Proteomic analysis of the entomopathogenic nematode Steinernema feltiae IS-6 IJs under evaporative and osmotic stresses

Abstract

In order to improve the storage capability under desiccation of the widely sold biological insecticides based on entomopathogenic nematodes (EPNs), we need to understand how these organisms respond to desiccation stress. As part of our studies to achieve this, we studied survival and protein expression in infective juveniles of the EPN Steinernema feltiae IS-6 when exposed to evaporative (exposure to 97% relative humidity (RH) for 3 days, followed by a 1-day exposure to 85% RH) and osmotic (exposure to 24% glycerol for 8 h) stresses. More than 400 protein spots that were detected by proteomic analysis showed reproducible abundance within replications. Of these, 10 spots and 7 spots showed detectable changes in abundance under evaporative and osmotic stress, respectively, compared to fully hydrated nematodes. Three spots exhibited a differential response pattern between evaporative and osmotic desiccation (one was down regulated and two were novel in evaporative desiccation). Peptide mass mapping with MALDI-TOF mass spectrometry (MS) identified 10 desiccation-response proteins, among which several are known to be stress responsive including heat shock protein 60, coenzyme q biosynthesis protein, inositol monophosphatase and fumarate lyase that were found in both stresses. Other identified proteins are known to be involved in the cell cycle regulation, regulation of gene transcription, organization of macromolecular structure and some currently have no known functions. Our results suggest that it is unlikely that improvement of desiccation tolerance in EPNs can be achieved through genetic transformation and addition of single genes and that selective breeding could be the best approach to generate desiccation resistant worms.

Introduction

Most animals and other life forms cannot survive without water. The changing environment may impose temporal lack of water, leading to dehydration, adversely affecting motility and survival [1]. However, certain organisms can survive exposure to extreme desiccation by entering into a state of suspended animation known as anhydrobiosis [2]. Anhydrobiotic organisms can survive in the dry state for indefinite periods of time, after which, upon rehydration, they are able to resume normal metabolic activity [3], [4]. These organisms are found across all biological kingdoms, with baker's yeast, Saccharomyces cerevisiae, being a familiar example. More complex animals, for instance, rotifers, tardigrades, cysts of the crustacean Artemia salina and nematodes [4], [5] also show this behaviour. The morphological and biochemical basis of anhydrobiosis have been studied extensively, but only recently has some emphasis been directed towards a better understanding of the genetic and molecular foundation of this biological phenomenon.

The entomopathogenic nematodes (EPNs) (families Heterorhabditidae and Stinernematidae) are possibly the most beneficial parasites for mankind [6]. These obligate lethal parasites infect a wide range of insects and have been commercialized in many countries as biological insecticides for use in a range of agricultural and horticultural crops. This has been most successful in the United States where approximately 25,000 ha of citrus crops are treated annually with nematodes to control the root weevil Diaprepes abreviatus [7]. This success has been attributed largely to the ease of mass cultivation of the EPNs in fermenters, the wide range of nematode species that can be matched to a large range of target pests, the quick kill they cause in hosts as a result of lethal entomopathogenic bacteria carried by the nematodes and released into the hosts’ haemocoel. In addition, these nematodes have the ability to move through soil and find insects in cryptic habitats that are difficult to reach with chemical insecticides. Despite these advantages, sales of EPNs are tiny compared to chemical insecticides, and it is generally recognized that the single most important constraint to their more widespread use is their poor storage capacity [1], [6], [8]. It has been accepted by the entomopathogenic nematologist community that prolongation of EPNs storage is best achieved by induction of a dormant state. This can be attained by evaporative or osmotic partial dehydration [1], [9]. Currently, the vast majority of commercial products comprise concentrated nematodes and an inert solid carrier, such as vermiculite or diatomaceous earth. In these formulations, nematodes are partially inactivated as a result of incomplete dehydration that induces a semi-dormant state considerably prolonging the nematodes’ lifespan and enabling them to withstand the rigors of a fluctuating temperature regime that is typical when commercial products are shipped and applied [9]. Understanding the molecular basis of this dormancy may help to develop strains that are better adapted to surviving in this state within commercial products. Such strains could be developed either through selective breeding [10] or by genetically engineering nematodes with specific genes to enhance survival. Transformation protocols for EPNs already exist [11] and genetically engineered EPNs have already been field-tested [12].

The morphological and biochemical changes in EPN associated with evaporative and osmotic desiccation stresses have been well studied [1] and the first steps for understanding the molecular basis of stress tolerance in these organisms has been recently instigated [13]. Slow dehydration results in shrinking of body size [14], [15], reduced levels of glycogen [16], and increased levels of trehalose [16], [17]. Moreover, down regulation of expression of Steinernema feltiae glycogen synthase (Sf-gsy-1), which is the rate-limiting enzyme in glycogen synthesis, suggested that dehydration initiates a shift from glycogen to trehalose synthesis, at least in part by suppression of glycogen synthase transcription [18]. Subtraction hybridization and differential display has identified several classes of S. feltiae genes that are induced during desiccation stress response [19]. These include transcription regulators that may regulate gene transcription in genetic networks, proteinous stress protectants and metabolic enzymes that are involved in the production of osmoregulants, which may be end products of the same or other genetic networks. Among the identified stress related genes were S. feltiae LEA protein (Sf-LEA), (Sf-NAP-1) and Casein kinase 2 (Sf-CK2) genes [19]. A functional role for LEA protein in response to stress conditions in Caenorhabditis elegans was recently demonstrated using RNAi technique [20]. Synthesis and accumulation of proteins during the desiccation process have been characterized among bacteria, fungi, yeasts and plant seeds [21], [22].

Proteomics is the study of the proteins comprising the proteome, the total set of proteins encoded by the genome of an organism [23], including the changes in structure and abundance in response to developmental and environmental cues. Thus, proteomic analyses can provide a broad view of EPN responses to stress at the level of proteins. This approach has increased in sensitivity and power as a result of improvements in two-dimensional polyacrylamide gel electrophoresis, protein detection and quantification, fingerprinting and partial sequencing of proteins by mass spectrometry (MS), bio informatics, and methods for gene isolation [24]. In nematology, proteomics has been applied to establish a 2-D map of the C. elegans proteome by means of two-dimensional polyacrylamide gel electrophoresis [25], but little is known about EPN proteins. Chen et al. [26] identified the proteins in osmotically stressed infective juveniles (IJs) of S. feltiae using two-dimensional electrophoresis. The results indicated that osmotic stress in desiccated IJs was associated with the induction of Actin, Proteasome regulatory particle (ATPase-like), GroEL chaperonin, GroES co-chaperonin and Transposase family member [26]. Solomon et al. [27], also using S. feltiae IS-6 identified a heat-stable, water stress-related protein with a molecular mass of 47 kDa (designated desc47).

As part of our studies to understand desiccation of EPNs with the long-term aim of developing nematodes with improved storage capacity, we report here a global proteome analysis of the evaporative and osmotic desiccation responses of an EPN. As our model nematode we selected a desiccation tolerant strain of S. feltiae (IS-6), isolated from the soil of a citrus orchard in the Negev desert, an arid region in Israel [15]. The main objectives of this research were to identify proteins for which expression was altered during desiccation and to compare the protein expression response of S. feltiae (IS-6) under evaporative and osmotic desiccation.