The expression and evaluation of CrpeNPV gp37 as a formulation additive for enhanced infectivity with CrleGV-SA and improved Thaumatotibia leucotreta control
- Authors: Muleya, Naho
- Date: 2024-10-11
- Subjects: Cryptophlebia leucotreta Biological control , False Codling Moth , Cryptophlebia leucotreta granulovirus , Cryptophlebia peltastica nucleopolyhedrovirus , Citrus Diseases and pests South Africa , Baculoviruses
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463919 , vital:76457
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant pest native to Africa, causing damage to citrus and posing a threat to the export of fresh citrus in South Africa. Classified as a phytosanitary risk by several South African export markets, this pest necessitates effective control measures. Baculoviruses emerge as promising biological control agents against T. leucotreta due to their inherent safety and eco-friendly characteristics. Among these, Cryptophlebia leucotreta Granulovirus (CrleGV-SA) and Cryptophlebia peltastica Nucleopolyhedrovirus (CrpeNPV) stand out, both causing larval mortality upon infecting T. leucotreta. CrleGV-SA has been formulated into the products Cryptogran™, CryptoMax™ and Cryptex®, while CrpeNPV has been formulated into the product Multimax™. Both viruses are used in integrated pest management programmes to reduce fruit damage in agricultural fields, with CrleGV-SA having been employed against T. leucotreta for nearly 20 years in South Africa. However, these control options are limited by factors such as virulence and the slow speed of kill. This limitation can be addressed by exploiting potential synergistic relationships between baculoviruses infecting the same host. Previous studies have demonstrated that the truncated CpGV gp37 can enhance the infectivity of NPVs on other lepidopteran pests, such as Spodoptera exigua (Hübner). Although the mechanism behind this phenomenon remains unclear, it presents an opportunity to enhance the effectiveness of baculovirus-based management strategies. Notably, the genome of CrpeNPV encodes gp37, while CrleGV-SA lacks this gene. The potential interaction between CrleGV-SA and CrpeNPV gp37 remains unexplored. Therefore, investigating whether they exhibit synergistic or antagonistic effects is essential for optimising baculovirus-based management of T. leucotreta. This study aims to express CrpeNPV gp37 in a bacterial system and then evaluate its effect on larval mortality when combined with CrleGV-SA in laboratory bioassays. The initial step involved extracting genomic DNA (gDNA) from occlusion bodies (OBs) of CrpeNPV. A modified Quick DNA Miniprep plus kit was utilised, which entailed pre-treatment with Na2CO3 followed by neutralisation with Tris-HCI before gDNA extraction using the kit. Subsequently, the concentration of the gDNA was estimated using a Nanodrop spectrophotometer. Oligonucleotides targeting the CrpeNPV gp37 gene were designed for PCR amplification, with the gDNA serving as a template. The gp37 amplicon was identified through agarose gel electrophoresis and then gel purified in preparation for cloning. Secondly, the purified PCR product was cloned into the intermediate vector pJET1.2/blunt and then subcloned into the bacterial expression vector pCA528 through DNA ligation. The construction of recombinant plasmids (pJET-gp37 and pCA-gp37) was conducted and verified using Colony PCR, plasmid extraction, restriction enzyme analysis, and Sanger sequencing. Thirdly, the recombinant protein (6×His-SUMO-gp37) was expressed and purified using Nickel affinity chromatography and analysed through SDS-PAGE and Western blot techniques. The expression of 6×His-SUMO-gp37 was carried out at both 25 °C and 18 °C. A time course induction study was conducted, inducing transformed cells for 0-, 3-, 5-, and 24-hours post induction (hpi). SDS-PAGE and Western blotting of samples collected at various time points revealed that 6×His-SUMO-gp37, approximately 42 kDa in size, was visible from 3 hpi, with maximal expression at 24 hpi. Solubility analysis of 6×His-SUMO-gp37 was performed at both temperatures, showing solubility at 18 °C but predominantly present in the insoluble fraction. The soluble protein was purified under native conditions, while the insoluble protein was purified under denaturing conditions. Despite being unable to elute 6×His-SUMO-gp37 under native conditions, successful elution was achieved under denaturing conditions, confirmed via Western blot analysis. No further experiments were conducted on the eluted 6×His-SUMO-gp37 under denaturing conditions. Lastly, a preliminary surface dose bioassay was conducted to evaluate the efficacy of pelleted bacteria expressing 6×His-SUMO-gp37 in combination with CrleGV-SA against T. leucotreta neonates. Two lethal concentration doses of CrleGV-SA were prepared: a low concentration (2.96×104 OBs/mL) capable of killing 40 % of the T. leucotreta population, and a high concentration (2.96×105 OBs/mL) capable of killing 90 % of the population. The target protein, 6×His-SUMO-gp37, and the control, pCA528, were obtained by lysing the cells, centrifuging the samples, and collecting the insoluble fractions in pellet form. These fractions were then resuspended in PBS and used as treatments in combination with the prepared CrleGV-SA concentration doses. The concentration of the pellets was estimated using a Nanodrop spectrophotometer by measuring the absorbance at 280 nm. The bioassay results revealed that the combination of 100 μg/mL of pelleted bacteria expressing 6×His-SUMO-gp37 with CrleGV-SA had no effect on T. leucotreta larval mortality compared to CrleGV-SA alone. A one-way ANOVA was performed to assess differences among the virus treatment groups, concluding that no statistically significant differences were observed among the groups. The experiments in this study provided valuable insights for future research, particularly in exploring the use of a protein-virus combination as a novel method for pest control. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
- Date Issued: 2024-10-11
- Authors: Muleya, Naho
- Date: 2024-10-11
- Subjects: Cryptophlebia leucotreta Biological control , False Codling Moth , Cryptophlebia leucotreta granulovirus , Cryptophlebia peltastica nucleopolyhedrovirus , Citrus Diseases and pests South Africa , Baculoviruses
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/463919 , vital:76457
- Description: Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Tortricidae) is a significant pest native to Africa, causing damage to citrus and posing a threat to the export of fresh citrus in South Africa. Classified as a phytosanitary risk by several South African export markets, this pest necessitates effective control measures. Baculoviruses emerge as promising biological control agents against T. leucotreta due to their inherent safety and eco-friendly characteristics. Among these, Cryptophlebia leucotreta Granulovirus (CrleGV-SA) and Cryptophlebia peltastica Nucleopolyhedrovirus (CrpeNPV) stand out, both causing larval mortality upon infecting T. leucotreta. CrleGV-SA has been formulated into the products Cryptogran™, CryptoMax™ and Cryptex®, while CrpeNPV has been formulated into the product Multimax™. Both viruses are used in integrated pest management programmes to reduce fruit damage in agricultural fields, with CrleGV-SA having been employed against T. leucotreta for nearly 20 years in South Africa. However, these control options are limited by factors such as virulence and the slow speed of kill. This limitation can be addressed by exploiting potential synergistic relationships between baculoviruses infecting the same host. Previous studies have demonstrated that the truncated CpGV gp37 can enhance the infectivity of NPVs on other lepidopteran pests, such as Spodoptera exigua (Hübner). Although the mechanism behind this phenomenon remains unclear, it presents an opportunity to enhance the effectiveness of baculovirus-based management strategies. Notably, the genome of CrpeNPV encodes gp37, while CrleGV-SA lacks this gene. The potential interaction between CrleGV-SA and CrpeNPV gp37 remains unexplored. Therefore, investigating whether they exhibit synergistic or antagonistic effects is essential for optimising baculovirus-based management of T. leucotreta. This study aims to express CrpeNPV gp37 in a bacterial system and then evaluate its effect on larval mortality when combined with CrleGV-SA in laboratory bioassays. The initial step involved extracting genomic DNA (gDNA) from occlusion bodies (OBs) of CrpeNPV. A modified Quick DNA Miniprep plus kit was utilised, which entailed pre-treatment with Na2CO3 followed by neutralisation with Tris-HCI before gDNA extraction using the kit. Subsequently, the concentration of the gDNA was estimated using a Nanodrop spectrophotometer. Oligonucleotides targeting the CrpeNPV gp37 gene were designed for PCR amplification, with the gDNA serving as a template. The gp37 amplicon was identified through agarose gel electrophoresis and then gel purified in preparation for cloning. Secondly, the purified PCR product was cloned into the intermediate vector pJET1.2/blunt and then subcloned into the bacterial expression vector pCA528 through DNA ligation. The construction of recombinant plasmids (pJET-gp37 and pCA-gp37) was conducted and verified using Colony PCR, plasmid extraction, restriction enzyme analysis, and Sanger sequencing. Thirdly, the recombinant protein (6×His-SUMO-gp37) was expressed and purified using Nickel affinity chromatography and analysed through SDS-PAGE and Western blot techniques. The expression of 6×His-SUMO-gp37 was carried out at both 25 °C and 18 °C. A time course induction study was conducted, inducing transformed cells for 0-, 3-, 5-, and 24-hours post induction (hpi). SDS-PAGE and Western blotting of samples collected at various time points revealed that 6×His-SUMO-gp37, approximately 42 kDa in size, was visible from 3 hpi, with maximal expression at 24 hpi. Solubility analysis of 6×His-SUMO-gp37 was performed at both temperatures, showing solubility at 18 °C but predominantly present in the insoluble fraction. The soluble protein was purified under native conditions, while the insoluble protein was purified under denaturing conditions. Despite being unable to elute 6×His-SUMO-gp37 under native conditions, successful elution was achieved under denaturing conditions, confirmed via Western blot analysis. No further experiments were conducted on the eluted 6×His-SUMO-gp37 under denaturing conditions. Lastly, a preliminary surface dose bioassay was conducted to evaluate the efficacy of pelleted bacteria expressing 6×His-SUMO-gp37 in combination with CrleGV-SA against T. leucotreta neonates. Two lethal concentration doses of CrleGV-SA were prepared: a low concentration (2.96×104 OBs/mL) capable of killing 40 % of the T. leucotreta population, and a high concentration (2.96×105 OBs/mL) capable of killing 90 % of the population. The target protein, 6×His-SUMO-gp37, and the control, pCA528, were obtained by lysing the cells, centrifuging the samples, and collecting the insoluble fractions in pellet form. These fractions were then resuspended in PBS and used as treatments in combination with the prepared CrleGV-SA concentration doses. The concentration of the pellets was estimated using a Nanodrop spectrophotometer by measuring the absorbance at 280 nm. The bioassay results revealed that the combination of 100 μg/mL of pelleted bacteria expressing 6×His-SUMO-gp37 with CrleGV-SA had no effect on T. leucotreta larval mortality compared to CrleGV-SA alone. A one-way ANOVA was performed to assess differences among the virus treatment groups, concluding that no statistically significant differences were observed among the groups. The experiments in this study provided valuable insights for future research, particularly in exploring the use of a protein-virus combination as a novel method for pest control. , Thesis (MSc) -- Faculty of Science, Biochemistry, Microbiology & Bioinformatics, 2024
- Full Text:
- Date Issued: 2024-10-11
Augmentation of Aphytis melinus DeBach (Hymenoptera: Aphelinidae) for the control of California red scale Aonidiella aurantii Maskell (Hemiptera: Diaspididae) on citrus
- De Beer, Ernst Friedrich Ludwig
- Authors: De Beer, Ernst Friedrich Ludwig
- Date: 2024-04-05
- Subjects: Aphytis melinus , Aphytis , Aonidiella aurantii , Citrus Diseases and pests South Africa , Pests Biological control
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435638 , vital:73175 , DOI 10.21504/10962/435638
- Description: Aphytis lingnanensis was reared and tested in South Africa in the early 2000s for augmentation against red scale on citrus and was found to be ineffective. Aphytis melinus is now commercially available and it is important that the efficacy of augmentation thereof on red scale is determined locally. Field trials, fitness assessments and molecular identification on A. melinus from two insectaries were done. Field trials was done in seven, five and six pairs of comparable release and control orchards across the Eastern and Western Cape during the seasons of 2019/2020, 2020/2021 and 2021/2022 respectively. Red scale infestation was monitored and a sample of 20 infested fruit from each orchard was randomly collected every four weeks. Aphytis spp. responsible for parasitism were identified and the percentage parasitism recorded. Results of this study of field trials suggest that the augmentation of A. melinus did not significantly increase the level of parasitism above that of the untreated control. Five repetitions with six replicates of flight and longevity tests were performed with wasps from each insectary. Wasps in the longevity test from two insectaries were kept at 23 °C and 65% RH with honey. Flight tests were performed in tubes of 16 by 30 cm, with a light above a clear, sticky ceiling at 23 °C and 65% RH. On average in five replicates, 65%, 33% and 17% A. melinus wasps were alive on day one, five and 10 respectively. The overall sex ratio was 1.58 for females to males, but 1.05, 2.19 and 2.66 for non-flyers, non-crawlers, crawlers, and flyers respectively. In flight tests for both insectaries combined, only 36.97% of wasps could initiate flight in 24 h while 56.96% remained on the tube floor, and 6.05% attempted to crawl upwards. No significant differences in flight performance were recorded between the two insectaries. Wasps from the local insectary lived significantly longer during the longevity tests but were shorter in transit than wasps from the overseas insectary. COI genes were sequenced and compared against Genbank sequences using BLAST. Molecular identifications did not confirm morphological identifications for all species, indicating unexpected genetic complexity. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Date Issued: 2024-04-05
- Authors: De Beer, Ernst Friedrich Ludwig
- Date: 2024-04-05
- Subjects: Aphytis melinus , Aphytis , Aonidiella aurantii , Citrus Diseases and pests South Africa , Pests Biological control
- Language: English
- Type: Academic theses , Doctoral theses , text
- Identifier: http://hdl.handle.net/10962/435638 , vital:73175 , DOI 10.21504/10962/435638
- Description: Aphytis lingnanensis was reared and tested in South Africa in the early 2000s for augmentation against red scale on citrus and was found to be ineffective. Aphytis melinus is now commercially available and it is important that the efficacy of augmentation thereof on red scale is determined locally. Field trials, fitness assessments and molecular identification on A. melinus from two insectaries were done. Field trials was done in seven, five and six pairs of comparable release and control orchards across the Eastern and Western Cape during the seasons of 2019/2020, 2020/2021 and 2021/2022 respectively. Red scale infestation was monitored and a sample of 20 infested fruit from each orchard was randomly collected every four weeks. Aphytis spp. responsible for parasitism were identified and the percentage parasitism recorded. Results of this study of field trials suggest that the augmentation of A. melinus did not significantly increase the level of parasitism above that of the untreated control. Five repetitions with six replicates of flight and longevity tests were performed with wasps from each insectary. Wasps in the longevity test from two insectaries were kept at 23 °C and 65% RH with honey. Flight tests were performed in tubes of 16 by 30 cm, with a light above a clear, sticky ceiling at 23 °C and 65% RH. On average in five replicates, 65%, 33% and 17% A. melinus wasps were alive on day one, five and 10 respectively. The overall sex ratio was 1.58 for females to males, but 1.05, 2.19 and 2.66 for non-flyers, non-crawlers, crawlers, and flyers respectively. In flight tests for both insectaries combined, only 36.97% of wasps could initiate flight in 24 h while 56.96% remained on the tube floor, and 6.05% attempted to crawl upwards. No significant differences in flight performance were recorded between the two insectaries. Wasps from the local insectary lived significantly longer during the longevity tests but were shorter in transit than wasps from the overseas insectary. COI genes were sequenced and compared against Genbank sequences using BLAST. Molecular identifications did not confirm morphological identifications for all species, indicating unexpected genetic complexity. , Thesis (PhD) -- Faculty of Science, Zoology and Entomology, 2024
- Full Text:
- Date Issued: 2024-04-05
Composition and physiological roles of gut microbiota in the False Coding Moth (Thaumatotibia leucotreta)
- Authors: Richardson, Perryn Heather
- Date: 2023-10-13
- Subjects: False codling moth , Microbiomes , Insect physiology , Citrus Diseases and pests South Africa , Biological pest control , Cryptophlebia leucotreta
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424457 , vital:72155
- Description: Gut microbiota can have a profound influence on host performance, behaviour and fitness. For False Codling Moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae), a major pest of citrus in South Africa, little work has been undertaken to date on gut microbe diversity or its influence on the host. This thesis aimed to i) characterise the gut microbiome of FCM under laboratory conditions and in FCM from the field, ii) and produce moths with reduced gut microbiota through egg dechorionation, which was followed by iii) the measurement of a suite of physiological traits, namely mass, survival and thermal stress in FCM from normal laboratory, dechorionated laboratory and field collected larvae that may be indicative of overall field performance. We aimed to directly test the hypothesis that gut microbial diversity partly determines insect performance and fitness by measuring its effects on growth, development, and tolerance to cold temperatures in FCM. FCM eggs that underwent dechorionation with sodium hypochlorite had an overall effect on larval survival, egg morphology and both larval and adult moth physiological measures. Increasing concentrations of sodium hypochlorite significantly decreased insect survival, (𝜒2(1, n = 10 850) = 21.724, p-value < 0.0001), with a concentration of ≈3.69% as the concentration limit (p-value < 0.001). Successful dechorionation of FCM was achieved with a wash of sodium hypochlorite at around 3.69% concentration and was visually confirmed by reduction of FCM egg surface area, (𝜒2(25, n = 260) p-value < 0.0001) and Scanning Electron Micrographs of the egg morphology. The gut microbiome of FCM from the different focus treatments was successfully characterized. Identification of the dominant bacterial families in these microbiomes revealed Xanthobacteraceae, Beijerinckiaceae and Burkholderiaceae in both the laboratory reared and field collected larvae, which suggests their systematic association with T. leucotreta. The most abundant genera were revealed as Bradyrhizobium, Methylobacterium and Burkholderia-Caballeronia-Paraburkholderia. Comparison of larval mass showed that treatment (dechorionated or not) had a significant effect on larval mass (𝜒2(2, n = 230) = 22.703, p-value < 0.001), field larvae were heavier than both control larvae and larvae with a disrupted gut microbiome. However, adult insects with a disrupted gut microbiome had more mass than individuals from the control and field-collected larvae with intact gut microbiomes (𝜒2(2, n = 230) = 39.074, p-value < 0.001). Despite the difference in mass between larval treatments, there was no significant difference in relative protein (𝜒2(2, n = 24) = 5.680, p-value = 0.06), carbohydrate (𝜒2(2, n = 24) = 3.940, p-value = 0.14) or lipid (𝜒2(2, n = 24) = 6.032, p-value = 0.05) content between individuals from the control and dechorionated treatments and field-collected individuals. Turning to thermal physiology, insects collected from the field took significantly longer to recover from chill coma than both laboratory treatments with intact and disrupted gut microbiomes (𝜒2(2, n = 129 = 39.659, p-value < 0.001). In addition, exposure to cold stress showed that treatment had a significant effect on insect mortality (𝜒2(2, n = 272) = 9.176, p-value = 0.01), with individuals from the control and dechorionated treatment being less likely to die after experiencing cold stress compared to field-collected individuals. Differences in the mass and thermal tolerance of insects with intact and disrupted gut microbiota suggest that gut microbiota may play an important role in the cold performance of T. leucotreta, and these findings constitute the basis for future molecular work on the functions of these bacterial taxa. This research highlights the need for consideration of the effects of T. leucotreta microbiome in current pest control programs. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
- Authors: Richardson, Perryn Heather
- Date: 2023-10-13
- Subjects: False codling moth , Microbiomes , Insect physiology , Citrus Diseases and pests South Africa , Biological pest control , Cryptophlebia leucotreta
- Language: English
- Type: Academic theses , Master's theses , text
- Identifier: http://hdl.handle.net/10962/424457 , vital:72155
- Description: Gut microbiota can have a profound influence on host performance, behaviour and fitness. For False Codling Moth (FCM), Thaumatotibia leucotreta (Lepidoptera: Tortricidae), a major pest of citrus in South Africa, little work has been undertaken to date on gut microbe diversity or its influence on the host. This thesis aimed to i) characterise the gut microbiome of FCM under laboratory conditions and in FCM from the field, ii) and produce moths with reduced gut microbiota through egg dechorionation, which was followed by iii) the measurement of a suite of physiological traits, namely mass, survival and thermal stress in FCM from normal laboratory, dechorionated laboratory and field collected larvae that may be indicative of overall field performance. We aimed to directly test the hypothesis that gut microbial diversity partly determines insect performance and fitness by measuring its effects on growth, development, and tolerance to cold temperatures in FCM. FCM eggs that underwent dechorionation with sodium hypochlorite had an overall effect on larval survival, egg morphology and both larval and adult moth physiological measures. Increasing concentrations of sodium hypochlorite significantly decreased insect survival, (𝜒2(1, n = 10 850) = 21.724, p-value < 0.0001), with a concentration of ≈3.69% as the concentration limit (p-value < 0.001). Successful dechorionation of FCM was achieved with a wash of sodium hypochlorite at around 3.69% concentration and was visually confirmed by reduction of FCM egg surface area, (𝜒2(25, n = 260) p-value < 0.0001) and Scanning Electron Micrographs of the egg morphology. The gut microbiome of FCM from the different focus treatments was successfully characterized. Identification of the dominant bacterial families in these microbiomes revealed Xanthobacteraceae, Beijerinckiaceae and Burkholderiaceae in both the laboratory reared and field collected larvae, which suggests their systematic association with T. leucotreta. The most abundant genera were revealed as Bradyrhizobium, Methylobacterium and Burkholderia-Caballeronia-Paraburkholderia. Comparison of larval mass showed that treatment (dechorionated or not) had a significant effect on larval mass (𝜒2(2, n = 230) = 22.703, p-value < 0.001), field larvae were heavier than both control larvae and larvae with a disrupted gut microbiome. However, adult insects with a disrupted gut microbiome had more mass than individuals from the control and field-collected larvae with intact gut microbiomes (𝜒2(2, n = 230) = 39.074, p-value < 0.001). Despite the difference in mass between larval treatments, there was no significant difference in relative protein (𝜒2(2, n = 24) = 5.680, p-value = 0.06), carbohydrate (𝜒2(2, n = 24) = 3.940, p-value = 0.14) or lipid (𝜒2(2, n = 24) = 6.032, p-value = 0.05) content between individuals from the control and dechorionated treatments and field-collected individuals. Turning to thermal physiology, insects collected from the field took significantly longer to recover from chill coma than both laboratory treatments with intact and disrupted gut microbiomes (𝜒2(2, n = 129 = 39.659, p-value < 0.001). In addition, exposure to cold stress showed that treatment had a significant effect on insect mortality (𝜒2(2, n = 272) = 9.176, p-value = 0.01), with individuals from the control and dechorionated treatment being less likely to die after experiencing cold stress compared to field-collected individuals. Differences in the mass and thermal tolerance of insects with intact and disrupted gut microbiota suggest that gut microbiota may play an important role in the cold performance of T. leucotreta, and these findings constitute the basis for future molecular work on the functions of these bacterial taxa. This research highlights the need for consideration of the effects of T. leucotreta microbiome in current pest control programs. , Thesis (MSc) -- Faculty of Science, Zoology and Entomology, 2023
- Full Text:
- Date Issued: 2023-10-13
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