Tribolium Castaneum)
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Characterisation of variables associated with the flight behaviour of the rust-red flour beetle (Tribolium castaneum) Komal Gurdasani Master of Biotechnology The University of Queensland A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2017 School of Biological Sciences ii Abstract Tribolium castaneum Herbst (Coleoptera: Tenebrionidae) causes significant losses to stored grain products. This problem is aggravated by the development and spread of resistance to various grain protectants, especially the fumigant phosphine. Studying the dispersal behaviour of T. castaneum will help in understanding the spread of resistance. Active dispersal in T. castaneum occurs by both walking and flight. Flight enables dispersal across a wider geographical area and increases the probability of spreading resistance genes geographically. Dispersal of pests also has implications for understanding the ecology of resident populations and thus also for improving food security effectively. Therefore, a good understanding of T. castaneum dispersal by flight is critical to managing this pest and its resistance to phosphine. The research reported here was designed to study factors that influence the flight of these long-lived beetles. Various techniques were employed, in both the laboratory and field, to investigate a broad range of factors that may influence T. castaneum flight behaviour. I also explored the use of nanoparticles as novel external markers for small insects, with an emphasis on their use in mark- recapture studies of T. castaneum. The ability to gauge the dispersal distance of these insects accurately would aid considerably in understanding the spatiotemporal dynamics (and gene flow) of this species across landscapes. Chapter 2 investigated the flight propensity of T. castaneum at two different ages at the same time (old (~12 weeks) and young (~2 weeks post-emergence)). First generation laboratory cultures of field-collected beetles were used to avoid the known effects of protracted laboratory culturing. Flying beetles were captured individually using a customized trap, and their age, sex, weight, size, fat content, fecundity, polyandry and lipofuscin intensity (as an estimate of age) were determined. Beetles were also collected from the culture medium and considered to be resident (non-flyers). The assays indicated that flight propensity declined significantly with age as more young beetles took to flight than old ones. Flight was not affected by sex, body size or weight but lipofuscin intensity of old beetles was similar to young beetles in culture, so it is not an indicator of age. Since, its levels were only significantly higher in young flying females than young resident females it may be related to the individual’s metabolic status. Fecundity assessments indicate that the total fecundity was not related to age or flight as no significant differences were found between resident and flying beetles across and between all age groups. Microsatellite markers were used to assess parentage of offspring. They confirmed that females of iii this species are polyandrous in the field (as expected from their polyandry in laboratory cultures). The number of males inseminating females in the field ranged from one to four, and this held too for first generation laboratory cultures (which is reported in Chapter 3 as part of a larger study of fecundity, polyandry and genetic diversity). The results from Chapters 2 & 3 were used to make predictions about the migration behaviour of T. castaneum in the field (Chapter 4). These predictions were tested by sampling beetles flying at various distances (2, 20 and 300 m) from a large field population (infesting cotton seed in storage). Beetles were also collected directly from the infested cotton seed (‘resident beetles’). The total fecundity of females until day 105, post emigration in all treatments was not statistically different. The females that emigrated are presumed to be relatively young and had mated multiple times as they had the potential to produce numerous offspring (mean = 307.3, range = 0 – 582) without having access to males. Difference in fat content was significant between flying beetles (both sexes) trapped at 2 m and flying beetles trapped at 20 m and 300 m from storage. However, no significant trends were observed between resident and flying beetles. The relationship of flight propensity with weight, body size, and lipofuscin intensity were similar to the trends documented in the laboratory flight experiment. In chapter 5, I tested green fluorescent nanoparticles (CdTe/CdS quantum dots) as external markers on T. castaneum and other small insects for mark-recapture studies. Quantum dots (QDs) proved to be effective markers in the laboratory for at least 48 hours when beetles were kept without food. The culture medium seemed to be abrasive and so removed the particles. In the laboratory, QDs had no negative effects on the flight behaviour or survival of T. castaneum. In a field test, beetles treated with QDs were captured in good numbers at 20 m from the release point but the fluorescent signals from these recaptures were low, perhaps because the QDs were quenched by the release of stress chemicals (benzoquinones). Four main conclusions are drawn from the research outlined above. (i) Flight propensity of T. castaneum is related to age, but is not affected by sex, weight, body size or fat content. (ii) Lipofuscin levels may be related to metabolic status rather than age. (iii) Females that fly have a high potential fecundity and considerable paternal genetic diversity in their spermathecae, as females are polyandrous before flight and this increases the chances of spreading phosphine resistance geographically. (iv) Quantum dots are non-toxic and safe as external markers in the laboratory, and further designing should make them effective in the field, so they can be used in large scale mark-recapture studies. iv Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. v Publications during candidature Peer-reviewed papers Rafter, M.A., McCulloch, G.A., Daglish, G.J., Gurdasani, K. &Walter, G.H. (2017). Polyandry, genetic diversity and fecundity of emigrating beetles: understanding new foci of infestation and selection. Journal of Pest Science. doi: 10.1007/s10340-017-0902-8 Publications included in this thesis Rafter, M.A., McCulloch, G.A., Daglish, G.J., Gurdasani, K. &Walter, G.H. (2017). Polyandry, genetic diversity and fecundity of emigrating beetles: understanding new foci of infestation and selection. Journal of Pest Science. doi: 10.1007/s10340-017-0902-8 Contributor Statement of contribution Gurdasani, K. Molecular genetics (50%) Maintenance of laboratory cultures (100%) Data analysis (10%) Rafter, M.A. Designed experiments (60%) Data analysis (60%) Wrote the paper (70%) Daglish, G.J. Designed experiments (10%) Wrote the paper (10%) McCulloch, G.A. Molecular genetics (50%) Designed experiments (20%) Data analysis (30%) Wrote the paper (10%) Walter, G.H. Designed experiments (10%) Wrote the paper (10%) vi Contributions by others to the thesis Professor Gimme Walter, Dr. Michelle Rafter and Dr. Gregory Daglish contributed significantly to the conception, designing and development of this research work. Statistical guidance, critical review, troubleshooting and advice for data analyses and interpretation were also provided. My colleague Dr. Graham McCulloch contributed significantly by providing training and guidance for molecular methods and computer analyses for polyandry detection in Tribolium castaneum and Rhyzopertha dominica. This work generated a jointly authored manuscript along with Dr. Michelle Rafter, Dr. Gregory Daglish and Dr. Gimme Walter as co-authors. This work has been listed as Chapter 3 of this thesis and is currently published in the Journal of Pest Science. My contribution was the characterization of polyandry in reference laboratory strains and newly established laboratory cultures of T. castaneum and Rhyzopertha dominica from