an insect enters the device, it could only go 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.

Assembling Process

Before the detailed description of how we design our device, we can show you a simple animation of the assembling process of our device. As you can see, our device is very easy to use. Just assemble the outer shell, inner shell (containing PBAN) and tenon together to complete the device. Then, you can put the device at where you want to attract the harmful insects.

Mechanism

As you can see, we divide our design chart into two parts－exterior and interior. The exterior is just like the appearance of pyramid, and the interior is designed to equip PBAN. At the beginning of using the device, we turn on the blue light to attract the target harmful insects. When the target female insects are stuck in the interior and eat the food mixed with PBAN solution, they will release pheromone, and attract the same species. Even we turn off the blue light, our device can attract many target male insects because the female insects inside our device is still in rut and release sex pheromone to attract their mates. After the harmful insects go into our device, the design of our device will take advantages of their habit. Insects always fly high to escape so they will be stuck in the top of 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. The outer shell is composed of 4 triangular acrylic planes which has a trapezoid entrance. When insects come inside and eat PBAN solution, it will release pheromone. With the pheromone smell, our device can trap and collect more and more insects of the same 　　　species.

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 at 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.

Advantages

1. We successfully trap the targeted female moth in our device, forcing them to release sex pheromone by eating PBAN, that results in attracting more insects of the same kind.

2. Inner shell is removable so that it’s easier to replenish new PBAN solution and the food of insect.

3. Compared to conventional light bulb, LED is much brighter and conserves more energy. It could powered by battery so 　　　it’s also easier to establish.

4. The PBAN system can run day and night. Its function won’t be affected by sunlight.

5. Pyramid is a good-looking form and can enrich the entire device with a technological feeling.