Elsevier

Journal of Insect Physiology

Volume 119, November–December 2019, 103965
Journal of Insect Physiology

Proteases and nucleases across midgut tissues of Nezara viridula (Hemiptera:Pentatomidae) display distinct activity profiles that are conserved through life stages

https://doi.org/10.1016/j.jinsphys.2019.103965Get rights and content

Highlights

  • The transcriptome reflects the biochemical profiles of midgut regions M1 to M3.

  • The distinct digestive profiles of M1 to M3 are conserved in adults and nymphs.

  • Serine and cysteine protease inhibitors are produced by multiple tissues.

  • Cytochrome P450s provide additional midgut metabolic flexibility.

Abstract

The southern green stink bug, Nezara viridula is a polyphagous pest of commercially important crops during both nymph and adult stages. This insect has recently transitioned from a secondary agricultural pest to one of primary concern. Novel management solutions are needed due to the limited effectiveness of current control strategies. We performed biochemical and transcriptomic analyses to characterize digestive enzymes in the salivary glands and along midgut tissues of N. viridula nymphs and adults fed on sweet corn. The digestive profiles were more distinct between midgut regions (M1 to M3) than between life stages. Aminopeptidase and chymotrypsin activities declined from the M1 (anterior) toward the M3 midgut region. Cysteine protease activity was higher in the M2 and M3 regions than in M1. Differences in sensitivity to chymotrypsin inhibitors between midgut regions suggest that distinct genes or isoforms are expressed in different regions of the gut. In nymphs, DNA and RNA degradation was higher in M1 than in M3. Adult nuclease activity was low across all midgut regions, but high in salivary glands. The differences in protease activities are reflected by transcriptomic data and functional enrichment of GO terms. Together, our results show that different regions of the digestive tract of N. viridula have specific and distinct digestive properties, and increase our understanding of the physiology of this organism.

Introduction

Although considered secondary pests in some crops of economic importance such as corn, soybean and cotton, the deleterious impact of stink bugs of the pentatomid complex has increased in recent years (Koch and Pahs, 2014). Stinkbug damage alters the appearance or taste of fruiting bodies, or induces premature maturation. Economic losses in 2008 from stinkbug damage to U.S. cotton alone were estimated at US$31 million (Pilkay et al., 2015). Current stink bug control strategies are limited to the spraying of chemical insecticides with inconsistent and diminishing effect, or the use of biological control (Jones, 1988). The increased pest status of stink bugs has been attributed to reduced insecticide use due to expanded adoption of transgenic crops (Greene et al., 1999), variable sensitivity to insecticides (Willrich et al., 2003), and reduced competition from controlled primary pest species (Zeilinger et al., 2016). Stink bugs rely on overlapping periods of flowering and fruiting to produce several generations of offspring in a year, with the distribution of life stages varying across species, cultivar, and seasons (Jones and Sullivan, 1982, Schumann and Todd, 1982, Tillman, 2010), which presents an additional challenge to their control. The southern green stinkbug, Nezara viridula, an important member of this pentatomid complex, is polyphagous and affects crops year round. N. viridula is widely distributed and is found in the Americas extending from northern Argentina to the eastern and southeastern United States (Panizzi et al., 2000). Effective control of N. viridula and related pest species will require novel and alternative methods, protein- (e.g. Bt toxin) and nucleic acid- (e.g. RNAi interference) based methods in particular, that should be tailored to the feeding habits and physiology of stink bugs (Chougule and Bonning, 2012).

N. viridula feeds by a piercing-sucking mechanism, whereby the insect injects saliva through its mouthparts into the plant to liquefy the tissue, and then ingests the pre-degraded material to complete digestion in the gut. Thorough degradation and absorption of the dietary components requires the action of a combination of enzymes produced by the digestive organs. Despite its economic importance, biochemical studies on the digestive physiology of this species are not as numerous. The overall digestive profiles of saliva, salivary glands, and guts of adult N. viridula (Lomate and Bonning, 2016) and of the invasive species Halyomorpha halys (Lomate and Bonning, 2018), have been characterized. Complementary profiles of nucleases and proteases with different pH optima and specific activity in salivary glands and gut were found. The dominance of cysteine protease activity in the gut (primarily cathepsins), is consistent with that of the Heteroptera infraorder of Hemiptera (Terra and Ferreira, 2012, Terra and Ferreira, 1994).

The N. viridula midgut is anatomically divided into 4 separate ventricles or regions (Hirose et al., 2006). The anatomical separation of organs in the digestive tract is often indicative of distinct physiological roles, as shown in heteropteran species such as Dysdercus peruvianus and Rhodnius prolixus (Terra and Ferreira, 2012). Despite different feeding habits, the ultrastructure of midgut tissues among Heteroptera is shared (Santos et al., 2017). Different regions of the midgut vary in the length of microvilli, number of mitochondria, and glycogen granule content for example, pointing to specific roles of each region in digestion and absorption (Fialho et al., 2009, Meguid et al., 2013). Differences in general cysteine protease activity were noted between N. viridula midgut compartments during a study of the potential role of gut microbiota in digestive capabilities (Medina et al., 2018). Slight differences were also observed for protease activities in whole gut extracts between adult and fifth instar nymphs of the predatory stink bug, Podisus maculoventris (Bell et al., 2005). Whether the digestive profile is conserved across N. viridula development (from nymph to adult) remains to be addressed. As feeding on plant material may start as early as the first instar (Esquivel and Medrano, 2014), knowledge of digestive profiles throughout development is important toward management of both nymph and adult N. viridula that both contribute to crop losses.

Building on our previous work, we sought to address the lack of information on nymphal gut physiology and the differential roles of N. viridula midgut regions in digestion. To increase understanding of the digestive profiles, we combined enzymatic assays and transcriptomic analysis to identify the activity of proteases and nucleases in the M1, M2 and M3 midgut regions in adults and nymphs of N. viridula, along with those of the salivary gland. By use of these two approaches, we discovered differences in the digestive enzymes of the gut tissues that are supported by changes in transcript abundance and are consistent with a distinct tissue pH in each midgut region.

Section snippets

Reagents

Azocasein, Nα-Benzoyl-D,L-arginine 4-nitroanilide hydrochloride (BApNA), N-Succinyl-Ala-Ala-Pro-Phe p-nitroanilide (SAAPFpNA), L-Leucine p-nitroanilide (LpNA), pGlu-Phe-Leu p-nitroanilide (pGFLpNA), Phenylmethylsulfonyl fluoride (PMSF), Nα-Tosyl-L-lysine chloromethyl ketone hydrochloride (TLCK), Nα-Tosyl-L-phenylalaninechloromethyl ketone (TPCK),E-64,Ethylenedintrilotetraecetic acid (EDTA), and Calf thymus DNA were purchased from Sigma-Aldrich (St. Louis, MO, USA). Baker’s Yeast RNA was

Different parts of the digestive tract have different general digestive properties

We sought to determine whether the digestive profiles of different gut regions of N. viridula differed and whether these profiles varied between life stages. We selected nymphs that were halfway through third instar for dissection because they were actively feeding on the plant material, and because earlier stage nymphs were too small for adequate acquisition of tissues by dissection. Newly molted adults were transferred to fresh diet for a further 24 h before dissection to allow insects to

Discussion

The goal of this study was to establish whether the anatomically distinct sections of the N. viridula gut had specialized enzymatic functions in digestion, and to address whether the digestive enzyme profiles are conserved across stink bug development. Insights into stink bug digestive physiology may in the future facilitate the development of targeted control measures to limit crop damage caused by both adults and nymphs.

From the results of this study, we conclude that the M1, M2 and M3

Conclusions

In this study we examined the digestive enzyme profiles of individual tissues of the digestive tract of N. viridula adults and nymphs, complementing and expanding previous findings. This more detailed description of enzyme activities across gut regions and life stages provides insight into the differing physiological roles of each gut compartment in nymph and adult N. viridula complementing ultrastructural observations. This work also highlights the tissue- and stage-specific enzymatic

Data accessibility

Raw reads sequenced for this project available through NCBI SRA accession SRP193118. The transcript abundance estimates used for differential expression analysis are accessible through GEO with ID GSE130097.

Funding

This material is based upon work supported by the National Science Foundation I/UCRC, the Center for Arthropod Management Technologies, under Grant Nos. IIP-1338775 and 1821914, and by industry partners.

Author contributions

PEC performed the experiments and bioinformatics analysis, PEC and BCB devised the experiments and wrote the manuscript.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgements

Dr. Ke Wu (University of Florida) kindly provided the pGlo plasmid and primer sequences to prepare GFP dsRNA. The authors acknowledge University of Florida Research Computing for computational resources and support for this study.

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