The transcriptome of anal papillae of Aedes aegypti reveals their importance in xenobiotic detoxification and adds significant knowledge on ion, water and ammonia transport mechanisms

https://doi.org/10.1016/j.jinsphys.2021.104269Get rights and content

Highlights

  • Identified ion transporter genes permit development of a more comprehensive model.

  • Results suggest anal papillae are major sites of ammonia detoxification.

  • Results suggest anal papillae are major sites of xenobiotic detoxification.

  • Aquaporin AaAQP2 expression is likely important during abrupt brackish water exposure.

Abstract

The anal papillae of mosquito larvae are osmoregulatory organs in direct contact with the external aquatic environment that actively sequester ions and take up water in dilute freshwater. In the disease vector Aedes aegypti mechanisms of ion, water and ammonia transport have only been partially resolved. Furthermore, A. aegypti larvae are known to reside in high ammonia sewage and high salt brackish waters, and understanding of anal papillae function in these conditions is in its infancy. The objective of this study was to identify the complement of ion and water transport genes expressed by the anal papillae of freshwater larvae by sequencing their transcriptome, and comparing their expression in anal papillae of larvae abruptly transferred to brackish water for 24 h. Results identified a number of ion and water transport proteins, ammonia detoxifying enzymes, a full suite of xenobiotic detoxifying enzymes and transporters, and G-protein coupled receptors of specific hormones. We identified a marked increase in transcript and protein abundance of aquaporin AaAQP2 in the anal papillae with abrupt transfer to brackish water. We present an updated and more comprehensive model for ion and water transport with additional putative transporters for Na+ and Cl- uptake in the anal papillae. These are organs which are actively engaged in Na+, Cl- and water uptake and regulation when the aquatic larvae encounter fluctuating salinities over the course of their development. Furthermore the transcriptome of the anal papillae includes a full set of xenobiotic detoxification genes suggesting that these are important detoxification organs which is particularly important when larvae reside in polluted water.

Introduction

The anal papillae of mosquito larvae are multifunctional organs that have important roles in osmoregulation and ammonia excretion. The larvae of Aedes aegypti have four papillae surrounding the anus. Each papilla is a sac-like structure protruding into the external environment and is composed of a simple syncytial epithelium covered by a thin, permeable cuticle (Sohal and Copeland, 1966). The base of each anal papilla opens into the hemocoel allowing hemolymph to fill the lumen of each anal papilla (Edwards and Harrison, 1983). The epithelium receives oxygen through large tracheal branches which give rise to tracheoles (Edwards and Harrison, 1983). No nervous tissue is present in the anal papillae of Aedes aegypti suggesting that modulation of epithelial functions may be controlled by autocrine or endocrine signalling; however, nothing is known about hormone and/or receptor expression in the anal papillae (Edwards and Harrison, 1983).

In recent years, the molecular mechanisms underpinning ammonia excretion by anal papillae have been well studied in both freshwater and high ammonia environments using a combination of laboratory and field studies (Chasiotis et al., 2016, Durant and Donini, 2018, Durant and Donini, 2019, Durant et al., 2017). This is important because Aedes aegypti inhabit ammonia-rich septic systems where they are sheltered from climate and predation allowing populations to persist and vector disease throughout the year (Barrera et al., 2008, Burke et al., 2010, Durant and Donini, 2019, Irving-Bell et al., 1987). Other habitats that Aedes aegypti are increasingly exploiting consist of brackish water, particularly in coastal regions where seawater inundation occurs (Ramasamy and Surendran, 2016, Ramasamy et al., 2011, Surendran et al., 2012, Surendran et al., 2018).

In the preferred freshwater habitats, the anal papillae of larvae have been shown to absorb water and actively transport ions from the external aquatic environment to the hemolymph (Donini and O’Donnell, 2005, Stobbart, 1971a, Stobbart, 1971b). The molecular mechanisms of ion transport in anal papillae have been only partially resolved. The primary ionomotive enzymes Na+/K+-ATPase (NKA) and V-type H+-ATPase (VA), which are thought to be responsible for energizing the transport of ions, have been localized to the basal and apical membranes of the epithelium, respectively, using antibodies and immunofluorescent techniques (Patrick et al., 2006). Pharmacological studies have suggested that Na+ uptake at the apical side occurs through a Na+ channel, while Cl- uptake may occur through a Cl-/HCO3 exchanger (Del Duca et al., 2011). Pharmacology also implicates the importance of carbonic anhydrase in supplying H+ to VA, and HCO3 to the Cl- exchanger (Del Duca et al., 2011). Studies with ion omission or substitution suggest that Na+ is exchanged for H+ and Cl- is exchanged for HCO3 (Stobbart, 1971b). The specific molecular identity of these secondary transporters is unknown.

When larvae encounter higher salinity, studies have shown ultrastructural changes in the epithelium that suggest a reduction in ion transport capacity, and these observations are consistent with decreased NaCl uptake by anal papillae of freshwater larvae exposed to brackish water for six hours (Donini et al., 2007, Sohal and Copeland, 1966). However, other studies with field collected mosquitoes suggest the anal papillae remain functional in brackish water; albeit, it is unclear at present whether that may be for ion uptake, ion secretion, ammonia transport and/or water flux (Surendran et al., 2018).

Water uptake by the anal papillae was documented by Wigglesworth and Marusalin (Marusalin et al., 2012, Wigglesworth, 1932). It was estimated that anal papillae are directly responsible for 30% of daily water uptake by mosquitoes living in dilute freshwater (Wigglesworth, 1932). Water uptake by anal papillae is particularly interesting because freshwater animals osmoregulate by limiting water uptake while absorbing ions, thereby preventing dilution of body fluids. Furthermore, the syncytial nature of the anal papillae epithelium seems naturally adapted to limit any paracellular water permeability. Therefore, the fact that anal papillae are quite permeable to water is perplexing. Relatively high transcript abundance of AaAQP2 and AaAQP6 were reported in the anal papillae and both of these aquaporins have been shown to transport water in heterlogous expression systems (Drake et al., 2015, Marusalin et al., 2012, Sreedharan, 2018). More recent studies have shown anal papillae expression of entomoglyceroporins (AaAQP4 and AaAQP5, aquaporins that transport solutes and water) which is dependent on the external salinity (Akhter et al., 2017). Further research on aquaporin expression in anal papillae is important to understand the physiological purpose of their permeability to water.

The anal papillae are structurally simple organs consisting of epithelial tissue with trachea making transcriptome sequencing an ideal tool to identify the molecular transport and regulatory mechanisms of the anal papillae which to date, remain mostly unknown. Transcriptome sequencing can also begin to identify how salinity impacts gene expression of the anal papillae, thus providing clues for salinity-induced functional changes. In this study, the transcriptomes of anal papillae from Aedes aegypti larvae that were in freshwater (FW) and short-term exposure to brackish water (BW, 30% artificial seawater) were sequenced and analyzed for differentially expressed genes. The results identify expressed genes that are important in ammonia/nitrogen excretion/regulation, ion and solute transport, water transport, and hormone regulation. Results also strongly suggest that the anal papillae are a major organ for xenobiotic detoxification.

Section snippets

Experimental animals and treatments

Aedes aegypti (Liverpool strain) eggs were obtained from a laboratory colony at York University, Toronto, ON, Canada. Eggs were hatched in dechlorinated tap water [in µmol l−1: Na+ 590; Cl- 920; Ca2+ 760; K+ 43], hereby referred to as freshwater (FW), and larvae were reared until they reached the 4th instar. Larvae were fed daily with a solution of liver powder and yeast and the rearing water was replaced every two days. For RNAseq studies, 3 groups of 100 larvae each were transferred into

Transcriptome of anal papillae and differentially expressed genes with BW treatment

The transcripts of 18,801 genes were detected in at least 1 of the 3 samples of anal papillae from FW larvae. There were 8133 genes detected with a mean of at least 1 transcript per million (TPM), and 4146 genes detected with a mean of at least 5 TPM. Of the 100 most abundantly expressed genes, 72 are ribosomal proteins. Several genes with unspecified products are in this group (Table 1). The heat shock protein 70 (AAEL019403-RA) is highly expressed and transcript abundance decreases with BW

Transcriptome of anal papillae

Sequencing the transcriptome of mosquito anal papillae has identified a complement of secondary ion transporters, protein metabolism enzymes, G-protein coupled receptors, and a full suite of xenobiotic detoxification genes. As a result, we have a greater understanding of the mechanisms of ion transport, nitrogen regulation, and hormonal control in the anal papillae as well as identifying another important role for these organs, specifically xenobiotic detoxification.

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