Recipient Organization
AUBURN UNIVERSITY
108 M. WHITE SMITH HALL
AUBURN,AL 36849
Performing Department
Biological Sciences
Non Technical Summary
In agricultural settings, arachnids (mites, spiders, ticks, scorpions) can play very diverse, often opposing, roles. They can either be agricultural pests or protectors. Spider mites, for example, can be a significant pests on grapes, hops, organic vegetables, ornamental nursery products, and a wide range of other crops. True spiders, on the other hand, are generalist predators that rarely damage plant material. Reports indicate that as main as 19 spider species are common in rice, 16 in soybeans, and 57 in sugarcane. In these environments, spiders often function as natural biological control agents against a wide array of pests including aphids, ants, moth larvae, and weevils. Gaining a better understanding of the relationship between energetics and arachnid ecology is important for both the development of targeted pesticides and conversely for better managment of spiders in IPM systems. Unravelling the causal relationship between cellular physiology and ecological performance is a complex undertaking requiring coordinated investigations of genetics, proteins, biochemistry, morphology, and behavior. Two sibling species of burrow dwelling trapdoor spiders are proposed as a novel experimental system. This system will be used to define the relationship between protein level variation, cellular energetics, and adaptive response in natural populations. The two spider species occur in close proximity yet exhibit substantial genetic and morphological differences, occupy distinctly different habitats, and are ecologically divergent. Preliminary data also indicate that protein patterns differ, in particular the well studied, ubiquitous trafficking protein SQSTM1/p62. Recent evidence argues that p62 plays critical physiological roles in autophagy, energy production, and mitochondrial dynamics. Based on its known functions and the preliminary data, it is hypothesized that p62 plays an essential role in the cellular physiology of trapdoor spiders that directly links to key morphological and ecological characteristics. The mechanisms proposed to underlie these links are p62's role in mitochondrial-based energy dynamics and p62's association with protein trafficking to vesicles, specifically a potential involvement in ommochrome granule dynamics.
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Goals / Objectives
We hypothesize that p62 plays an essential role in the cellular physiology of trapdoor spiders that directly links to key morphological and ecological characteristics. The mechanisms proposed to underlie these links are p62's role in mitochondrial-based energy dynamics and p62's association with protein trafficking to vesicles, specifically a potential involvement in ommochrome granule dynamics. To study these relationships, two specific Aims are proposed:Aim 1 - Identify the role of p62 in energetics, autophagy, and mitochondrial functionAim 2 - Relate p62 variation, cellular energetics, and mitochondrial dynamics to spider ecology.Completion of these two Aims will provide unique insight into the path between cellular physiology and ecology in this system.
Project Methods
Specific Aims - To better understand the relationships p62, cellular physiology, and the ecology of trapdoor spiders, two specific Aims are proposed:Aim 1 - Identify the role of p62 in energetics, autophagy, and mitochondrial function within spider cells.Aim 2 - Relate p62 variation, cellular energetics, and mitochondrial dynamics to spider ecology. Research DesignAim 1 - Identify the role of p62 in energetics, autophagy, and mitochondrial function within spider cells. Work under this aim will involve two objectives: 1) Creation of primary cell cultures; and 2) examining the contributions of p62 and associated proteins to trapdoor spider cell physiology. The initial protocol for primary cell cultures will follow the method of Sicaeros et al. (2007) as described for Drosophila. Cell cultures will be created from neural tissue harvested from spiderlings. Young spiders and silk sacs containing developing juveniles are readily available from maternal burrows during late spring and early summer (Bond 2012). Cells harvested from these cultures will provide lysates for Western blot analyses. Antibodies for proteins associated with p62, autophagy, mitochondria, and vesicle membranes will be evaluated to determine their function in spider cell physiology. Proteins of primary interest will be p62, HDAC6, TRAF6, LC3, TOM40, ATPase, and proteins involved with the mTOR pathway (complete protocols will be provided in the full proposal). Transcriptome and genomic sequence data generated using the Illumina Hi-Seq platform will be used to further define the forms of proteins present (bioinformatics pipelines will be described in the full proposal). Evidence for p62's physical association with organelles including lysosomes, mitochondria, and ommochrome granules will be evaluated using immuno-electron microscopy. Mitochondrial morphology and location will be determined using immuno-cytochemical techniques with MitoTracker Red stain under fluorescent microscopy (Seibenhener 2013). p62 protein levels will be manipulated in the cell lines using Cas9-guideRNA and/or siRNA knockdown methods. Comparison of these cells to native cells will provide unique information on p62's physiological role. Collectively, these data will directly address the hypotheses that p62 plays a role in spider energetics and coloration. Aim 2 - Relate p62 variation, cellular energetics, mitochondrial dynamics to spider ecology. Field investigations and common garden experiments will be used to establish p62's role in spider ecophysiology. Field studies will focus on establishing ecological conditions experienced by both species, specifically relating to burrow construction and prey availability. Soil characteristics including texture, structure, organic content, and compaction will be assessed at multiple sites with samples submitted for analysis to the Auburn University Soils Testing Center. To estimate volume and displacement effort, foam casts will be made for 5-10 burrows in each soil type and 3-dimensional models generated. Prey item availability will be determined by field observation and collections. Calorimetry, protein, and lipid analyses will be conducted to estimate energy availability and nutritional value of key prey. Field derived soil and nutrition data will be used to set conditions for laboratory trials. Two main questions will be addressed experimentally: 1) excavation energetics and 2) physiological responses to prey quality. Excavation energetics will follow established protocols (Suter et al. 2011; Hils and Hembree 2015). Briefly, size matched adult female spiders (n≈5 per species x 4 trials; actual sample size details will be provided in the full proposal) will be placed in controlled environments (temperature, humidity etc.) containing common substrates. Burrowing behavior will be monitored using video and total metabolic costs will be determined using O2 and CO2 analyzers (Canals et al. 2015). Arthur Appel, Professor - Department of Entomology, Auburn University, will provide expertise, guidance, and equipment for these experiments. Soil samples from the field sites will be used as controls; subsequent trials will employ artificial substrates of varying sizes and densities. Responses to prey quality will be assessed using modifications of the approach of Schmidt et al. (2012). High and low quality populations of crickets will be cultured and presented as prey. Feeding responses of the spiders will be monitored to determine consumption rates and prey choice. After two weeks, the spiders will be euthanized and protein / mitochondrial analyses outlined in Aim 1 will be conducted. Completion of this Aim will provide unique insight into the path between cellular level physiology and ecology. It is anticipated that this research will establish a model for wider investigations of burrowing species across multiple taxonomic levels.