Running title: Soldier fly salivary gland transcriptome Title for author: K. Etebari et al. Correspondence: Kayvan Etebari, School of Biological Sciences, The University of Queensland, Brisbane, QLD 4072, Australia. Email: [email protected] Original article Australian sugarcane soldier fly’s salivary gland transcriptome in response to starvation and feeding on sugarcane crops Kayvan Etebari1, Karel. R. Lindsay2 Andrew L. Ward3and Michael J. Furlong1 1 School of Biological Sciences, The University of Queensland, Brisbane QLD, Australia, 2 Sugar Research Australia, 26135 Peak Downs Highway, Te Kowai QLD 4740 Australia, 3 Sugar Research Australia, 50 Meiers Rd, Indooroopilly QLD 4068 Abstract The soldier fly is an endemic pest of sugarcane in Australia. Small numbers of larvae can cause significant damage to roots and reduce the crop yields. Little is known about the composition and function of the soldier fly salivary gland, its secretions, and their roles in insect‒plant interactions. In This is an Accepted Article that has been peer-reviewed and approved for publication in the Insect Science but has yet to undergo copy-editing and proof correction. Please cite this article as doi: 10.1111/1744-7917.12676. This article is protected by copyright. All rights reserved. this study, we performed transcriptome analysis of the salivary glands of starved and sugarcane root-fed soldier fly larvae. A total of 31 119 highly expressed assembled contigs were identified in the salivary glands and almost 50% of them showed high levels of similarity to known proteins in Nr databases. Of all the obtained contigs, only 9727 sequences contain an open reading frame of over 100 amino acids. Around 31% of contigs were predicted to encode secretory proteins, including some digestive and detoxifying enzymes and potential effectors. Some known salivary secreted peptides such as serine protease, cysteine proteinase inhibitors, antimicrobial peptides and venom proteins were among the top 100 highly expressed genes. Differential gene expression analysis revealed significant modulation of 850 transcripts in salivary glands upon exposure to plant roots or starvation stress. Here, we identified some venom proteins which were significantly upregulated in the salivary glands of soldier fly larvae exposed to sugarcane roots. In other insects and nematodes some of these proteins have been used to manipulate host plant defense systems and facilitate the invasion of the host plant. These findings provide a further insight into the identification of potential effector proteins involved in soldier fly‒sugarcane interactions. Keywords: Inopus, salivary gland, soldier fly, sugarcane pest, transcriptome Introduction: The soldier flies, Inopus rubriceps Macquart (Diptera: Stratiomyidae) and Inopus flavus (James) are endemic to Australia where they are economically important insect pests of sugarcane (Saccharum officinarum L.) (Allsopp & Robertson, 1988). The distribution of I. rubriceps extends throughout eastern Queensland and New South Wales and there are also established populations in New Zealand and California, USA (Robertson, 1986). The known distribution of I. flavus is more limited to local areas in eastern central Queensland (Hitchcock, 1976) and the damage they cause has increased recently. Soldier fly larvae are slow growing and most individuals develop through 8‒9 larval instars after hatching and reach the adult stage within one year. If larvae fail to pupate in the autumn (March to May in Queensland) they complete their development over two years after passing through an additional 3 or 4 instars (Hitchcock, 1976). The adult lifespan for both males and females is less than five days. In sugarcane crops, the larvae of both species cause economic damage by feeding on sugarcane roots, resulting in poor sugarcane yields, inhibition of bud development and germination, and reduced ratoons after harvest (Samson, 2001). Soldier fly pest management is difficult in sugarcane crops as insecticides are ineffective and no crop varieties are tolerant to 2 This article is protected by copyright. All rights reserved. feeding. The development of improved pest management strategies requires a better understanding of the relationship between soldier fly and its sugarcane host plants. The underlying mechanisms responsible for the detrimental effects of solider fly feeding on sugarcane remain very poorly understood. Hitchcock (1976), attributed poor germination and ratooning to the mechanical damage caused by larvae excavating cavities in roots (Hitchcock, 1976). In barley, Fellowes (1975) demonstrated that increasing densities of soldier fly larvae feeding on the roots caused decreased shoot production and root mass but considered the cause to be a loss of nutrients (Fellowes, 1975). The high levels of damage that the above ground parts of sugarcane crops can suffer following apparently low levels of feeding damage to roots has led to speculation that soldier fly feeding leads to the introduction of toxic chemicals or plant growth inhibitors into the roots but up to now there has been no evidence to support such hypotheses (Samson, 2001). Sugarcane soldier larvae have specialized mouthparts consisting of two hooked mandible-like structures that operate on the vertical plane and excavate asymmetrical cone shaped cavities in roots (Fellowes, 1975; Hitchcock, 1976) where larvae bury their heads to feed on fluids. Interactions between plants and insects with piercing-sucking mouthparts are similar to those between plants and plant pathogens during the infection process, and the interplay between host plants and larvae corresponds the plant–pathogen model proposed by Jones and Dangl (Jones & Dangl, 2006). This model has been examined in many other sap sucking insects and host plant interactions (Stuart, 2015; Zhang et al., 2017). Plant pathogens and insects with sucking mouthparts can deliver effectors into the host plant to manipulate plant immunity. Salivary glands release proteins that aid feeding, external digestion and the suppression of host plant defenses (Rivera-Vega et al., 2017). In the Diptera, human blood feeding disease vectors such as Anopheles gambiae (Culicidae) emit salivary proteins that function as anti-collagens in their hosts and aid digestion (Arca et al., 2005). Agricultural pests including Mayetiola destructor (Cecidomyiidae) (Shukle et al., 2009) and Sitodiplosis mosellana (Cecidomyiidae) (Al-Jbory et al., 2018b) produce effector proteins to manipulate host growth and metabolism to form plant outgrowths (galls) which benefit the insect with enhanced nutrition and reduced plant defenses (Chen et al., 2010). The salivary glands in insects are also used for the transmission of plant pathogens including viruses, bacteria, mycoplasma-like bodies, and phytoplasmas, which severely impact agricultural industries (Sugio et al., 2015; Kaur et al., 2016). Soldier flies have a pair of salivary glands positioned ventro-laterally in the thorax. These are known to secrete the digestive enzyme invertase, which suggests that larvae are feeding on the phloem 3 This article is protected by copyright. All rights reserved. (Fellowes, 1975). However, there is no information on other proteins that may be produced by soldier fly salivary glands or on micro-organisms that might be associated with them and transmitted to host plants during feeding. In this study, a transcriptomic approach was developed to characterize the composition of salivary glands in soldier fly larvae. This cutting-edge approach improved our understanding of the insect-plant interaction as it enabled the global gene expression profile in soldier fly salivary glands to be investigated. Comparison of the gene expression profiles of salivary glands from insects that had been exposed to host plants with those of salivary glands from insects that had been held in isolation of host plant roots produced a list of differently expressed genes. We also screened all soldier fly transcripts to identify genes that potentially encode a secretory protein. Proteins with hypothetical secretory signal peptides and without any transmembrane domains that are overexpressed during root feeding are more likely to pass through the cellular membrane and show potential effector activity. Our results provided greater understanding of the molecular mechanism behind soldier fly feeding on sugarcane roots and provide data for future identification of possible effector proteins in closely related insects. Material and methods Experiment set up Soldier fly (Inopus falvus) larvae were collected from an infested sugarcane field near Hay Point, Queensland (21°18' 5"S, 149 °14' 7"E). In February 2018, stools were dug from the ground and large larvae were manually collected from the roots and associated soil. Larvae were transferred to aerated 480 mL polypropylene containers with soil collected from the same sugarcane field and transported to the laboratory and used in experiments within 2 days. To examine direct interactions between larvae and plant roots, individual sugarcane seedlings (cultivar Q208) were transplanted into six 50 ml Falcon tubes filled with 70% peat and 30% sand and grown in sunlight for three weeks to develop a root system. To avoid exposure of the roots to light, all falcon tubes were wrapped with aluminum foil. Seedlings were irrigated with 5ml water every second day. Determination of larval instar is difficult in this species. We used larvae that were ~10 mm long, this is typically the size of later, 5th and 6th