Safe and fast, two simple words, but vital for an effective biopesticide. The Food and Agriculture Organisation (FAO) and the national government of Kenya, a country infested by locusts, highlighted the pressing need for a pesticide with these attributes to solve the actual locust crisis. Chemical pesticides are effective but not specific to desert locust [1], and a safer alternative used, the fungus Metarhizium anisopliae, is too slow [2]. Throughout the process of designing our biopesticide, we conducted interviews with experts from different disciplines. They helped us understand the complexities associated with designing a safe and fast solution to the locust crisis. This led us to design PHOCUS: a bacteriophage based biopesticide that is fast and safe.

Figure 1. PHOCUS: a bacteriophage-based pesticide that is fast and safe.

PHOCUS is a biopesticide based on engineered bacteriophages that infect the gut bacteria of the locust. Upon infection, the bacteria produce molecules that are specifically toxic to locusts: the crystal protein, Cry7Ca1, and an RNA interference (RNAi) precursor. The Cry7Ca1 toxin, from Bacillus thuringiensis strain BTH-13, has been described to have specific toxicity against locust [3, 4]. Cry7Ca1 punctures the gut lining facilitating the RNAi precursor to reach the hemolymph, where it silences the expression of locust essential genes.

To gain more insight into the feasibility of PHOCUS as a safe and fast biopesticide, we developed two mathematical models. With them we aimed to (1) predict the speed at which the toxins would be produced, and (2) acquire insights on how the spatial distribution of the locusts gut affect the host-phage population dynamics. From the former, we learned that phage infection and toxin production is a fast process. We also learned which strategies were the best to improve the production of the toxin. For the latter model, we could determine that phage mobility is a crucial property that influences toxin production.

Our target: the desert locusts

We target the desert locust (Schistocerca gregaria) as this is the locust species that causes the most economic and environmental damage. This desert locust threatens the livelihood of one-tenth of the global population [5] and is considered to be the most important species of locust to tackle due to its ability to migrate over large distances and rapidly multiply [6].

Throughout the past months, desert locusts have been found in Northern Africa, the Arabian peninsula and the South of Asia (Figure 2). During a plague, locust swarms can spread out to over 60 countries and cover up to one-fifth of the Earth’s land [5]. After selecting the desert locust as our target, we worked towards designing a biopesticide that is specific and therefore safe for other insects and animals. Our biopesticide also needs to work fast enough to eliminate swarms.

Figure 2. Areas affected by desert locust plagues [5].

Engineering bacteriophages instead of bacteria

In order to deliver the toxins to the desert locust, lytic bacteriophages will be used as a vector. Bacteriophages are a type of virus that infect and replicate within bacteria [7]. Lytic bacteriophages infect and reproduce fast as the cellular machinery of the host is taken over after infection and all energy is put towards expressing the viral genetic information.

Engineering bacteriophages instead of bacteria

We decided to engineer bacteriophages and not the bacteria. There are several reasons why using engineered bacteriophages is a superior delivery strategy than engineered bacteria:

  • After phage infection, bacteriophages hijack the bacterial transcription/translation system to redirect all resources to the production of new phage particles. If we would use engineered bacteria, upon delivery not all of the cell's energy and resources would be dedicated to producing our toxic molecules.
  • By using phages we take advantage of the fact that the bacteria are already present in the gut. In contrast to bacteria, which first needs to settle inside the gut and out-compete the bacteria that are already present, our factories are already available.
  • In the locust gut, the phages are expected to produce higher levels of toxin and faster than bacteria. We do not need to deliver many phages to reach high toxin levels as they propagate and spread upon infection. If we would use bacteria they would need to take over the gut microbiome or we would have to deliver very high numbers of bacteria inside the gut to produce enough toxin.

The target of the bacteriophage

The bacteriophages will target the gut bacteria of the locust (Figure 5). The bacterial community composition of the desert locust gut has been studied thoroughly to investigate the interactions between the locust and locust gut bacteria [12, 13, 14, 15]. Most of these studies looked at the role and abundance of Pantoea agglomerans, as there were reasons to believe that this bacterium was involved in the production of phenolic components of a locust cohesion pheromone. However, a recent study shows that neither P. agglomerans, nor any other specific bacterial species, is consistently present in the gut of the desert locust [16]. Nevertheless, this study shows that gregarious, field-collected, desert locusts maintain a core population of the bacterial Enterobacter genus [Dr. O. Lavy, personal interview]. 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.

Figure 5. The bacteriophages encounter their target bacteria inside the locust gut.

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 contain the DNA coding for the Cry7Ca1 toxin from Bacillus thuringiensis (Bt), as it has been shown to be effective against locusts [3, 4], as well as a short-hairpin RNA (shRNA) that will specifically target unique sequences of locust essential genes.

Although both these options have been shown to work against locusts, they are not yet a good fit to be implemented in the field. For instance, the Cry7Ca1 toxin, specific to locust, would have to be provided to them. However, when exposed to the expected harsh environmental conditions, the protein could quickly lose its activity. In order to circumvent this issue, we propose to produce them together with the bacteriophages. Furthermore, the dsRNA would have to be injected into the locust, rendering the control of swarms unfeasible. To overcome this problem, we designed a method to deliver dsRNA to the necessary location through the locust gut. By combining existing toxins, and a novel delivery method, we can develop an effective biopesticide.

After further research we realised that co-expressing Cry7Ca1 and shRNA together would enhance their effectiveness. The Cry7Ca1 toxin will poke holes into the locust gut [3, 4]. These holes will favour the shRNA produced to reach the hemolymph, the location where shRNA has shown to work best. This combined approach will also decrease the change of resistance formation (see Safety).

Mathematical models of phage infection and toxin production

Sufficient amounts of toxin need to be produced inside the locust gut for our biopesticide to be effective. In the previously mentioned Cry7Ca1 study, they indicated that approximately 0.87 µg/ml of activated Cry7Ca1 toxin is required for a 50% lethality in Locusta migratoria manilensis [4]. Similarly, the injection of 30 ng of dsRNA in the hemolymph of the desert locust is required for a maximum knockdown effect [27]. Before testing our biopesticide on actual locusts, we decided to use mathematical models to gain insights on how our biopesticide would function and on the speed at which the toxins are produced. From this, we obtained knowledge about which phage properties are relevant to consider for an effective functioning of our biopesticide.

Host-phage interactions

First, we adapted a model of phage infection and toxin production based on the work of Beretta and Kuang (1998) [37]. This model describes the temporal evolution of susceptible bacteria, infected bacteria, phages and toxin concentration (Figure 13A). The aim of this model is to investigate how host-phage population dynamics influence the concentration profile of the produced toxin and which parameters are most important to consider for optimal toxin production. Furthermore, this model is extended (Figure 13B) to investigate if the rate at which bacteria turn resistant against phages could potentially be a threat to the effectiveness of our biopesticide.

Figure 13. A. Schematic representation of our phage infection and toxin production model. B. Schematic representation of our phage infection and toxin production model including phage resistance

Influence of spatial effects on host-phage interactions

Our first model describes host-phage interaction dynamics as an isolated system (i.e. the spatial organization of the bacteria is not taken into account). Most likely, the bacteria in the locust gut reside in so-called microcolonies, which are small groups of cells packed close to each other [38, 13]. The spatial organization of cells in these microcolonies can influence phage propagation and toxin production in the locust gut. We hypothesized that spatial effects, such as phage mobility and substrate diffusivity, may have an important influence on the performance of our biopesticide. To investigate these effects on phage infection and toxin production, we modelled the temporal evolution of the number of bacteria, phages and produced toxin with a more detailed 2D biofilm simulation framework. Furthermore, as our biopesticide does not target all bacteria of the locust gut, we investigated the influence of biofilm heterogeneity on the resulting toxin production.

Figure 14. Visual representation of the output created with our 2D biofilm simulation model

Future perspectives