DNA-based identification of forensically significant beetles from Southern Africa
- Authors: Collett, Isabel Judith
- Date: 2015
- Subjects: Carrion insects , Forensic entomology , Cleridae , Dermestidae , Silphidae , Staphylinidae , Scarabaeidae , Histeridae
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5923 , http://hdl.handle.net/10962/d1017801
- Description: Necrophilous insects, if correctly identified, can provide useful forensic information. Research in this area has focussed on flies and beetles remain comparatively under-studied, partly because some adult carrion beetles are difficult to identify morphologically, as are their juvenile stages, often requiring specialist expertise in both cases. Molecular taxonomy has been proposed as a solution to these problems. DNA “barcodes" are short fragments of mitochondrial cytochrome oxidase I (COI) DNA that are anticipated to delineate species. This approach is becoming increasingly popular, but has been met with varying enthusiasm from taxonomists. This thesis examines their use in identifying forensically significant beetles.The DNA barcodes of 234 specimens of 25 forensically significant southern African beetle species from seven families (Cleridae, Dermestidae, Silphidae, Staphylinidae, Scarabaeidae, Trogidae and Histeridae) were obtained. Thirty-three initial barcode amplification failures were overcome by using primers other than the standard Folmer pair, undermining the barcode concept’s hope of universal primers that would allow even non-specialists to produce barcodes. Another 150 specimens (64%) entirely failed to yield barcodes, including 18 fresh specimens of three species of Trogidae, implying another lack of universality of the barcoding protocol. The majority of the beetles clustered with confamilials on neighbour-joining and maximum likelihood trees, but 1.3% of the barcodes failed to cluster with their respective families, raising questions concerning the associating power of barcodes. The identification tools of the GenBank and BOLD on-line DNA sequence databases identified 21% of the specimens to the species level, 6% of them correctly. There was evidence of a paralogous sequence in the Cleridae that, while supporting identification now that it has been associated with a morphological identification, would hamper attempts at identification by clustering or phylogenetic analysis.Distance and haplotype network analyses of the barcodes of six widespread species showed that they are not geographically structured. Barcodes are thus unlikely to be indicators of the region of origin of a species and will not determine whether a corpse has been relocated after death. To assess whether a different mitochondrial DNA fragment might address (some of) these problems, a 2.2 kb fragment extending from the 5’ end of the COI gene to the 3’ end of the Cytochrome Oxidase II (COII) gene was analysed for nine species. It was found that, for Dermestidae, Scarabaeidae and Histeridae, higher degrees of diversity occurred downstreamof the barcode region, but the region of highest diversity in the Cleridae was in the barcode region. Thus, finding a more reliable fragment along the COI-COII region for each family may make robust and guaranteed DNA-based identification of these beetles more likely. The possibility of a forensic specimen being incorrectly or not identified based on its barcode alone exists in about 40% of cases, even with the new barcodes reported here. Forensic science sets a very high bar in assessing the performance of its techniques, and it is concluded that barcodes currently have unsettling failure rates as court-worthy evidence.
- Full Text:
- Date Issued: 2015
Gene expression analysis of Thaumatotibia leucotreta in response to the Cryptophlebia leucotreta granulovirus
- Authors: Ridgeway, Jaryd Antony
- Date: 2015
- Subjects: Gene expression , Insects -- Viruses , Tortricidae -- Viruses
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:5931 , http://hdl.handle.net/10962/d1017809
- Description: Gene expression studies provide baseline information on the interactions of insects with their environment. Despite the importance of this information, limited gene expression data are available for most insect pests, including the family Tortricidae (Lepidoptera), which includes Thaumatotibia leucotreta (Meyr), an important agricultural pest in Africa. Because T. leucotreta can be controlled successfully by a granulovirus, this system is a good model for exploring insect-virus susceptibility. The main aim of this study was to investigate gene expression of T. leucotreta in responce to virus infection. However, before pursuing this aim, two objectives required completion. First, the most suitable RNA extraction method for insects needed to be determined and second, the most suitable reference genes for qPCR for Tortricidae pests needed to be identified. Once these objectives were accomplished, the response of T. leucotreta to its granulovirus was evaluated at different temperatures and points after infection.Four RNA extraction methods, the RNeasy® Mini Kit, SV Total RNA isolation system, TRIzol® reagent, and a CTAB-based method, were compared using two beetle and two moth species, including T. leucotreta. The quality of extracted RNA was similar for all four species for all extraction methods. Based on several criteria, the best RNA extraction method was the SV Total RNA isolation system. Six candidate reference genes were evaluated for qPCR using different tissue types of T. leucotreta and two other Tortricidae pests. Additionally, reference genes were evaluated for T. leucotreta with and without its granulovirus at different temperatures. Reference gene stability was found to be dependent on species and tissue type. Overall the most suitable combination of reference genes for T. leucotreta were α-actin, arginine kinase and elongation factor 1-α.Gene expression of T. leucotreta in response to granulovirus infection at different temperatures and intervals after infection was evaluated by qPCR using 13 target genes associated with the infection process. Most genes were down-regulated after 24 and 48 h.p.i. However, after 72 h.p.i most genes were up-regulated. The same trend was observed at different temperatures, where most genes were down-regulated at 15°C and 25°C but up-regulated at 35°C. These results show that there is a dynamic gene expression response in T. leucotreta due to granulovirus infection under different conditions. Not only do these findings provide insight into the control of this tortricid pest, they also contribute further to our knowledge of insect-virus interactions.
- Full Text:
- Date Issued: 2015