Isolation, identification and genetic characterisation of a microsporidium isolated from the carob moth, Ectomyelois ceratoniae (Lepidoptera: Pyralidae)
- Authors: Lloyd, Melissa
- Date: 2018
- Subjects: Pyralidae , Pyralidae -- Genetics , Pyralidae -- Phylogeny , Pyralidae -- Pathogens , Cladistic analysis , Transmission electron microscopy , Carob moth (Ectomyelois ceratoniae)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/61894 , vital:28075
- Description: Carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) is an economically important pest, yet its biology and pest status on citrus in South Africa was, until recently, poorly understood. A study was initiated to determine the cause of collapse of a laboratory carob moth colony that was established to investigate the biology of carob moth on citrus and to develop integrated management strategies for the pest. An organism was isolated from deceased larvae and was morphologically identified as a microsporidium, based on transmission electron microscopy. Microsporidia are obligate intracellular parasites that have been found to infect almost all eukaryotes. Several Nosema species have been isolated from economically important insect pests, yet little genetic information is available from online databases for identification. Mature spores were recovered and measured using transmission electron microscopy. Spores were ovocylindrical with a wrinkled exospore, and had a length of 2.8 ± 0.02 pm and a width of 1.6 ± 0.04 pm. The identity of the microsporidium was confirmed by PCR amplification, sequencing and analysis of the regions encoding the ribosomal RNA. BLAST analysis of the different rRNA regions amplified showed that the microsporidium shared a 96 - 99 % identity with Nosema sp. M-Pr, Nosema carpocapsae, Nosema oulemae, Nosema sp. CO1, Microsporidium 57864, and Nosema bombi. Phylogenetic analysis of the SSU and LSU rRNA genes showed that the microsporidium clustered with the Nosema / Vairimorpha clade, supported by a bootstrap value of 100. The organisation of the RNA cistron was determined by PCR amplification using the primer set 18f and L1328r to be 5’-SSU-ITS-LSU-IGS-5S-3’, which confirms the placement of the microsporidium within the Nosema / Vairimorpha clade. Because the BLAST results showed a close relationship with Nosema carpocapsae, a microsporidium infecting codling moth, the pathogenicity of the microsporidium was tested against codling moth by inoculating artificial diet with a high spore concentration of 1.1 x 107 spores/ml and a low spore concentration of 1.1 x 104 spores/ml. DNA was extracted from deceased larvae inoculated with the high concentration, and PCR of the SSU rRNA gene and bacterial 16S region was performed. Mortality in the high concentration experiment was significant (p = 0.05), but the cause of infection was determined to be a bacterium, through sequencing and BLAST analysis of the bacterial 16S rDNA. The bacterium shared a 99 % identity with Bacillus cereus. Percentage mortality (p = 0.09), larval mass (p = 0.09) and instar (p = 0.24) did not differ significantly between treatments in the low concentration experiment. DNA was extracted from the larvae and PCR amplification of the SSU rRNA gene was performed to determine whether microsporidia were present. No SSU bands were observed in any of the treatments and percentage mortality was not significant, thus it was determined that no infection occurred. This is the first study to report the genetic characterisation of a microsporidium isolated from carob moth and provides important genetic information for classification of microsporidia within the Nosema / Vairimorpha clade. It is also one of few studies in which the complete rRNA cistron of a species within the Nosema / Vairimorpha clade has been sequenced. The identification of a microsporidium from a laboratory colony of carob moth is important as it provides information about pathogens infecting the carob moth and constraints to carob moth rearing, which is useful for further studies on rearing carob moth and for establishment of a clean colony for research purposes.
- Full Text:
- Date Issued: 2018
- Authors: Lloyd, Melissa
- Date: 2018
- Subjects: Pyralidae , Pyralidae -- Genetics , Pyralidae -- Phylogeny , Pyralidae -- Pathogens , Cladistic analysis , Transmission electron microscopy , Carob moth (Ectomyelois ceratoniae)
- Language: English
- Type: text , Thesis , Masters , MSc
- Identifier: http://hdl.handle.net/10962/61894 , vital:28075
- Description: Carob moth, Ectomyelois ceratoniae (Zeller) (Lepidoptera: Pyralidae) is an economically important pest, yet its biology and pest status on citrus in South Africa was, until recently, poorly understood. A study was initiated to determine the cause of collapse of a laboratory carob moth colony that was established to investigate the biology of carob moth on citrus and to develop integrated management strategies for the pest. An organism was isolated from deceased larvae and was morphologically identified as a microsporidium, based on transmission electron microscopy. Microsporidia are obligate intracellular parasites that have been found to infect almost all eukaryotes. Several Nosema species have been isolated from economically important insect pests, yet little genetic information is available from online databases for identification. Mature spores were recovered and measured using transmission electron microscopy. Spores were ovocylindrical with a wrinkled exospore, and had a length of 2.8 ± 0.02 pm and a width of 1.6 ± 0.04 pm. The identity of the microsporidium was confirmed by PCR amplification, sequencing and analysis of the regions encoding the ribosomal RNA. BLAST analysis of the different rRNA regions amplified showed that the microsporidium shared a 96 - 99 % identity with Nosema sp. M-Pr, Nosema carpocapsae, Nosema oulemae, Nosema sp. CO1, Microsporidium 57864, and Nosema bombi. Phylogenetic analysis of the SSU and LSU rRNA genes showed that the microsporidium clustered with the Nosema / Vairimorpha clade, supported by a bootstrap value of 100. The organisation of the RNA cistron was determined by PCR amplification using the primer set 18f and L1328r to be 5’-SSU-ITS-LSU-IGS-5S-3’, which confirms the placement of the microsporidium within the Nosema / Vairimorpha clade. Because the BLAST results showed a close relationship with Nosema carpocapsae, a microsporidium infecting codling moth, the pathogenicity of the microsporidium was tested against codling moth by inoculating artificial diet with a high spore concentration of 1.1 x 107 spores/ml and a low spore concentration of 1.1 x 104 spores/ml. DNA was extracted from deceased larvae inoculated with the high concentration, and PCR of the SSU rRNA gene and bacterial 16S region was performed. Mortality in the high concentration experiment was significant (p = 0.05), but the cause of infection was determined to be a bacterium, through sequencing and BLAST analysis of the bacterial 16S rDNA. The bacterium shared a 99 % identity with Bacillus cereus. Percentage mortality (p = 0.09), larval mass (p = 0.09) and instar (p = 0.24) did not differ significantly between treatments in the low concentration experiment. DNA was extracted from the larvae and PCR amplification of the SSU rRNA gene was performed to determine whether microsporidia were present. No SSU bands were observed in any of the treatments and percentage mortality was not significant, thus it was determined that no infection occurred. This is the first study to report the genetic characterisation of a microsporidium isolated from carob moth and provides important genetic information for classification of microsporidia within the Nosema / Vairimorpha clade. It is also one of few studies in which the complete rRNA cistron of a species within the Nosema / Vairimorpha clade has been sequenced. The identification of a microsporidium from a laboratory colony of carob moth is important as it provides information about pathogens infecting the carob moth and constraints to carob moth rearing, which is useful for further studies on rearing carob moth and for establishment of a clean colony for research purposes.
- Full Text:
- Date Issued: 2018
A systematic study of Berkheya and allies (Compositae)
- Authors: Phaliso, Ntombifikile
- Date: 2013
- Subjects: Compositae -- Africa, Southern , Daisies -- Africa, Southern , Compositae -- Phylogeny -- Africa, Southern , Compositae -- Geographical distribution -- Africa, Southern , Compositae -- Classification -- Africa, Southern , Cladistic analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4179 , http://hdl.handle.net/10962/d1003054
- Description: Berkheya Ehrh. is a genus of daisies in the tribe Arctotideae, subtribe Gorteriinae with over 80 species, most of which occur in southern Africa. This genus has centres of diversity associated with the montane regions of South Africa, including the Drakensberg Alpine Centre and Mpumalanga escarpment regions. Previous molecular and morphological studies indicate that Berkheya is paraphyletic. I present phylogenies based on nrDNA (ITS; Internal Transcribed Spacer) and cpDNA (psbA-trnH) sequence data analysed with Bayesian Inference and Parsimony. A phylogeny of combined cp- and nrDNA is also presented. These phylogenies are used to assess generic limits and to investigate the biogeographic patterns of Berkheya and its allies. The ITS phylogeny shows five well supported clades of Berkheya, two of which (Clades I and 2) are monophyletic summer rainfall region clades. Clades 3 to 5 are all paraphyletic winter rainfall clades with Cullumia occurring in the third clade, Cuspida occurring in the fourth and Didelta in Clade 5. Both psbA-trnH and combined phylogenies show concordance with the clade distribution shown in the ITS phylogeny. The ITS phylogeny was used to analyse correspondence with Roessler’s (1959) Series. It was found that the phylogeny showed considerable agreement with Roessler’s series, but B. bipinnatifida and B. spinosa of Series Speciosae may require some revision as well as taxa of monotypic series Cruciatae and Angustae. It is suggested that the latter series be merged with Cullumia species to form a single series. Some consideration should be taken to include Didelta species into Series Fruticosae as Didelta occurs in subclade 5b of Clade 5 with other Series Fruticosae taxa. Achene morphology was examined from species from each of the five clades to investigate the relationships of Berkheya and its allies, as well as to determine if there were any consistent achene features for each clade. The structure of surface cells on the fruit, the presence, absence and morphology of twin hairs as well as the structure of the pappus scales were found to be most useful in reflecting phylogenetic relationships within the clades. When compared with the clades of the ITS phylogeny, achene morphology showed consistent characters between taxa occurring in the same clades. As the most comprehensive study involving Berkheya, this phylogenenetic investigation was able to confirm that Berkheya is a paraphyletic genus with Didelta, Cullumia and Cuspida needing to be subsumed into Berkheya. An alternative classification is that taxa of Clade 5 could possibly be erected as an expanded Didelta, separate and sister to Berkheya.
- Full Text:
- Date Issued: 2013
- Authors: Phaliso, Ntombifikile
- Date: 2013
- Subjects: Compositae -- Africa, Southern , Daisies -- Africa, Southern , Compositae -- Phylogeny -- Africa, Southern , Compositae -- Geographical distribution -- Africa, Southern , Compositae -- Classification -- Africa, Southern , Cladistic analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4179 , http://hdl.handle.net/10962/d1003054
- Description: Berkheya Ehrh. is a genus of daisies in the tribe Arctotideae, subtribe Gorteriinae with over 80 species, most of which occur in southern Africa. This genus has centres of diversity associated with the montane regions of South Africa, including the Drakensberg Alpine Centre and Mpumalanga escarpment regions. Previous molecular and morphological studies indicate that Berkheya is paraphyletic. I present phylogenies based on nrDNA (ITS; Internal Transcribed Spacer) and cpDNA (psbA-trnH) sequence data analysed with Bayesian Inference and Parsimony. A phylogeny of combined cp- and nrDNA is also presented. These phylogenies are used to assess generic limits and to investigate the biogeographic patterns of Berkheya and its allies. The ITS phylogeny shows five well supported clades of Berkheya, two of which (Clades I and 2) are monophyletic summer rainfall region clades. Clades 3 to 5 are all paraphyletic winter rainfall clades with Cullumia occurring in the third clade, Cuspida occurring in the fourth and Didelta in Clade 5. Both psbA-trnH and combined phylogenies show concordance with the clade distribution shown in the ITS phylogeny. The ITS phylogeny was used to analyse correspondence with Roessler’s (1959) Series. It was found that the phylogeny showed considerable agreement with Roessler’s series, but B. bipinnatifida and B. spinosa of Series Speciosae may require some revision as well as taxa of monotypic series Cruciatae and Angustae. It is suggested that the latter series be merged with Cullumia species to form a single series. Some consideration should be taken to include Didelta species into Series Fruticosae as Didelta occurs in subclade 5b of Clade 5 with other Series Fruticosae taxa. Achene morphology was examined from species from each of the five clades to investigate the relationships of Berkheya and its allies, as well as to determine if there were any consistent achene features for each clade. The structure of surface cells on the fruit, the presence, absence and morphology of twin hairs as well as the structure of the pappus scales were found to be most useful in reflecting phylogenetic relationships within the clades. When compared with the clades of the ITS phylogeny, achene morphology showed consistent characters between taxa occurring in the same clades. As the most comprehensive study involving Berkheya, this phylogenenetic investigation was able to confirm that Berkheya is a paraphyletic genus with Didelta, Cullumia and Cuspida needing to be subsumed into Berkheya. An alternative classification is that taxa of Clade 5 could possibly be erected as an expanded Didelta, separate and sister to Berkheya.
- Full Text:
- Date Issued: 2013
A taxonomic revision of the genera of the subtribe Dracophilinae (Aizoaceae: Ruschioideae)
- Authors: Mannheimer, Coleen Anne
- Date: 2006
- Subjects: Aizoaceae , Plants -- Classification , Cladistic analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4247 , http://hdl.handle.net/10962/d1007461 , Aizoaceae , Plants -- Classification , Cladistic analysis
- Description: Namibia, Juttadinteria and Dracophilus, the three genera belonging to the subtribe Dracophilinae Schwantes (Aizoaceae: Ruschioideae) were revised. Macro-morphology, leaf anatomy and micromorphology of leaf epidermides, pollen, tapetal orbicules and seed were studied in order to test taxon limits, to determine relationships between taxa, and to improve knowledge of their characteristics as well as the taxonomy of the subtribe. The investigation was based on herbarium specimens, field observations and cultivated, living plants. Phenetic cluster analyses were used to confirm species while intergeneric and interspecific relationships of the taxa so indicated were later tested by means of a phylogenetic analysis. The existence of the three genera was provisionally upheld by this study although phylogenetic analysis showed Namibia nested as a strongly supported monophyletic group within a poorly supported luttadinteria. Further work is needed to clarify whether Namibia should be sunk into Juttadinteria. The latest treatments of Juttadinteria and Dracophilus by Hartmann (2001) were supprted. However, in contrast to her latest treatment of Namibia, N. pomonae was sunk into N. cinerea and N. ponderosa was reinstated and typified. Juttadinteria was found to be a poorly resolved genus with many intergrading and overlapping characters, possible due to recent speciation. Possible subspecific groups within J. deserticola and J. simpsonii were indicated by the phenetic study but further work is needed before any formal infraspecific taxonomic rank can be assigned to them. Detailed descriptions of the nine elucidated species and the three genera as well as new keys for their identification are provided in order to facilitate further work in this group. Distribution maps are provided for the genera and species, and the subtribe. Variation of character states within the subtribe, distribution area, ecology and biology are discussed.
- Full Text:
- Date Issued: 2006
- Authors: Mannheimer, Coleen Anne
- Date: 2006
- Subjects: Aizoaceae , Plants -- Classification , Cladistic analysis
- Language: English
- Type: Thesis , Masters , MSc
- Identifier: vital:4247 , http://hdl.handle.net/10962/d1007461 , Aizoaceae , Plants -- Classification , Cladistic analysis
- Description: Namibia, Juttadinteria and Dracophilus, the three genera belonging to the subtribe Dracophilinae Schwantes (Aizoaceae: Ruschioideae) were revised. Macro-morphology, leaf anatomy and micromorphology of leaf epidermides, pollen, tapetal orbicules and seed were studied in order to test taxon limits, to determine relationships between taxa, and to improve knowledge of their characteristics as well as the taxonomy of the subtribe. The investigation was based on herbarium specimens, field observations and cultivated, living plants. Phenetic cluster analyses were used to confirm species while intergeneric and interspecific relationships of the taxa so indicated were later tested by means of a phylogenetic analysis. The existence of the three genera was provisionally upheld by this study although phylogenetic analysis showed Namibia nested as a strongly supported monophyletic group within a poorly supported luttadinteria. Further work is needed to clarify whether Namibia should be sunk into Juttadinteria. The latest treatments of Juttadinteria and Dracophilus by Hartmann (2001) were supprted. However, in contrast to her latest treatment of Namibia, N. pomonae was sunk into N. cinerea and N. ponderosa was reinstated and typified. Juttadinteria was found to be a poorly resolved genus with many intergrading and overlapping characters, possible due to recent speciation. Possible subspecific groups within J. deserticola and J. simpsonii were indicated by the phenetic study but further work is needed before any formal infraspecific taxonomic rank can be assigned to them. Detailed descriptions of the nine elucidated species and the three genera as well as new keys for their identification are provided in order to facilitate further work in this group. Distribution maps are provided for the genera and species, and the subtribe. Variation of character states within the subtribe, distribution area, ecology and biology are discussed.
- Full Text:
- Date Issued: 2006
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