Differential transcriptomics in the regulation of digestion and uptake of protease inhibitors in the desert locust, Schistocerca gregaria

Insects are among the most successful organisms on the planet. One explanation for their success is their extraordinary ability to successfully consume a wide range of foods. Like all heterotrophic organisms, insects need to acquire vital nutrients from their diet. The central organ for food digestion and absorption of nutrients is the gastrointestinal tract. This organ’s principal functions are to mediate the efficient digestion of food and to protect the organism against harmful chemicals, microorganisms, and mechanical damage from the food. These functions are achieved through regional differentiation of the alimentary canal, as well as highly flexible adaptations to the consumed diets, both at anatomical and molecular levels. This central role of the digestive tract in the insect’s life history has made it an important subject of study. Numerous studies describing the general gut morphology and associated digestive mechanisms of various insects exist. In contrast, the molecular patterns underlying digestion and nutrient uptake in insects are still poorly and only partially characterized.

Insects have a high socio-economic impact. Many are considered to be pests, endangering the health and livelihood of a large proportion of the world’s population. These pest insects are generally combated in different ways, mostly including a variety of biological and chemical insecticides. However, as many chemical insecticides pose threats to human health and the environment, development of new and eco-friendly alternatives is essential. Moreover, an increasing number of studies are reporting on resistance of insect populations against widely applied insecticides, both in the laboratory and on the field. A promising alternative would be to disrupt essential molecules within the insect gut, ideally resulting in consequent mortality of the pest insect. However, in order to find such target sites, more research on the gut physiology of insects is pivotal.

In this context, we decided to examine the changes in the midgut transcriptome of the desert locust, Schistocerca gregaria, during the digestive process. The relative size of the desert locust together with its polyphagous nature makes it a highly favorable organism for studying gut physiology. We performed RNA sequencing (RNA-Seq) analysis of the messenger RNA (mRNA) content of midguts dissected at various time points following food uptake. The midgut is a key part of the digestive tract of insects, which is typically the site of enzymatic digestion and nutrient uptake, and it was therefore selected for deep sequencing. The RNA-Seq data were used to create a S. gregaria midgut reference transcriptome, which was consulted to study the specific transcript profile of this tissue. Moreover, this transcriptome database is a useful resource to further study the digestive process in insects in general, and additionally represents an excellent database for future midgut-associated studies in this insect species in particular. Furthermore, a differential expression analysis was performed to investigate differences in the midgut transcript profiles between two hours after feeding and twenty-four hours after feeding, in order to find genes mediating the digestive process in the desert locust. A total of 569 and 212 transcripts were found to be significantly up- and downregulated in the midgut two hours after feeding, respectively. Briefly, this analysis clearly demonstrated the desert locust’s ability to swiftly induce the expression of a large array of genes during the digestive process in response to food availability in the gut.

The list of transcripts upregulated two hours after feeding was further subjected to a detailed analysis in search for putatively lethal candidate targets for future pest management. Consequently, two upregulated transcripts were further investigated in vivo by means of RNA interference (RNAi). A vacuolar-type H+-ATPase (H+ V-ATPase) subunit a encoding transcript, denoted Sg-VAHa_1, and a Niemann-Pick C1 b (NPC1b) encoding transcript, denoted Sg-NPC1b, were selected based on their expected pivotal role in the intestine of the desert locust. In general, insect H+ V-ATPases are well-known for supporting transepithelial molecular transport by generating favorable membrane potentials, while insect NPC1b is probably responsible for the dietary sterol uptake in the midgut. Silencing each of these transcripts resulted in developmental defects and high mortality rates within two weeks after the first injections with double-stranded (ds) RNA, indicating their vital importance for the desert locust. These RNAi experiments demonstrated the possibility of discovering essential genes by analyzing the S. gregaria midgut transcriptome, hence emphasizing their promising potential as candidate targets for combating insect pests.

This doctoral research provides for the first time an insight into the midgut transcriptome of S. gregaria during the digestive process. This information was then used to further investigate the regulation of feeding and digestion in this insect species. In addition, this study also generated a broad and promising list of possible novel insecticide targets present in the midgut of the desert locust.