Tissue-specific transcription of proteases and nucleases across the accessory salivary gland, principal salivary gland and gut of Nezara viridula

Abstract

The phytophagous stink bug, Nezara viridula (L.) infests multiple plant species and impacts agricultural production worldwide. We analyzed the transcriptomes of N. viridula accessory salivary gland (ASG), principal salivary gland (PSG) and gut, with a focus on putative digestive proteases and nucleases that present a primary obstacle for the stability of protein-or nucleic acid-based stink bug control approaches. We performed high throughput Illumina sequencing followed by de novo transcriptome assemblies. We identified the sequences of 141 unique proteases and 134 nucleases from the N. viridula transcriptomes. Analysis of relative transcript abundance in conjunction with previously reported proteome data (Lomate and Bonning, 2016) supports high levels of serine protease expression in the salivary glands and high cysteine protease expression in the gut. Specifically, trypsin and chymotrypsin transcripts were abundant in the PSG, and cathepsin L-like cysteine protease transcripts were abundant in the gut. Nuclease transcript levels were generally lower than those of the proteases, the exception being abundant transcripts of ribonuclease-C20 in the PSG. The abundance of chymotrypsin, trypsin, and some carboxypeptidase transcripts suggests a significant role for the PSG in production of digestive enzymes. This result is at odds with the premise that the ASG produces watery saliva, which is high in enzymatic activity, while the PSG produces only sheath saliva. We have generated a comprehensive transcriptome sequence dataset from the digestive organs of N. viridula, identified major protease and nuclease genes and confirmed expression of the most abundant enzymes thereby providing greater insight into the digestive physiology of N. viridula.

Transcriptomic and proteomic analysis of putative digestive proteases in the salivary gland and gut of Empoasca (Matsumurasca) onukii Matsuda

“…In addition, certain numbers of the transcripts identi ed from the proteomic pro les were associated with the increase of the FPKM (expected number of Fragments Per Kilobase of transcript sequence per Million base pairs sequenced) value of the transcripts (Fig. 2b), which were similar to the previous observations in the investigation of proteases of stinkbugs [21,26].…”

“…Transcriptomic analysis coupled with proteomic pro ling is an effective strategy for identi cation of functional proteins in the SG and gut of hemipterans [21,26]. For example, proteases in the SG and gut of the two devastating pentatomid stink bugs, Halyomorpha halys and Nezara viridula, were investigated to identify proteases highly expressed in each tissue [21,26]. Furthermore, mapping of SG transcripts of H. halys to the protein pro les of watery saliva, which is the saliva secreted into the host plants, revealed 22 abundant digestive proteases.…”

Prospects, challenges and current status of RNAi through insect feeding

“…It has been also reported that the transcriptome of the stink bug Nezara viridula contains around 134 nucleases secreted by the gut and salivary gland. These studies provided an insight into the abundance of nucleases in insects, their broad substrate specificity, and their highly evolved physiology …”

“…These studies provided an insight into the abundance of nucleases in insects, their broad substrate specificity, and their highly evolved physiology. 25 There are also a large number of insects with extra-oral digestion. They inject into or salivate externally on food to get it to break down before consumption.…”

Transcription and Activity of Digestive Enzymes of Nezara viridula Maintained on Different Plant Diets

“…Most proteolytic activity occurs in M2 and M3 (Cantón and Bonning, 2019) mediated by the cysteine proteases Cathepsin B and L, while M4 functions to house endosymbiotic bacteria (Hosokawa et al, 2016). The proteases and nucleases present in saliva, salivary glands, and midgut tissues of N. viridula have been cataloged (Liu et al, 2018). The manner in which these myriad digestive enzymes are employed for digestion of plant material of varied composition is unclear.…”