Project Description

A problem of biblical proportions

Locust plagues have afflicted human civilisations throughout history. Drawings in ancient Egyptian pyramids already display the devastation caused by the locusts' appetite and the ensuing famine [1]. Unfortunately, this problem of biblical proportion has not been left in the past. Recently, humongous swarms of the desert locust, Schistocerca gregaria, have threatened the livelihood of communities, endangering food security and economic stability [2]. The Food and Agriculture Organization (FAO) of the United Nations predicts that a staggering 10% of the world population will be affected by the current plague [3]. Although there are 19 species classified as locusts [4], our target is the desert locust because this is the species that inflicts the most damage [5].

Normally, desert locusts live solitary lives, but under the right environmental conditions they come together. When a critical locust density is reached, the phenotype of these insects changes from a solitary to a gregarious phase [4]. This phenomenon is referred to as phase polyphenism (Figure 1), and is key to the swarming behaviour observed during plagues. The switch to the gregarious phase is triggered when the locusts touch each other’s hind legs, or when they see or smell each other. They change size and colour, have a less restricted diet and become voracious [6], eating their bodyweight (2 grams) every day. Although this may seem insignificant, a swarm of one square kilometre consumes the same amount of food as 35,000 people a day [3]. Recently, a swarm in Kenya has been spotted with a size of 2,400 square kilometres, containing between 4 and 8 billion locusts. This swarm consumes the daily equivalent of 3.5 million people [2].

Phage polyphenism
Figure 1. Phase polyphenism of the desert locust (Schistocerca gregaria). Alterations of the desert locusts from a solitary phage to a gregarious phase.

The FAO and national governments are responsible for locust control. These organisations have two control methods available: chemicals and biopesticides. The chemical pesticides, although effective, are not specific to the desert locust. Therefore, they are also harmful to other animals, humans and the environment [7]. The alternative, a bio-pesticide called Metarhizium anisopliae, is a fungus that is specific to locusts. However, it works rather slow, with a killing time of 7 to 14 days [2]. Because of this lack of speed, it cannot be used in emergency responses during sudden outbreaks.
In essence, the people fighting the desert locusts are in need of a safe and fast solution to end these troubles once and for all. We as a team were shocked about these events and discovered an underlying passion in all of us to helping fight the desert locusts.

“Our mission is to tackle the locust crisis by developing a fast and safe biopesticide through responsible innovation and collaboration.”

PHOCUS; Target locusts from within

PHOCUS is a targeted bacteriophage-based biopesticide used to control the desert locust crisis. Our aim is to kill the locusts by producing toxic molecules in their gut. These molecules will be produced by the gut bacteria after infection with our engineered bacteriophage. The toxins we want to produce are the locust specific Cry7Ca1 toxin and short-hairpin RNAs (shRNAs) that target essential locust genes. This process is illustrated in Figure 2.

Project Overview
Figure 2. Project graphical overview. 1. Bacteriophages are engineered to contain DNA coding for our toxic molecules. 2. Engineered bacteriophages will be sprayed on vegetation in affected areas. 3. Locusts will eat the crops and ingest the bacteriophages. 4. Bacteriophages infect host bacteria and produce Cry7Ca1 and shRNA. Upon lysis, the cell bursts and the toxins and shRNAs are released. 5. Toxin and shRNA release causes locusts to die, swarms disappear.

Delivery: Engineered bacteriophages

Our delivery method consists of bacteriophages, as they are viruses that specifically infect bacteria [8]. PHOCUS targets the bacteria in the locust gut. Although the microbiota of different sections of the locust gut vary among individuals and species, the Enterobacter genus has been reported to be the most abundant [9]. To account for any variety in the core Enterobacter population in the locust gut, our biopesticide consists of a cocktail of phages targeting a variety of bacteria from the Enterobacter genus. To reach the locust gut target bacteria, our engineered bacteriophages will be sprayed on vegetation that will be eaten by gregarious locusts from the swarms. The relatively mild pH (7-8) [10] of their gut allows the bacteriophages to survive there [11].

We have used Escherichia coli (E. coli) BL21 (DE3), a bacteria from the genus Enterobacter, as our model organism, together with the bacteriophage T7. We have developed a proof of concept to choose the position where we would insert our desired gene in the engineered bacteriophage by using GFP as a reporter. We successfully engineered T7 phages to contain GFP using the Bacteriophage Recombineering of Electroporated DNA (BRED) method [12] (Figure 3).

Proposed implementation
Figure 3. The BRED method was used to engineer T7 bacteriophages to replace a non-essential gene with GFP. Linear double-stranded DNA (dsDNA) and T7 phages were co-electroporated in BL21 (DE3) cells. Recombination of the dsDNA and the non-essential gene resulted in engineered T7 phages containing GFP.

Mode of action: killing the gregarious locusts

Our goal is to kill the gregarious locusts present in the swarms by using a specific and unique complementary approach that combines the insecticidal activity of the crystal toxin Cry7Ca1 and the gene silencing effect of RNA interference (RNAi). The bacteriophage will carry the DNA coding for the Cry7Ca1 toxin from Bacillus thuringiensis (Bt), as it has been shown to be effective against locusts [13, 14], as well as shRNA that specifically targets essential locust genes. We successfully expressed the Cry7Ca1 toxin under a T7 promoter in E. coli BL21 (DE3) and purified it (see Results). Moreover, we designed and transcribed shRNA in vitro with T7 polymerase. As the shRNA needs to be transported from the locust gut to the hemolymph to be effective (see RNA interference (RNAi)), we designed a cargo consisting of different proteins. We validated the binding of a key protein of this proposed delivery cargo to the designed shRNA. In order to be converted into short silencing RNA (siRNA), the shRNA has to be cleaved by Dicer, an essential enzyme of the RNAi maturation pathway (see RNA interference (RNAi)). We have demonstrated that the designed and in vitro transcribed shRNA can be cleaved by Dicer (Results).

Mathematical models of phage infection and toxin production

Once a bacteriophage finds its bacterium, it injects its DNA into it. Upon infection, the bacteriophage hijacks the cellular machinery of the bacterium and turns it into a factory expressing and amplifying the injected DNA. The Cry7Ca1 and shRNA will be produced inside these bacteria together with new engineered bacteriophages. We decided to use lytic bacteriophages in our project to ensure enough production of our toxin. Lytic bacteriophages rapidly take over the host, make the host produce phages and toxins and cause release of toxins after lysis occurs [8]. When lysis happens, the bacteria burst and the produced compounds and bacteriophages are released inside the locust gut. Those bacteriophages then begin a new infective cycle (Figure 6).

Proposed implementation
Figure 6. Schematic of the bacteriophage-host interaction. Lytic bacteriophage infects its bacterium, the cellular machinery is taken over and new phages and toxins are produced. Consecutively the cell lyses, the toxins and phages are released. The newly made phages infect bacteria; the cycle begins anew.

To gain a better insight into this infection process, as well as feasibility of our biopesticide, we modelled the evolution over time of the number of bacteria, phages, and produced toxin. Furthermore, since speed is one of the design requirements of PHOCUS, we used modelling to gain insight in the speed at which the toxins are produced inside the locust gut. To determine the speed of our biopesticide we developed a model of phage infection and toxin production based on the work of Beretta and Kuang [26]. With this model we demonstrated the capability of our biopesticide to produce lethal quantities of toxins to kill the locust within 7 days. Furthermore, we demonstrated which design strategies are most effective in improving the effectiveness of our biopesticide. At last, we concluded that the rate at which bacteria acquire resistance does not directly pose a threat to our biopesticide.

However, in the desert locust gut, bacteria most likely reside in micro-colonies [Dr. O. Lavy, personal interview, 27, 28]. To understand how the spatial distribution of the bacteria in the gut influences phage propagation and toxin production, we modelled the bacterial population as a 2D biofilm with an agent-based description of phages based on the work of Simmons et al. [29]. This model allowed us to study the influence of the spatial organisation of bacteria on the effectiveness of our biopesticide. With this model we demonstrated the importance of phage mobility inside the biofilm for effective toxin production. Additionally, we showed how the spatial organization of bacteria in such biofilms promotes toxin production. At last, we demonstrated how biofilm heterogeneity (fraction of target and non-target bacteria) must be taken into account in the design of our biopesticide.


In short, we designed PHOCUS, a specific and safe biopesticide targeting the desert locust. The biopesticide consists of a cocktail of engineered bacteriophages targeting the Enterobacter genus in the gut of the locusts. The engineered bacteriophages contain the DNA coding for the locust specific Cry7Ca1 toxin and shRNAs. The feasibility of our biopesticide against locusts has been analysed through mathematical models. We showed that the toxic molecules are produced fast. Therefore, we conclude that our biopesticide has the potential to kill the locusts fast.

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