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<h3 class="hook" align="left"><a href="#">Modeling</a> > <a href="https://2014.igem.org/Team:Valencia_UPV/Modeling/diffusion">Pheromone Diffusion</a></h3></p></br>
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<div align="center"><span class="coda"><roja>P</roja>heromone <roja>D</roja>iffusion <br/><br/> and <roja>M</roja>oths <roja>R</roja>esponse</span> </div>
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</br></br>
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<div class="tabs">
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    <ul class="tab-links">
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        <li class="active"><a href="#tab1">Introduction</a></li>
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<li><a href="#tab2">Diffusion Equation</a></li>
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<li><a href="#tab3">Moth Response</a></li>
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        <li><a href="#tab4">Simulation</a></li>
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        <li><a href="#tab5">Results</a></li>
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        <div id="tab1" class="tab active">
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<ul class="tab-links">
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        <li class="active"><a class="normal-link-page" href="#tab3">Sos un volutto :)</a></li>
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<p>Sexually communication among moths is accomplished chemically by the release of an "odor" into the air. This "odor" is the <span class="black-bold">sexual pheromones</span>.</p><br/>
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<div align="center"><img width="540px" src="https://static.igem.org/mediawiki/2014/9/9d/VUPVIntro_sexpheromone.png" alt="female_sex_pheromones" title="Female and Male Moths"></img></div><br/>
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<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.8em; width: 700px;"><span class="black-bold">Figure 1</span>. Female moth releasing sex pheromones and male moth.</p></div><br/>
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<p>Pheromones are molecules that can undergo a diffusion process in which the random movement of gas molecules transport the chemical away from its source (Sol I. Rubinow, Mathematical Problems in the Biological Sciences, Lecture 9). However, diffusion processes are complex, and modelling them analytically and with accuracy is difficult, even more when the geometry is not simple.</p><br/>
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<p>For this reason, we decided to consider a simplified model in which pheromone chemicals obey to the heat diffusion equation. Then, it is solved by the Euler numeric approximation in order to obtain the spatial and temporal distribution of pheromone concentration. <a class="normal-link-page" href="#">See more about heat equation and mathematical expressions for Euler method.</a></p><br/>
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<p> Moths seem to respond to gradients of pheromone concentration attracted towards the source, although there are other factors that lead moths sexually to pheromone sources such as optomotor anemotaxis (J. N. Perry and C. Wall , A Mathematical Model for the Flight of Pea Moth to Pheromone Traps Through a Crop). However, increasing the pheromone concentration to unnaturally high levels may disrupt male orientation (W. L. Roelofs and R. T. Carde, Responses of Lepidoptera to synthetic sex pheromone chemicals and their analogues). <a class="normal-link-page" href="#"> See more about the modeling of moth flight paths.</a></p><br/>
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<p>Using a modeling environment called <a class="normal-link-page" href="#">Netlogo</a>, we simulate the approximate moths behavior during the pheromone dispersion process. So, this will help us to predict moth response when they are also in presence of our synthetic plants.</p><br/>
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        </div>
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        <div id="tab2" class="tab">
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            <p>Since pheromones are chemicals released into the air, we have to consider both the motion of the fluid and the one of the particles suspended in the fluid.</p><br/>
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<p>The motion of fluids can be described by the <span class="black-bold">Navier–Stokes equations</span>. But the frequent nonlinearity of these equations makes most problems difficult or impossible to solve, since it may exists turbulences in the air flow [1]. Now attending to the particles suspended in the fluid, an option for pheromone dispersion modeling consists in the assumption of pheromones diffusive-like behavior.
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That is: pheromones are molecules that can undergo a diffusion process in which the random movement of gas molecules transport the chemical away from its source [2].</p><br/>
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<p>There are two ways to introduce the notion of diffusion: either a phenomenological approach starting with <span class="black-bold"> Fick's laws of diffusion</span>  and their mathematical consequences, or a physical and atomistic one, by considering the <span class="black-bold"> random walk</span>  of the diffusing particles.</p><br/>
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<p>In our case, we decided to hold our diffusion process by the <span class="black-bold">Fick's laws</span>. So it is postulated that the flux goes from regions of high concentration to regions of low concentration, with a magnitude that is proportional to the concentration gradient. However, diffusion processes are complex, and modelling them analytically and with accuracy is difficult, even more when the geometry is not simple (final distribution of our plants in the crop field). For this reason, we decided to consider a simplified model in which pheromone chemicals obey to the <span class="black-bold"> heat diffusion equation</span>.</p><br/><br/>
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<h3>Approximation</h3><br/><br/>
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The '''diffusion equation''' is a partial differential equation which describes density dynamics
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in a material undergoing diffusion. It is also used to describe processes exhibiting
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diffusive-like behavior, like in our case.
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The equation is usually written as:
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 +
$$\frac{\partial \phi (r,t) }{\partial t} = \nabla · [D(\phi,r)  \nabla \phi(r,t)]$$
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 +
where $\phi(r, t)$ is the density of the diffusing material at location r and time t and
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$D(\phi, r)$ is the collective diffusion coefficient for density $\phi$ at location $r$; and
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$\nabla$ represents the vector differential operator.
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If the diffusion coefficient does not depend on the density then the equation is linear and
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$D$ is constant. Thus, the equation reduces to the following linear differential equation:
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$$\frac{\partial \phi (r,t) }{\partial t} = D \nabla^2 \phi(r,t)$$
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also called the '''heat equation'''.
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Making use of this equation we can write pheromones chemicals diffusion equation with no
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wind effect consideration as:
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$$\frac{\partial c }{\partial t} = D \nabla^2 C = D \Delta c$$
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where c is the pheromone concentration,  $\Delta$ is the Laplacian operator, and $D$ is
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the pheromone diffusion constant in air.<br/><br/>
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If we consider the wind, we face a diffusion system with drift and an advection term is
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added to the equation above.
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$$\frac{\partial c }{\partial t} = D \nabla^2 c - \nabla \cdot (\vec{v} c )$$
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where $\vec{v}$ is the average ''velocity'' that the quantity is moving. Thus, $\vec{v}$
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would be the velocity of the air flow.<br/>
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For simplicity, we are not going to consider the third dimension. In $2D$ the equation
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would be:
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$$\frac{\partial c }{\partial t} = D \left(\frac{\partial^2 c }{\partial^2 x} + \frac{\partial^2 c }{\partial^2 y}\right) – \left(v_{x} \cdot \frac{\partial c }{\partial x} + v_{y} \cdot \frac{\partial c }{\partial y} \right) = D \left( c_{xx} + c_{yy}\right) - \left(v_{x} \cdot c_{x} + v_{y} \cdot c_{y}\right) $$
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<html><br/>
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<div align="center"><img width="650px" src="https://static.igem.org/mediawiki/2014/1/11/VUPVDiffusion_purple.png" alt="modeling_equations_solving" title="Netlogo Screen"></img></div><br/><br/>
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For determining a numeric solution for this partial differential equation are
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used the so-called finite difference methods. The technic consists in
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approximating differential ratios as $h$ is closer to zero, so they are useful to
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approximate differential equations.
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With finite difference methods, partial differential equations are replaced by
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its approximations in finite differences, resulting in an algebraic equations
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system. The algebraic equations system is solved in each node
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$(x_i,y_j,t_k)$. These discrete values describe the temporal and spatial
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distribution of the unknown function.<br/><br/>
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Although implicit methods are unconditionally stable so time steps could be larger and
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make the calculus process faster, the tool we have used to solve our heat equation is the
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Euler explicit method.
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Euler explicit method is the simplest option to approximate spatial derivatives, in which all
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values are assumed at the beginning of Time.<br/><br/>
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The equation gives the new value of the pheromone level in terms of initial values in that
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node and its immediate neighbors. Since all these values are known the process is called
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explicit.
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$$c(t_{k+1}) = c(t_k) + dt \cdot c'(t_k),$$
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Now applying this method for the first case (with no wind consideration) we followed the
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next steps.
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1. Split time $t$ into $n$ slices of equal length <i>dt</i>:
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$$ \left\{ \begin{array}{c} t_0 &=& 0 \\ t_k &=& k \cdot dt \\ t_n &=& t
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\end{array} \right. $$
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2. Considering the backward difference for the Euler explicit method implies that the
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expression that refers to the current pheromone level each time step is:
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$$c (x, y, t) \approx c (x, y, t - dt ) + dt \cdot c'(x, y, t)$$
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3. And now considering the spatial dimension, it is applied central differences to the Laplace operator $\Delta$, and the backward differences to the vector differential operator $\nabla$ ( in 2D and assuming equal steps in x and y directions):
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$$c (x, y, t) \approx c (x, y, t - dt ) + dt  \left( D \cdot \nabla^2 c (x, y, t)  - \nabla \vec{v} c (x, y, t)  \right)$$
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$$ D \cdot \nabla^2 c (x, y, t) = D \left( c_{xx} + c_{yy}\right) = D \frac{c_{i,j-1} + c_{i,j+1} + c_{i-1,j } + c_{i+1,j} – 4 c_{I,j}}{s} $$
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$$ \nabla \vec{v} c (x, y, t) = v_{x} \cdot c_{x} + v_{y} \cdot c_{y} = v_{x} \frac{c_{i,j} – c_{i-1,j}}{h} + v_{y} \frac{c_{i,j} – c_{i,j-1}}{h} $$
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With respect to the boundary conditions, they are null since we are considering an opened-space. Attending to the implementation and simulation of this method, <i>dt</i> must be small enough to avoid instability.
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<p><h3 class="encabezado" align="center">Home</h3></p><hr/><br/>
 
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<p align="center"><h4>Project overview (Draft)</h4></p><br/>
 
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<p>Pests are one of the major problems in agriculture because their effect cause huge losses in this sector. Since pest control techniques relying on the use of pesticides are becoming less and less popular, because of the environmental damages they can cause, treatment with pheromones are gaining ground among pest management.  
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Nowadays insect pheromones are chemically synthesised, which happens to be an expensive and complex process because of manufacturing costs and purification steps. In pursuit this line of thinking, our main efforts are aimed at avoiding the damage produced by pests in crops, specifically moths, which cause significant damage in crops all around the world.  
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Moths lay eggs on the crops after mating, then the larvae eat the crops causing considerable damage. Our objective is to find an alternative method of pest control using pheromones.</p><br/>
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<p>Male moths find the females by tracing the pheromones they release into the air. For the attraction to be effective, female of a certain species must release the exact proportion of pheromone components. However, the strategy to be used is not based in attraction but in confusion.  
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<div id="tab3" class="tab">
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When the major pheromone component concentration in the environment reaches high levels, males are not able to find the females. This strategy is called mate disruption.</p><br/>
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<p>Our final objective is to create a synthetic plant which can produce a determined pheromone specifically in their trichomes, allowing its diffusion and secretion to the environment, causing mating disruption among moths (Lepidoptera).
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<h3>The Idea</h3><br/>
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These plants would allow pest control simply sowing some of them around a crop field and inducing the pheromone production by applying a copper-rich nutrient solution before the insects mating season.  
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In addition, due to their induced sterility, these plants would be completely safe to cultivate in the field and easily recognizable due to their anthocyanin production which would turn them purple.</p><br/>
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            <p>When one stares at moths, they apparently move with erratic flight paths. It is possibly because of predator avoiding reasons.</p><br/>
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<p align="center">PHEROMONE BIOSYNTHESIS</p><br/>
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<p>The first step in the creation of this synthetic plant is to define and construct the metabolic pathway of pheromone production.</p><br/>
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<p> In this frame, sex pheromones influence in moth behavior is also considered. Since these are pheromones released by females in order to attract an individual of the opposite sex, it makes sense that males respond to <span class="purple-bold">gradients of sex pheromone concentration</span>, being attracted towards the source. As soon as a flying male <span class="green-bold">randomly</span> comes into conical pheromone-effective sphere of sex pheromone released by a virgin female, the male begins to seek the females in a zigzag way, approaches to the females and finally copulates with her [1].</p><br/><br/><br/>
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<p>"Most moth species utilize Type I pheromones that consist of straight-chain compounds 10-18 carbons in length with a functional group of a primary alcohol, aldehyde, or acetate ester, and usually with several double bonds, de novo synthesized in the pheromone gland through modifications of fatty acid biosynthetic pathways" [1].</p><br/>
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<p>Following the principle of modularity in synthetic biology, Hagström et al [2] produced the insect pheromone Z-11:160H in yeast from palmitic acid by introducing a delta-11 fatty-acyl desaturase and a fatty-acyl reductase from Agrotis segetum. Recently, Ding et al [3] had already done the same work taking into account that palmitate is synthesised in the chloroplast, leading to successful results.</p><br/>
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<h3>Approximation</h3><br/>
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<p>In Ding's work they use a delta 11 desaturase mRNA from Amyelois transitella (accession number JX964774); HarFAR_KKYR, an improved version of HarFAR-3 fatty acid reductase from Helicoverpa armigera (accession number JF709978) with an almost five folds conversion rate, and 1,2-diacyl-sn-glycerol:acetyl-CoA acetyltransferase from Euonymus alatus (accession
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number GU594061). With these three enzymes we would obtain two pheromones: Z-11:16OH and Z-11:16OAc.</p><br/>
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<img width="150px" style="float:left; margin-right: 15px; margin-bottom: 15px;" src="https://static.igem.org/mediawiki/2014/1/17/VUPVPolillita_con_vectores_v1.png" alt="moth_array"></img>
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<p>Moreover, inspired by Hagström's discussion we also thought about using an alcohol oxidase in order to try to obtain pheromone Z11-16:Ald from Z11-16:OH. According to the SolGenomics database, there is already a fatty-acid alcohol oxidase in Nicotiana benthamiana but there was no certainty that the enzyme would work in in vivo conditions.  
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On the other hand, we learned that Candida tropicalis had a fatty alcohol oxidase that catalysed the conversion from Z11-16:Ald to Z11-16:O [4,5,6,7]. (At the moment it is not clear from which organism will the fatty acid alcohol oxidase be obtained).</p><br/>
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<p>In this project we approximate the resulting moth movement as a vectorial combination of a <span class="purple-bold">gradient vector</span> and a <span class="green-bold">random vector</span>. The magnitude of the gradient vector comes from the change in pheromone concentration level among points separated by a differential stretch in space. More precisely, the gradient points in the direction of the <b>greatest rate of increase</b> of the function and its magnitude is the slope of the graph in that direction. The <span class="green-bold">random vector</span> is restricted in this ‘moth response’ model by a fixed angle, assuming that the turning movement is relatively continuous and for example the moth can’t turn 180 relative degrees at the next instant.</p><br/>
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<p>At the same time we were planning which pheromones could we produce, we also took into account on which insects will they have effect. We did a thorough search in the Pherobase database (http://www.pherobase.com) and we checked which insect met the conditions of having one of those as a major pheromone component and being a plague.</p><br/>
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<div align="center"><img src="https://static.igem.org/mediawiki/2014/d/de/Ivllobel_pathway.png" alt="pheromone_pathway" title="Pheromone Pathway"></img></div><br/>
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            <p>Since the objective of this project consists in avoiding pest damage by reaching the mating disrupting among moths, our synthetic plants are supposed to release enough sexual pheromone so as to <span class="red-bold">saturate moth perception</span>. In this sense the resulting moth vector movement will depend ultimately on the pheromone concentration levels in the field and the moth ability to follow better or worse the gradient of sex pheromone concentration.</p><br/>
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<div align="center"><p style="font-size: 0.8em; width: 70%;">Biosynthetic pathway of pheromone production. A continuous line shows the substrate and a discontinuous line shows the resulting pheromones.</p></div><br/>
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<p align="center">TRICHOMES</p><br/>
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<p>At this point, let’s highlight the three main aspects we consider for the characterization of males moth behavior:</p><br/>
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<p>Once the metabolic pathway needed to synthetize the desired pheromone has been properly described and characterized, a method that allows pheromone diffusion from the plant to the exterior environment must be implemented. With this objective in mind, we decided to express the enzymes required to produce the pheromones specifically in glandular trichomes, which are secretory structures naturally present in the plant.  
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For doing so, the enzyme coding sequence would be under the control of CSP2 promoter from Nicotiana tabacum [8], inducing their specific expression in trichomes and allowing the pheromones secretion.</p><br/>
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<p align="center">SWITCH</p><br/>
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[[File:Table_behavior.png|600px|center|Male moths behaviour characterization.]]
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<p>The pheromone production would suppose an additional energy cost to the plant, therefore the objective is to activate this pathway and the subsequent pheromone production only when necessary, upon Lepidoptera mating season. The regulation mechanism chosen with that purpose has been the one naturally controlling the CUP1 metallothionein gene in S. cervisiae [9].
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CUP1 function is to chelate copper ions to avoid its toxicity to the yeast; therefore it is only expressed under high copper concentration. This regulation mechanism relies on the expression of a transcription factor (ACE1 or CUP2) that changes its conformation under high copper concentrations allowing its interaction with a regulating region (Metal Responsive Element, MRE) in the promoter of the CUP1 gene.</p><br/>
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<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.9em; width: 700px;"><span class="black-bold">Table 1</span>. Male moths behaviour characterization.</p></div>
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<p>Taking as a reference this mechanism, Mett et al adapted it so it could be used in whole plants. Takanori Saijo and Akitsu Nagasawa [10] posteriorly improved its efficiency. We decided to take their work as an example and use a system in which the transcription factor CUP2 or ACE1 is expressed under the constitutive promoter P35S, and the enzymes responsible for the pheromone production are expressed under the regulation of a inducible promoter, composed of the MRE domain fused with the P35S minimal promoter.
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Therefore, CUP2 transcription factor would only interact with MRE and the pheromone will be produced under high copper concentrations. A copper-rich solution will be applied to the plant by foliar spraying or soil drenching during Lepidoptera mating season allowing pheromone production on demand.</p><br/>
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<p>In this context, this ensemble of behaviors could be translated in a sum of vectors in which the random vector has a constant module and changeable direction inside a range, whereas the gradient vector module is a function of the gradient in the field. The question now is: <b>how do we include the saturation effect in the resulting moth shift vector?</b></p><br/>
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<p align="center">BIOSAFETY</p><br/>
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<p>One of the concerns about having transgenic plants in the field involves its uncontrolled spread. It is being developed a biosecurity module containing two parts: a male sterility submodule and an identity preservation submodule.</p><br/>
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<p>With this in mind and focusing on the implementation process, our approach consists on the following:</p><br/>
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<p>The male sterility submodule is based on a barnase, an RNase gene from Bacillus amyloliquefaciens, specifically expressed in anthers under the regulation of the TA29 tapetum-specific promoter. As result, pollen from these plants will not be capable of fertilize plants.  
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The identity preservation submodule will express Rosea1, a transcription factor that activates anthocyanin synthesis pathways, leading to their accumulation, so that plants containing the biosafety module will be easily differentiated from non-transgenic plants.</p><br/>
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<p>The <span class="purple-bold">gradient vector</span> instead of experiencing a change in its <b>magnitude</b>, this will be always the <b>unit</b> and its <b>direction</b> that of the <span class="purple-bold">greatest rate of increase</span> of the pheromone concentration. A <span class="green-bold">random direction vector</span> with constant module will not be literally considered, but a <span class="green-bold">random turning angle</span> starting from the gradient vector direction.</p></br/>  
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<p align="center">FINAL PLANT</p><br/>
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<p>Finally, our purpose is to find an ideal plant which gathers the special characteristics to lay out the implementation of the plant as a real solution of the problem. This ideal plant is still to be determined, and will probably vary depending on the geographical situation of the crop.</p><br/>
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<p>Attending to the previous question <i>how do we include the saturation effect in the resulting moth shift vector?</i>, here the answer: <b>the dependence on the</b> <span class="red-bold">moth saturation level</span> (interrelated with the pheromone concentration in the field) <b>will state in the random turning angle</b>.</p><br/>
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<p align="center">References</p><br/>
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<div style="position: relative; left: 3%; width: 96%;"><ol>
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</html>
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<li>Matsumoto S (2010) Molecular mechanisms underlying sex pheromone production in moths. Biosci Biotechnol Biochem 74: 223-231.</li>
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[[File:Moth_vector.png|600px|center|Approximation of the male moths behaviour.]]
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<li>Hagström A, Wang HL, Lienard MA, Lassance JM, Johansson T, et al. (2013) A moth pheromone brewery: production of (Z)-11-hexadecenol by heterologous co-expression of two biosynthetic genes from a noctuid moth in a yeast cell factory. Microb Cell Fact 12: 125.</li>
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<li>Ding BJ, Hofvander P, Wang HL, Durrett TP, Stymne S, et al. (2014) A plant factory for moth pheromone production. Nat Commun 5: 3353.</li>
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<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.9em; width: 700px;"><span class="black-bold">Table 1</span>. Approximation of the male moths behaviour.</p></div>
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<li>Vanhanen S, West M, Kroon JT, Lindner N, Casey J, Cheng Q et al (2000) A consensus sequence for long-chain fatty-acid alcohol oxidases from Candida identifies a family of genes involved in lipid omega-oxidation in yeast with homologues in plants and bacteria. J Biol Chem. 275, 4445-52.</li>
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<li>Eirich LD, Craft DL, Steinberg L, Asif A, Eschenfeldt WH, et al. (2004) Cloning and characterization of three fatty alcohol oxidase genes from Candida tropicalis strain ATCC 20336. Appl Environ Microbiol 70: 4872-4879.</li>
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<p>This random turning angle will not follow a uniform distribution, but a Poisson distribution in which the <b>mean is zero </b>(no angle detour from the gradient vector direction) and the <b>standard-deviation</b> will be <b>inversely proportional to the intensity of the gradient of sex pheromone concentration in the field</b>. This approach will drive to a ‘sexual confusion’ of the insect as the field homogeneity increases, since the moth in its direction of displacement will fit the gradient direction with certain probabilities which depend on how saturated they are.</p><br/>
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<li>Kemp GD, Dickinson FM, Ratledge C (1991) Activity and substrate specificity of the fatty alcohol oxidase of Candida tropicalis in organic solvents. Appl Microbiol Biotechnol. 34(4), 441-445.</li>
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<li>Kemp GD, Dickinson FM, Ratledge (1988) Inducible long chain alcohol oxidase from alkane-grown Candida tropicalis. Appl Microbiol Biotechnol. 29(4), 370-374.</li>
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<h3>References:</h3>
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<li>Sallaud C, Giacalone C, Topfer R, Goepfert S, Bakaher N, et al. (2012) Characterization of two genes for the biosynthesis of the labdane diterpene Z-abienol in tobacco (Nicotiana tabacum) glandular trichomes. Plant J 72: 1-17.</li>
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<ol style="position: relative; left: 4%; width: 90%;">
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<li>Mett VL, Lochhead LP, Reynolds PH (1993) Copper-controllable gene expression system for whole plants. Proc Natl Acad Sci U S A 90: 4567-4571.</li>
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<li>Yoshitoshi Hirooka and Masana Suwanai. Role of Insect Sex Pheromone in Mating Behavior I. Theoretical Consideration on Release and Diffusion of Sex Pheromone in the Air. </li>
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</ol></div>
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<li>W. L. Roelofs and R. T. Carde. Responses of  Lepidoptera to Synthetic Sex Pheromone Chemicals and their Analogues. </li>
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</ol>
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<p></p><br/>
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        </div>
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<div id="tab4" class="tab">
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<br/>
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            <p>Using a modeling environment called Netlogo, we try to simulate the approximate moth population behavior when pheromone diffusion processes are given.</p><br/>
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<p> <a href="http://ccl.northwestern.edu/netlogo/">Netlogo</a> simulator can be find in its website at Northwestern University.</p>
 +
<p> To download the source file of our <span class="red-bold">Sexyplant</span> simlation in Netlogo click here:
 +
<a href="https://2014.igem.org/Team:Valencia_UPV/Modeling/sexyplants.nlogo" download>sexyplants.nlogo</a>
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<br/>
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<h3>SETUP:</h3>
 +
<ul style="list-style: disc; position: relative; left: 4%; width: 96%;">
 +
<li>We consider three  <span class="black-bold">agents</span>: <span class="marron-bold">male</span> and  <span class="fucsia-bold">female</span> moths, and  <span class="red-bold">sexyplants</span>.</li>
 +
<li>We have two kind of sexual pheromone emission sources: <span class="fucsia-bold">female</span> moths and <span class="red-bold">sexyplants</span>. </li>
 +
<li>Our scenario is an opened crop field where <span class="red-bold">sexyplants</span> are intercropped and moths fly following different patterns depending on its sex.</li>
 +
</ul>
 +
<p><span class="fucsia-bold">Females</span>, apart from emitting sexual pheromones, they move with erratic random flight paths. After mating, females are 2 hours in which they are not emitting pheromone.</p>
 +
 
 +
<p><span class="marron-bold">Males</span> also move randomly while they are under its detection threshold. But when they detect a certain pheromone concentration, they start to follow the pheromone concentration gradients until its saturation threshold is reached. </p>
 +
 
 +
<p> <span class="red-bold">sexyplants</span> act as continuously- emitting sources and their activity is regulated by a <span class="black-bold">Switch</span>.</p><br/>
 +
 
 +
<p> On the side of <span class="black-bold">pheromone diffusion</span> process, it is simulated in Netlogo by implementing the <span class="black-bold">Euler explicit</span> method. </p><br/>
 +
</html>
 +
[[File:Upv_simu1.png|600px|center|Figure 1. NETLOGO Simulation environment.]]
 +
<html>
 +
<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.9em; width: 700px;"><span class="black-bold">Figure 1</span>. NETLOGO Simulation environment.</p></div>
 +
 
 +
<h3>GO!</h3>
 +
<p>When <span class="red-bold">sexyplants</span> are switched-off, <span class="marron-bold">males</span> move randomly until they detect pheromone traces from <span class="fucsia-bold">females</span>, in that case they follow them. </p>
 +
<p>When <span class="red-bold">sexyplants</span> are switched-on, the pheromone starts to diffuse from them, rising up the concentration levels in the field. At first, the <span class="red-bold">sexyplants</span> have an effect of pheromone traps on the <span class="marron-bold">male</span> moths.</p><br/>
 +
 
 +
 
 +
</html>
 +
[[File:VUPV_Polillas.png|600px|center|Figure 2. On the left: sexyplants are switched-off and a male moth follows the pheromone trace from a female. On the right: sexyplants are switched on and a male moth go towards the static source like it happens with synthetic pheromone traps.]]
 +
<html>
 +
<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.9em; width: 700px;"><span class="black-bold">Figure 2</span>. On the left: sexyplants are switched-off and a male moth follows the pheromone trace from a female. On the right: sexyplants are switched on and a male moth go towards the static source like it happens with synthetic pheromone traps.</p></div>
 +
 
 +
<p>As the concentration rises in the field, it becomes more homogeneous. Remember that the <span class="green-bold">random turning angle</span> of the insect follows a Poisson distribution, in which the standard-deviation is inversely proportional to the intensity of the <span class="purple-bold">gradient</span>. Thus, the probability of the insect to take a <span class="black-bold">bigger</span> detour from the faced gradient vector direction is higher. This means that it is <span class="black-bold">less able</span> to follow pheromone concentration gradients, so <span class="black-bold">‘sexual confusion’ is induced</span>.</p>
 +
 
 +
</html>
 +
[[File:Upv_simu3.png|450px|center|Figure 3. NETLOGO Simulation of the field: sexyplants, female moths, pheromone diffusion and male moths.]]
 +
<html>
 +
<div align="center"><p style="text-align: justify; font-style: italic; font-size: 0.9em; width: 700px;"><span class="black-bold">Figure 3</span>. NETLOGO Simulation of the field: sexyplants, female moths, pheromone diffusion and male moths.</p></div>
 +
 
 +
      <h3> Parameters</h3>
 +
<p>
 +
The parameters of this model are not as well-characterized as we expected at first. Finding the accurate values of these parameters is not a trivial task, in the literature it is difficult to find a number experimentally obtained.</p>
 +
<p>So we decided to take an <span class="black-bold">inverse engineering approach</span>. Doing a model parameters swept, we simulate many possible scenarios, and then we come up with values of parameters corresponding to our desired one: insects get confused. This will be useful to know the limitations of our system and to help to decide the final distribution of our plants in the crop field. </p> <br/>
 +
 
 +
<dl>
 +
<dt>Diffusion coefficient</dt>
 +
<dd>Range of search: 0.01-0.2 cm^2/s <br/>
 +
References: [1], [2], [3], [5]</dd>
 +
<dt>Release rate (female)</dt>
 +
<dd>Range of search: 0.02-1 µg/h <br/>
 +
References: [4], [5]</dd>
 +
<dt>Release rate (sexyplant)</dt>
 +
<dd>The range of search that we have considered is a little wider than the one for the release rate of females. <br/>
 +
References: It generally has been found that pheromone dispensers releasing the
 +
chemicals above a certain emission rate will catch fewer males. The optimum release
 +
rate or dispenser load for trap catch varies greatly among species [4].  This certain emission rate above which male start to get confused could be the release rate from females.</dd>
 +
<dt>Detection threshold</dt>
 +
<dd>Range of search: 0.001-1 [Mass]/[Distante]^2</dd><br/>
 +
<dt>Saturation threshold </dt>
 +
<dd>Range of search: 1-5[Mass]/[ Distante]^2</dd><br/>
 +
<dt>Moth sensitivity</dt>
 +
<dd>This is a parameter referred to the capability of the insect to detect changes in pheromone concentration in the patch it is located and the neighbor patch. When the field becomes more homogeneous, an insect with higher sensitivity will be more able to follow the gradients.<br/>
 +
Range: 0-0.0009 <br/>
 +
(The maximum level of moth sensitivity has to be less than the minimum level of release rate of females, since this parameter is obtained from the difference)</dd>
 +
<dt>Wind force</dt>
 +
<dd>Range: -0.1 – 0.1 cm/sec <br/>
 +
References: [7] (700cm/sec!!!)</dd>
 +
<dt>Population</dt>
 +
<dd>The number of males and females can be selected by the observer.</dd>
 +
</dl>
 +
 
 +
<br/>
 +
<br/>
 +
<h3>Ticks (time step) !!!</h3>
 +
<p>We’ll consider the equivalence 20 ticks= 1 hour.  That is 1 tick = 3 minutes.</p>
 +
<br/>
 +
<h3>Patches !!!</h3>
 +
<p>The approximate velocity of a male moth flying towards the female in natural environment is 0.3 m/sec [6].  Each moth moves 1 patch per tick, so if 1 tick is equal to 3 minutes (180 sec), the patch is 54 meter long to get that velocity.</p>
 +
<p>One can modify the number of patches that conform the field so as to analyze its own case. </p>
 +
 
 +
<br/>
 +
 +
 +
<h3>References:</h3>
 +
<ol style="position: relative; left: 4%; width: 90%;">
 +
<li>Wilson et al.1969, Hirooka and Suwanai, 1976.</li>
 +
<li>Monchich abd Mauson, 1961, Lugs, 1968.</li>
 +
<li>G. A. Lugg. Diffusion Coefficients of Some Organic and Other Vapors in Air.</li>
 +
<li>W. L. Roelofs and R. T. Carde. Responses of  Lepidoptera to Synthetic Sex Pheromone  Chemicals and their Analogues, Page 386. </li>
 +
<li>R.W. Mankiny, K.W. Vick, M.S. Mayer, J.A. Coeffelt and P.S. Callahan (1980) Models For Dispersal Of Vapors in Open and Confined Spaces: Applications to Sex Pheromone Trapping in a Warehouse, Page 932, 940.</li>
 +
<li> Tal Hadad, Ally Harari, Alex Liberzon, Roi Gurka (2013) On the correlation of moth flight to characteristics of a turbulent plume. </li>
 +
<li> Average Weather For Valencia, Manises, Costa del Azahar, Spain. </li>
 +
</ol>
 +
<br/><br/>
 +
</div>
 +
 
 +
 +
        <div id="tab5" class="tab">
 +
<br/>
 +
   
 +
<p>
 +
The aim consists of reducing the possibility of meeting among moths of opposite sex. Thus, we will analyze the number of meetings in the three following cases:
 +
</p>
 +
 
 +
<ol style="position: relative; left: 4%; width: 90%;">
 +
<li>When sexyplants are switched-off and males only interact with females.</li>
 +
<li>When sexyplants are switched-on and have an effect of trapping males.</li>
 +
<li>When sexyplants are swiched-on and males get confused when the concentration of pheromone level is higher than their saturation threshold.</li>
 +
</ol>
 +
 
 +
<p>
 +
It is also interesting to analyze a fourth case, what does it happen if females wouldn’t emit pheromones and males just move randomly through the field? :</p>
 +
<ol start="4" style="position: relative; left: 4%; width: 90%;">
 +
<li>Males and females move randomly. How much would our results differ from the rest of cases? </li>
 +
</ol>
 +
 
 +
<p>
 +
What is important is that between the first and the third case, the number of meetings should be less in the latter than in the former. Then we are closer to our objective fulfillment.
 +
</p><br/>
 +
</div>
 +
</div>
 +
<br/><br/><br/>
 +
<a class="button-content" id="goto-left" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Modeling/overview"><strong>Go to Modeling Overview</strong></a>
 +
<a class="button-content" id="goto-right" align="center" href="https://2014.igem.org/Team:Valencia_UPV/Modeling/fba"><strong>Go to Pheromone Production</strong></a></br></br></br>
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Latest revision as of 14:57, 17 October 2014

Modeling > Pheromone Diffusion


Pheromone Diffusion

and Moths Response


Sexually communication among moths is accomplished chemically by the release of an "odor" into the air. This "odor" is the sexual pheromones.


female_sex_pheromones

Figure 1. Female moth releasing sex pheromones and male moth.


Pheromones are molecules that can undergo a diffusion process in which the random movement of gas molecules transport the chemical away from its source (Sol I. Rubinow, Mathematical Problems in the Biological Sciences, Lecture 9). However, diffusion processes are complex, and modelling them analytically and with accuracy is difficult, even more when the geometry is not simple.


For this reason, we decided to consider a simplified model in which pheromone chemicals obey to the heat diffusion equation. Then, it is solved by the Euler numeric approximation in order to obtain the spatial and temporal distribution of pheromone concentration. See more about heat equation and mathematical expressions for Euler method.


Moths seem to respond to gradients of pheromone concentration attracted towards the source, although there are other factors that lead moths sexually to pheromone sources such as optomotor anemotaxis (J. N. Perry and C. Wall , A Mathematical Model for the Flight of Pea Moth to Pheromone Traps Through a Crop). However, increasing the pheromone concentration to unnaturally high levels may disrupt male orientation (W. L. Roelofs and R. T. Carde, Responses of Lepidoptera to synthetic sex pheromone chemicals and their analogues). See more about the modeling of moth flight paths.


Using a modeling environment called Netlogo, we simulate the approximate moths behavior during the pheromone dispersion process. So, this will help us to predict moth response when they are also in presence of our synthetic plants.


Since pheromones are chemicals released into the air, we have to consider both the motion of the fluid and the one of the particles suspended in the fluid.


The motion of fluids can be described by the Navier–Stokes equations. But the frequent nonlinearity of these equations makes most problems difficult or impossible to solve, since it may exists turbulences in the air flow [1]. Now attending to the particles suspended in the fluid, an option for pheromone dispersion modeling consists in the assumption of pheromones diffusive-like behavior. That is: pheromones are molecules that can undergo a diffusion process in which the random movement of gas molecules transport the chemical away from its source [2].


There are two ways to introduce the notion of diffusion: either a phenomenological approach starting with Fick's laws of diffusion and their mathematical consequences, or a physical and atomistic one, by considering the random walk of the diffusing particles.


In our case, we decided to hold our diffusion process by the Fick's laws. So it is postulated that the flux goes from regions of high concentration to regions of low concentration, with a magnitude that is proportional to the concentration gradient. However, diffusion processes are complex, and modelling them analytically and with accuracy is difficult, even more when the geometry is not simple (final distribution of our plants in the crop field). For this reason, we decided to consider a simplified model in which pheromone chemicals obey to the heat diffusion equation.



Approximation



The diffusion equation is a partial differential equation which describes density dynamics in a material undergoing diffusion. It is also used to describe processes exhibiting diffusive-like behavior, like in our case.

The equation is usually written as:

$$\frac{\partial \phi (r,t) }{\partial t} = \nabla · [D(\phi,r) \nabla \phi(r,t)]$$

where $\phi(r, t)$ is the density of the diffusing material at location r and time t and $D(\phi, r)$ is the collective diffusion coefficient for density $\phi$ at location $r$; and $\nabla$ represents the vector differential operator.

If the diffusion coefficient does not depend on the density then the equation is linear and $D$ is constant. Thus, the equation reduces to the following linear differential equation: $$\frac{\partial \phi (r,t) }{\partial t} = D \nabla^2 \phi(r,t)$$

also called the heat equation.

Making use of this equation we can write pheromones chemicals diffusion equation with no wind effect consideration as:

$$\frac{\partial c }{\partial t} = D \nabla^2 C = D \Delta c$$

where c is the pheromone concentration, $\Delta$ is the Laplacian operator, and $D$ is the pheromone diffusion constant in air.

If we consider the wind, we face a diffusion system with drift and an advection term is added to the equation above.

$$\frac{\partial c }{\partial t} = D \nabla^2 c - \nabla \cdot (\vec{v} c )$$

where $\vec{v}$ is the average velocity that the quantity is moving. Thus, $\vec{v}$ would be the velocity of the air flow.

For simplicity, we are not going to consider the third dimension. In $2D$ the equation would be:


$$\frac{\partial c }{\partial t} = D \left(\frac{\partial^2 c }{\partial^2 x} + \frac{\partial^2 c }{\partial^2 y}\right) – \left(v_{x} \cdot \frac{\partial c }{\partial x} + v_{y} \cdot \frac{\partial c }{\partial y} \right) = D \left( c_{xx} + c_{yy}\right) - \left(v_{x} \cdot c_{x} + v_{y} \cdot c_{y}\right) $$


modeling_equations_solving


For determining a numeric solution for this partial differential equation are used the so-called finite difference methods. The technic consists in approximating differential ratios as $h$ is closer to zero, so they are useful to approximate differential equations.

With finite difference methods, partial differential equations are replaced by its approximations in finite differences, resulting in an algebraic equations system. The algebraic equations system is solved in each node $(x_i,y_j,t_k)$. These discrete values describe the temporal and spatial distribution of the unknown function.

Although implicit methods are unconditionally stable so time steps could be larger and make the calculus process faster, the tool we have used to solve our heat equation is the Euler explicit method.

Euler explicit method is the simplest option to approximate spatial derivatives, in which all values are assumed at the beginning of Time.

The equation gives the new value of the pheromone level in terms of initial values in that node and its immediate neighbors. Since all these values are known the process is called explicit.

$$c(t_{k+1}) = c(t_k) + dt \cdot c'(t_k),$$

Now applying this method for the first case (with no wind consideration) we followed the next steps.

1. Split time $t$ into $n$ slices of equal length dt: $$ \left\{ \begin{array}{c} t_0 &=& 0 \\ t_k &=& k \cdot dt \\ t_n &=& t \end{array} \right. $$

2. Considering the backward difference for the Euler explicit method implies that the expression that refers to the current pheromone level each time step is:

$$c (x, y, t) \approx c (x, y, t - dt ) + dt \cdot c'(x, y, t)$$

3. And now considering the spatial dimension, it is applied central differences to the Laplace operator $\Delta$, and the backward differences to the vector differential operator $\nabla$ ( in 2D and assuming equal steps in x and y directions):

$$c (x, y, t) \approx c (x, y, t - dt ) + dt \left( D \cdot \nabla^2 c (x, y, t) - \nabla \vec{v} c (x, y, t) \right)$$ $$ D \cdot \nabla^2 c (x, y, t) = D \left( c_{xx} + c_{yy}\right) = D \frac{c_{i,j-1} + c_{i,j+1} + c_{i-1,j } + c_{i+1,j} – 4 c_{I,j}}{s} $$ $$ \nabla \vec{v} c (x, y, t) = v_{x} \cdot c_{x} + v_{y} \cdot c_{y} = v_{x} \frac{c_{i,j} – c_{i-1,j}}{h} + v_{y} \frac{c_{i,j} – c_{i,j-1}}{h} $$


With respect to the boundary conditions, they are null since we are considering an opened-space. Attending to the implementation and simulation of this method, dt must be small enough to avoid instability.


The Idea


When one stares at moths, they apparently move with erratic flight paths. It is possibly because of predator avoiding reasons.


In this frame, sex pheromones influence in moth behavior is also considered. Since these are pheromones released by females in order to attract an individual of the opposite sex, it makes sense that males respond to gradients of sex pheromone concentration, being attracted towards the source. As soon as a flying male randomly comes into conical pheromone-effective sphere of sex pheromone released by a virgin female, the male begins to seek the females in a zigzag way, approaches to the females and finally copulates with her [1].




Approximation


moth_array

In this project we approximate the resulting moth movement as a vectorial combination of a gradient vector and a random vector. The magnitude of the gradient vector comes from the change in pheromone concentration level among points separated by a differential stretch in space. More precisely, the gradient points in the direction of the greatest rate of increase of the function and its magnitude is the slope of the graph in that direction. The random vector is restricted in this ‘moth response’ model by a fixed angle, assuming that the turning movement is relatively continuous and for example the moth can’t turn 180 relative degrees at the next instant.


Since the objective of this project consists in avoiding pest damage by reaching the mating disrupting among moths, our synthetic plants are supposed to release enough sexual pheromone so as to saturate moth perception. In this sense the resulting moth vector movement will depend ultimately on the pheromone concentration levels in the field and the moth ability to follow better or worse the gradient of sex pheromone concentration.


At this point, let’s highlight the three main aspects we consider for the characterization of males moth behavior:


Male moths behaviour characterization.

Table 1. Male moths behaviour characterization.

In this context, this ensemble of behaviors could be translated in a sum of vectors in which the random vector has a constant module and changeable direction inside a range, whereas the gradient vector module is a function of the gradient in the field. The question now is: how do we include the saturation effect in the resulting moth shift vector?


With this in mind and focusing on the implementation process, our approach consists on the following:


The gradient vector instead of experiencing a change in its magnitude, this will be always the unit and its direction that of the greatest rate of increase of the pheromone concentration. A random direction vector with constant module will not be literally considered, but a random turning angle starting from the gradient vector direction.


Attending to the previous question how do we include the saturation effect in the resulting moth shift vector?, here the answer: the dependence on the moth saturation level (interrelated with the pheromone concentration in the field) will state in the random turning angle.


Approximation of the male moths behaviour.

Table 1. Approximation of the male moths behaviour.

This random turning angle will not follow a uniform distribution, but a Poisson distribution in which the mean is zero (no angle detour from the gradient vector direction) and the standard-deviation will be inversely proportional to the intensity of the gradient of sex pheromone concentration in the field. This approach will drive to a ‘sexual confusion’ of the insect as the field homogeneity increases, since the moth in its direction of displacement will fit the gradient direction with certain probabilities which depend on how saturated they are.


References:

  1. Yoshitoshi Hirooka and Masana Suwanai. Role of Insect Sex Pheromone in Mating Behavior I. Theoretical Consideration on Release and Diffusion of Sex Pheromone in the Air.
  2. W. L. Roelofs and R. T. Carde. Responses of Lepidoptera to Synthetic Sex Pheromone Chemicals and their Analogues.



Using a modeling environment called Netlogo, we try to simulate the approximate moth population behavior when pheromone diffusion processes are given.


Netlogo simulator can be find in its website at Northwestern University.

To download the source file of our Sexyplant simlation in Netlogo click here: sexyplants.nlogo

SETUP:

  • We consider three agents: male and female moths, and sexyplants.
  • We have two kind of sexual pheromone emission sources: female moths and sexyplants.
  • Our scenario is an opened crop field where sexyplants are intercropped and moths fly following different patterns depending on its sex.

Females, apart from emitting sexual pheromones, they move with erratic random flight paths. After mating, females are 2 hours in which they are not emitting pheromone.

Males also move randomly while they are under its detection threshold. But when they detect a certain pheromone concentration, they start to follow the pheromone concentration gradients until its saturation threshold is reached.

sexyplants act as continuously- emitting sources and their activity is regulated by a Switch.


On the side of pheromone diffusion process, it is simulated in Netlogo by implementing the Euler explicit method.


Figure 1. NETLOGO Simulation environment.

Figure 1. NETLOGO Simulation environment.

GO!

When sexyplants are switched-off, males move randomly until they detect pheromone traces from females, in that case they follow them.

When sexyplants are switched-on, the pheromone starts to diffuse from them, rising up the concentration levels in the field. At first, the sexyplants have an effect of pheromone traps on the male moths.


Figure 2. On the left: sexyplants are switched-off and a male moth follows the pheromone trace from a female. On the right: sexyplants are switched on and a male moth go towards the static source like it happens with synthetic pheromone traps.

Figure 2. On the left: sexyplants are switched-off and a male moth follows the pheromone trace from a female. On the right: sexyplants are switched on and a male moth go towards the static source like it happens with synthetic pheromone traps.

As the concentration rises in the field, it becomes more homogeneous. Remember that the random turning angle of the insect follows a Poisson distribution, in which the standard-deviation is inversely proportional to the intensity of the gradient. Thus, the probability of the insect to take a bigger detour from the faced gradient vector direction is higher. This means that it is less able to follow pheromone concentration gradients, so ‘sexual confusion’ is induced.

Figure 3. NETLOGO Simulation of the field: sexyplants, female moths, pheromone diffusion and male moths.

Figure 3. NETLOGO Simulation of the field: sexyplants, female moths, pheromone diffusion and male moths.

Parameters

The parameters of this model are not as well-characterized as we expected at first. Finding the accurate values of these parameters is not a trivial task, in the literature it is difficult to find a number experimentally obtained.

So we decided to take an inverse engineering approach. Doing a model parameters swept, we simulate many possible scenarios, and then we come up with values of parameters corresponding to our desired one: insects get confused. This will be useful to know the limitations of our system and to help to decide the final distribution of our plants in the crop field.


Diffusion coefficient
Range of search: 0.01-0.2 cm^2/s
References: [1], [2], [3], [5]
Release rate (female)
Range of search: 0.02-1 µg/h
References: [4], [5]
Release rate (sexyplant)
The range of search that we have considered is a little wider than the one for the release rate of females.
References: It generally has been found that pheromone dispensers releasing the chemicals above a certain emission rate will catch fewer males. The optimum release rate or dispenser load for trap catch varies greatly among species [4]. This certain emission rate above which male start to get confused could be the release rate from females.
Detection threshold
Range of search: 0.001-1 [Mass]/[Distante]^2

Saturation threshold
Range of search: 1-5[Mass]/[ Distante]^2

Moth sensitivity
This is a parameter referred to the capability of the insect to detect changes in pheromone concentration in the patch it is located and the neighbor patch. When the field becomes more homogeneous, an insect with higher sensitivity will be more able to follow the gradients.
Range: 0-0.0009
(The maximum level of moth sensitivity has to be less than the minimum level of release rate of females, since this parameter is obtained from the difference)
Wind force
Range: -0.1 – 0.1 cm/sec
References: [7] (700cm/sec!!!)
Population
The number of males and females can be selected by the observer.


Ticks (time step) !!!

We’ll consider the equivalence 20 ticks= 1 hour. That is 1 tick = 3 minutes.


Patches !!!

The approximate velocity of a male moth flying towards the female in natural environment is 0.3 m/sec [6]. Each moth moves 1 patch per tick, so if 1 tick is equal to 3 minutes (180 sec), the patch is 54 meter long to get that velocity.

One can modify the number of patches that conform the field so as to analyze its own case.


References:

  1. Wilson et al.1969, Hirooka and Suwanai, 1976.
  2. Monchich abd Mauson, 1961, Lugs, 1968.
  3. G. A. Lugg. Diffusion Coefficients of Some Organic and Other Vapors in Air.
  4. W. L. Roelofs and R. T. Carde. Responses of Lepidoptera to Synthetic Sex Pheromone Chemicals and their Analogues, Page 386.
  5. R.W. Mankiny, K.W. Vick, M.S. Mayer, J.A. Coeffelt and P.S. Callahan (1980) Models For Dispersal Of Vapors in Open and Confined Spaces: Applications to Sex Pheromone Trapping in a Warehouse, Page 932, 940.
  6. Tal Hadad, Ally Harari, Alex Liberzon, Roi Gurka (2013) On the correlation of moth flight to characteristics of a turbulent plume.
  7. Average Weather For Valencia, Manises, Costa del Azahar, Spain.



The aim consists of reducing the possibility of meeting among moths of opposite sex. Thus, we will analyze the number of meetings in the three following cases:

  1. When sexyplants are switched-off and males only interact with females.
  2. When sexyplants are switched-on and have an effect of trapping males.
  3. When sexyplants are swiched-on and males get confused when the concentration of pheromone level is higher than their saturation threshold.

It is also interesting to analyze a fourth case, what does it happen if females wouldn’t emit pheromones and males just move randomly through the field? :

  1. Males and females move randomly. How much would our results differ from the rest of cases?

What is important is that between the first and the third case, the number of meetings should be less in the latter than in the former. Then we are closer to our objective fulfillment.





Go to Modeling Overview Go to Pheromone Production