Biological Sciences Undergraduate Works

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    Open Access
    Evaluating different double-stranded RNA structures for their ability to control pest flea beetles
    (2023-04) Verhaeghe, Lauren; Belmonte, Mark (Biological Sciences); Whyard, Steve
    Canola is an economically important Canadian crop that suffers significant annual losses by damage from feeding flea beetles (Phyllotreta cruciferae and P. striolata). Increasing incidences of resistance and concerns about off-target effects with current insecticides demands new methods of control. Recently, environmentally safer approaches to pest control have been investigated using RNA interference (RNAi), a sequence-specific gene silencing mechanism triggered by exogenous double-stranded RNA (dsRNA), to selectively induce the mortality of targeted species. This study examined three different dsRNA structures for their ability to kill P. striolata flea beetles and thereby reduce feeding damage on canola leaves. Long linear dsRNAs (212-214 bp), short hairpin RNAs (hpRNAs) (21-24 bp), and short paperclip RNAs (pcRNAs) (21-24 bp) targeting mRNAs of three essential genes, Ras opposite (Rop), Sec23, and Snf7 in P. striolata were investigated. P. striolata adults were fed dsRNA-treated canola leaf disks, and impacts on insect survivorship and leaf material consumption were recorded over an eight-day period. pcRNAs targeting Sec23 and Snf7, and long dsRNAs targeting Sec23 and Rop effectively killed flea beetles, resulting in the reduced consumption of treated canola leaf tissues. Consumption of both the Sec23-specific long dsRNA and pcRNAs caused similar levels of flea beetle mortality (68% and 76%, respectively), whereas only the Rop-specific long dsRNA and the Snf7 pcRNA were effective at killing the beetles (76% and 84%, respectively). hpRNAs proved the least effective across all gene targets, killing at most, between 40-52% of flea beetles, depending on the gene target, and in general, these values were not significantly different relative to the negative controls. While this study examined dsRNAs specific for only three target genes, it provides evidence that RNAi-based pesticides have the potential to control these economically important pests and that short pcRNAs can be as effective as conventional long linear dsRNAs.
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    Open Access
    Sex-sorting Aedes aegypti for sterile insect technique (SIT) by knocking down four different gene targets, expressed in the guts of female larvae
    (2023-04) Felix, Sharon
    Mosquitoes are vectors for diseases that affect approximately 0.7 billion people globally. Most of these diseases have a high incidence in tropical often developing countries, where they prove either fatal or severely debilitating. Despite huge investments of time and money to find solutions, we have been unable to control this disease vector. Additionally, some of the techniques previously and currently in use to prevent these diseases, have serious drawbacks such as detrimental effects on non-target species, humans and the environment. We need eco-friendly alternatives to these harmful chemicals, that are just as efficient while also being species-specific to avoid harmful effects on other beneficial insect species. Sterile insect technique (SIT) is a creative technology that does just that. It involves the mass release of sterile males of a species to outcompete wild males and mate with wild females, eventually resulting in the decline of a targeted insect population. However, before these sterile males are released, they must be separated from females. An efficient way to sex-sort males from females can use RNA interference (RNAi). RNAi is a natural defence mechanism that exists in eukaryotic cells, that can be used to knock down female-biased or female-specific genes. Targeted females will develop slower than their male counterparts due to this knockdown and this will enable their separation from a pool of male individuals. For my honours thesis, I targeted four such female-biased genes - AAEL014604, AAEL014797, AAEL009313 and AAEL005884 and measured development (in days), survival and pupal size for each of these treatments for both male and female mosquitoes. None of my treatments resulted in significant results but one of these (AAEL014797) showed great promise for an improved SIT.
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    Open Access
    Improving mosquito (Aedes aegypti) sex-sorting methods for sterile insect technique using RNAi gene knockdowns
    (2023-04) Rempel, Kadri
    Doublesex (dsx) is an alternatively spliced mosquito (Aedes aegypti) gene that controls sex development by producing male (DSXM) or female (DSXF) transcription factors that regulate gene expression in a sex-specific manner. As both DSXF and DSXM bind the same DNA sequence, their ability to regulate differential gene expression is predicted to be modulated by other proteins that interact with DSX. A previously conducted protein-protein interaction study identified several proteins that bind to DSXF and their role in altering female development was explored in this study by knocking down their corresponding transcripts using RNA interference during the larval stages of development. Knockdowns of the genes nop-14, wdr-48, and rnmt was achieved by feeding mosquito larvae HT115 strain E. coli transformed with a pl4440 plasmid containing a dsRNA template specific to each of three target genes. The knockdowns were carried out at their normal rearing temperature, 28oC, and at 22oC to slow mosquito development. Knockdown of one of the genes, wdr-48, at either temperature, significantly increased the time to pupation of females compared to males. This delayed development of females could prove useful in the sex sorting of males and females during the pupal stage by providing a method to select only males for a sterile male insect (SIT) population control program for Ae aegypti. Of note, the 22oC growth conditions provided a larger difference in the pupation times between the sexes than the 28oC conditions did, and thus may provide even better sex-sorting for mosquito SIT.
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    Open Access
    Exploring the role of ammonia transporters (AMTs) in the branchial tissue of the horseshoe crab, Limulus polyphemus
    (2023-05) Whiting, Holly; Campbell, Kevin (Biological Sciences); Treberg, Jason (Biological Sciences); Weihrauch, Dirk
    All organisms must manage ammonia as it is highly toxic and a product of many essential biochemical processes. One group of proteins that facilitates the movement of ammonia across cell membranes is the Ammonia Transport Protein family which are generally sorted into three groups—ammonia transporters (AMTs), Rhesus glycoproteins (Rh proteins), and methylamine permeases (MEPs)—expressed in plants, animals, and fungi, respectively. Recently, transcripts of AMTs have also been found in invertebrates, where experimental evidence suggest that they play a role in both ammonia excretion and ammonia sensing. The American horseshoe crab, Limulus polyphemus, expresses at least two AMT and two Rh proteins within the epithelia of their book gills which is the primary surface for ammonia excretion. Each gill lamellae have a ventral ammonia permeable side and a dorsal ammonia impermeable side. mRNA transcripts for both proteins LpAMT-1 and LpAMT-3 were found on the dorsal and ventral surfaces, bringing into question their function in direct ammonia excretion. In oocyte expression trials both AMTs failed to mediate the transport of radiolabeled methylamine, while transport was detected for both a coral and a human Rh protein. Direct evidence of ammonia transport by invertebrate AMTs has not been previously found and further research should be conducted into the function of these proteins in invertebrates.
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    Open Access
    Size matters: Host body mass overshadows climate change in parasite prevalence among semi-arid ground squirrels (Xerus inauris)
    (2023-04) Guarino, Andrea; Davoren, Gail (Biological Sciences); Garroway, Colin (Biological Sciences); Waterman, Jane
    Climate change is a phenomenon in which global temperatures are rising, and animals respond by undergoing thermal stress, which may be linked to increased susceptibility to ectoparasites. Environmental temperature greatly influences ectoparasites as they rely on external heat sources to regulate their body temperature. This study investigated the effects of increasing maximum and minimum temperatures on ectoparasites abundance (number of parasites on an individual) and prevalence (number of infected individuals) of adult female African Cape ground squirrels (Xerus inauris). This study also investigated the effects of host body mass and host body condition on ectoparasite abundance and prevalence. We did not find a relationship between rising temperatures and ectoparasite loads (abundance and prevalence), nor did we find a relationship between body condition and ectoparasite loads. We speculate that the thermoregulatory behaviours of Cape ground squirrels mitigate ectoparasite loads. We did not find evidence for a relationship between host body mass and abundance; however, we found a significant negative relationship between host body mass and parasite prevalence. Our results show that a higher proportion of individuals are infected with fleas compared to lice. It remains uncertain whether the inverse relationship between host body mass and prevalence is due to increased skin strength or other traits in larger individuals. The different life history traits of the parasites could explain why fleas are more prevalent than lice. Fleas, being facultative parasites, can avoid the grooming behaviour of hosts. In contrast, lice, being obligate parasites, are bound to remain on their host, thus, are more vulnerable to being removed. As climate change persists, the host-parasite relationship between Cape ground squirrels and ectoparasites may be worth revisiting.