Entomology

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Development of Beauveria brongniartii as a bio-insecticide to control white grub (Coleoptera: Scarabaeidae) species attacking sugarcane in South Africa.
(2016) Kheswa, Nozipho.; Conlong, Desmond Edward.; Laing, Mark Delmege.; Shuttleworth, Adam.
Abstract available in PDF file.
Investigations into the incidence and ecology of bilobata subsecivella (zeller) (lepidoptera: gelechidae) : a new pest of groundnut in South Africa.
(2015) Buthelezi, Nokubekezela Makhosi.; Conlong, Desmond Edward.; Olckers, Terence.
The leaf-mining moth, Bilobata subsecivella (Zeller) (Lepidoptera: Gelechiidae), thought to be an invasion from Indo-Asia (where it is known as Aproaerema modicella (Deventer); but hereafter referred to as B. subsecivella) has become a major pest of groundnut (Arachis hypogaea L.) and soya bean (Glycine maxi (L.) Merr.) in South Africa and Africa as a whole. Following the sudden outbreaks of B. subsecivella as a new pest of groundnut in a number of African countries, the continent has been confronted with the problem of having no information on the biology and ecology of the pest that can be used for its management/control. In this context, the main aim of the research for this thesis was to study the biology and ecology of B. subsecivella in South Africa with the main objective of obtaining information that will assist in its management as a novel pest of groundnut. To achieve this objective, several studies were carried out. First, a detection survey of B. subsecivella infestation was conducted on groundnut, soya bean and lucerne (Medicago sativa L.), the common host crops for B. subsecivella in India, at six widely separated sites in South Africa during the 2009/2010 growing season. The sites included the Agricultural Research Council research stations at Potchefstroom and Brits as well as the farms surrounding the Brits research farm in the North West province, Vaalharts Research Station in the Northern Cape province, the Department of Agriculture Lowveld Agricultural Research Station near Nelspruit in Mpumalanga province, and Bhekabantu and Manguzi in the northern part of the KwaZulu-Natal province. The study had three objectives. The first was to build a complete host crop/plant list and record damage symptoms caused by B. subsecivella in South Africa. The second was to identify the pest to species level. The third was to determine its inter- and intra-population genetic diversity by analysing in, both cases, the mitochondrial DNA (mtDNA) COI gene of specimens collected from these sites. Sixty specimens comprising 24 larvae, 24 pupae and 12 moths were collected from the six survey sites, and their mtDNA COI were sequenced and compared with those from the Barcode of Life Data System (BOLD) gene bank. Infestation by B. subsecivella was observed on groundnut and soya bean, but not on lucerne. The mtDNA COI from all specimens of the pest, irrespective of whether they were from groundnut or soya bean, matched 100% with the sequences in BOLD belonging to a B. subsecivella population occurring in Australia (referred to as Aproaerema simplexella (Walker)) and known as the soya bean moth in that country). There was very little genetic diversity between and within the populations from the six sites, which suggested that the populations were maternally of the same origin. Further molecular and phylogenetic studies were also completed to determine the evolutionary relationships between B. subsecivella populations collected from Australia, Africa and India. These studies involved sequencing and analysing five gene regions of mitochondrial and nuclear DNA, including COI, cytochrome oxidase II (COII), cytochrome b (cytb), 28 ribosomal DNA (28S rDNA), and intergenic spacer elongation factor-1 alpha (EF-1 ALPHA). The mtDNA COI analysis also included B. subsecivella (but called A. simplexella) sequences downloaded from the National Center for Biotechnology Information (NCBI) GeneBank collected from different areas in Australia. In four phylogenetic trees (COI, COII, cytb and EF-1 ALPHA), sequences of B. subsecivella personally sampled from Australia were grouped separately from the others, whereas sequences of B. subsecivella from South Africa, India and Mozambique were clustered in one group in most cases. Furthermore, in the mtDNA COI phylogenetic tree, one Australian sequence of B. subsecivella that was downloaded from the NCBI GeneBank was grouped with other sequences from South Africa, India and Mozambique. Moreover, one sequence of B. subsecivella personally sampled from Australia was grouped with the other two sequences of B. subsecivella from Australia that were downloaded from the NCBI GeneBank. Based on these results, it could be hypothesized that there is genetic diversity within B. subsecivella populations in Australia. The mtDNA COI gene analysis in the current study revealed that there are B. subsecivella populations in Australia that are similar to the B. subsecivella populations in South Africa, Mozambique and India. Phylogenetic analysis of the 28S gene region revealed a lack of genetic diversity between sequences of B. subsecivella from India, South Africa, Mozambique and Australia. Genetic pairwise distances between the experimental sequences ranged from 0.97 to 3.60% (COI), 0.19% to 2.32% (COII), 0.25 to 9.77% (cytb) and 0.48 to 6.99% (EF-1 ALPHA). Field experiments were then conducted at Vaalharts, Brits, Nelspruit, Manguzi and Bhekabantu during the 2010/2011 and 2011/2012 growing seasons. These experiments pursued three objectives. The first one was to determine B. subsecivella infestation levels on groundnut, soya bean, lucerne, pigeon pea (Cajanus cajan L.) and lablab bean (Lablab purpureus L.) under field conditions. The second was to develop a host plant list for B. subsecivella and the third was to determine the effect of cypermethrin application on damage by B. subsecivella to groundnut and soya bean plants. In the 2010/2011 season, larval infestation was monitored on groundnut crops planted in November 2010 and January 2011. In the 2011/2012 season, larval infestation was monitored on groundnut, soya bean, lucerne, pigeon pea and lablab bean planted in November 2011 and January 2012. Wild host plants were inspected for damage symptoms and the presence of larvae. An experiment which examined the effect of cypermethrin application on B. subsecivella damage to groundnut and soya bean plants was completed in the 2011/2012 season at Vaalharts and Nelspruit. A survey for wild plant hosts of B. subsecivella was conducted in the proximity of the field experiments during the 2011/2012 growing season, as well as in winter. Amongst the host crops tested, soya bean was highly infested by B. subsecivella followed by groundnut, at all sites. The pest was also observed on pigeon pea at all sites, but the infestation was very low, while lucerne had very low larval infestation. No infestation was observed on lablab bean across these sites. Groundnut and soya bean crops planted in January were severely infested by B. subsecivella, compared to the crops planted in November; however, B. subsecivella infestation on crops was observed 5-6 weeks after crop emergence. Sprays of cypermethrin on groundnut and soya bean reduced larval infestation in both crops to very low levels. Wild plant hosts identified were from five families which included three species in the Leguminosae, two species in the Convolvulaceae, two species in the Malvaceae and one species each in the Lamiaceae and Asteraceae. Seasonal monitoring of the flight activity of B. subsecivella moths was completed at Manguzi, Bhekabantu, Nelspruit, Brits and Vaalharts over a two-year period (from November 2010 to December 2012). The objective of this study was to monitor the flight activity of B. subsecivella in order to understand its dispersal and off-season survival tactics and to predict its initial occurrence. Pheromone traps were used to monitor the moths’ flight activity. Information collected included climatic data (rainfall, temperature and humidity) that were obtained from ARC weather stations placed at four planting sites. Pearson’s test for correlation was performed to assess the relationship between B. subsecivella moth catches and environmental factors (rainfall, temperature and humidity). Results from this study showed variation in B. subsecivella populations throughout the monitoring period. The highest peak in B. subsecivella catches was between January and April/May for both seasons. Though low in numbers, B. subsecivella moths were caught in winter at Manguzi, Nelspruit, Vaalharts and Bhekabantu. No B. subsecivella moths were trapped during the winter months at Brits. Pearson’s test for correlation indicated that there was a significant negative association between temperature and B. subsecivella catches in pheromone traps at Nelspruit, whereas at Vaalharts there was a significant positive association between humidity and B. subsecivella catches. There was no correlation between environmental factors and B. subsecivella catches at Manguzi and Brits. Furthermore, it was found that B. subsecivella in Australia (moths collected for DNA analysis in the current study) responded to the species-specific lure that was developed from the sex pheromone of B. subsecivella, referred to as A. modicella in India. Overall, the study revealed important ecological and genetic information on B. subsecivella populations occurring in southern Africa. More importantly, this study established the genetic connection between B. subsecivella populations from Australia, India and Africa. Hence, the species conforming to these populations were tentatively synonymized as B. subsecivella in this thesis.
Molecular diagnostics and phylogenetics of white grubs in sugarcane.
(2008) Dittrich-Schröder, Gundrun.; Conlong, Desmond Edward.; Mitchell, Andrew.
Scarabaeid pests in South Africa and especially KwaZulu-Natal are characterised by a very long larval life cycle and short pupal and adult periods. However, it has nearly always been the adults of the species that have been identified, with very little attention paid to the larval identification of the species. This is unfortunate as it is nearly always the larval stage that is found to be associated with crop damage. Accurate identification of the species of these larvae is important for the management of scarabaeid pest species, as it unlocks the necessary information on the biology and ecology of many species, which allows the adaptation of control methods for different species. Inadequate keys for the taxonomy of larvae of these groups, as well as the lack of morphological taxonomists working on these groups have been identified as constraints. When a species is difficult to identify using traditional taxonomic methods, DNA diagnostic tools can be useful. Chapter 2 investigated the feasibility of identifying scarabaeid larvae using mitochondrial DNA data. Variation in the base pair sequence of the mitochondrial cytochrome c oxidase sub unit I (cox 1) gene was used. DNA sequences of cox 1 from scarabaeid larvae collected from sugarcane fields were compared with sequences from scarabaeid adults of known species in order to identify the species attacking sugarcane. Neighbour-joining and maximum parsimony analyses of 658 bp cox 1 sequences identified groups of larvae that linked to adult specimens. The major groupings delimited specimens belonging to the subfamilies Dynastinae, Melolonthinae and Rutelinae. Within-group sequence divergence ranged from 0 - 3.4 % and divergence between sister groups ranged from 2.6 - 25.1 %. The recorded divergence range within and between tribes was 0 - 21.3 % and 17.3 - 28.5% respectively. Similarly, the divergence range observed within and between genera was 0 - 19.2 % and 17.1 - 25.4% respectively. The maximum sequence divergence observed within subfamilies was 23.7 % and divergence between subfamilies ranged from 16.8 - 26.7 %. Examination of pairwise sequence divergence levels as well as node support allowed 68% of the unidentified larval specimens to be associated with identified adult specimens. Phylogenetic analysis matched identified adult mtDNA with unidentified larval mtDNA. This allowed the identification of those larvae through morphological characteristics unique to certain species. To create a field key to the subfamilies of Dynastinae, Melolonthinae and Rutelinae the most useful character distinguishing larvae of different species was the raster but additional morphological characteristics were included. These relationships between larval and adult scarabaeid specimens from sugarcane were examined using various phylogenetic tools. The data set included a total of 19 morphological characters as well as 166 partial cox 1 gene sequences. Maximum parsimony analyses were performed on morphological, molecular and combined data. The same morphological and molecular data sets were run both separately and as a combined analysis with MrBayes. In both types of analyses the morphological data performed poorly and crude groupings resulted, dividing taxa to tribe level only. Molecular data showed greater resolution than the morphological data and taxa were separated into groups equivalent to species and morphospecies designated in Chapter 2. A partition homogeneity test indicated that both data types could be combined. It is recommended that both morphological and molecular data be utilised in identification of scarabaeid sugarcane pests and that a character-based approach be implemented. Further molecular data from other genes should be included to test the accuracy of these results. The keys produced during this study will allow workers to focus on a single species biology, and subsequently allow an analysis of between species interactions, and within species control. These advances are a start to the improvement of knowledge of the species composition of scarabaeid larvae in sugarcane fields, thus making management and biological control of these pests a greater possibility. Further recommendations for future work are discussed in Chapter 5.
Sugarcane stem borers in Ethiopia : ecology and phylogeography.
(2006) Assefa, Yoseph.; Conlong, Desmond Edward.; Mitchell, Andrew.
Eldana saccharina Walker (Lepidoptera: Pyralidae) is an indigenous insect widely distributed throughout Sub-Saharan Africa that is a major pest of sugarcane in southern Africa. Studies have shown that populations from West Africa have distinct behavioural differences compared to populations from eastern and southern Africa. In addition, the parasitoid guilds attacking populations in these regions are markedly different. This marked behavioural and parasitoid guild variation evoked a hypothesis of genetic diversification. To evaluate this hypothesis a project on the phylogeography of E. saccharina was initiated. The project was planned to include sampling of as many regions as possible in its known range in Africa, to obtain specimens of E. saccharina for genetic analysis. When these surveys were initiated in Ethiopia, it was found that there was no published literature available on the occurrence of stem borers in Ethiopian sugarcane. It was thus clear that no stalk borer/parasitoid surveys had been completed in either sugarcane or any large grass and sedge indigenous hosts in Ethiopia. The study was thus expanded beyond the investigation of only the genetic diversity of E. saccharina, to include area-wide surveys to determine ecological aspects of the borer complex in suspected host plants, including sugarcane, in Ethiopia. In this way the host plant range and distribution of E. saccharina and other sugarcane borers in Ethiopia in particular could be determined, samples for a larger phylogeography project could be collected, and the insect's impact on sugarcane could be assessed. Quantified area-wide surveys of the sugarcane estates and small-scale farmer fields of Ethiopia were conducted between December 2003 and February 2004. The surveys verified the presence of four lepidopteran stem borer species on Ethiopian sugarcane. These were Chilo partellus Swinhoe (Lepidoptera: Crambidae), Sesamia calamistis Hampson (Lepidoptera: Noctuidae), Busseola fusca Fuller (Lepidoptera: Noctuidae) and Busseola phaia Bowden (Lepidoptera: Noctuidae). The surveys indicated that Busseola species are the major and most widely distributed sugarcane stem borers in sugarcane farms of Ethiopia. Over 70% of the peasant sugarcane fields visited were infested by these borers, with the highest levels of infestation (35% and 50%) being in the northern and western part of the country, respectively. Busseola was also the predominant stem borer of sugarcane in two of the three estates (Wonji and Finchawa). Chilo partellus and S.calamistis were recovered in very low numbers at all the commercial estates and from peasant farms in the western part of Ethiopia. However, C. partellus was the predominant sugarcane stem borer in lowland areas of northern, southern and eastern parts of the country. Eldana saccharina was recovered from large sedges in waterways of Metehara and Wonji sugar estates in the central part of the country, and sedges growing around lakes in northern and southern Ethiopia, but not from sugarcane anywhere in Ethiopia. The phylogeographic study conducted on E. saccharina populations from eleven countries of Africa clearly showed the population structure of the insect within the continent. Five hundred and two base pairs of the mitochondrial DNA (mtDNA), corresponding to the Cytochrome Oxidase subunit I (COl) region, were sequenced to clarify phylogenetic relationships between geographically distant populations from eastern, northern, southern and western Africa. Results revealed that E. saccharina is separated into four major populations corresponding to their geographical location, i.e. West African, Rift Valley and two southern African populations. Sequence divergence between the four populations ranged from 1% to 4.98%. The molecular data are congruent with an isolation by distance pattern except for some of the specimens from eastern and southern Africa where geographically close populations are genetically distant from each other. Geographical features such as the Rift Valley and large water bodies in the continent seem to have a considerable impact on the genetic diversity in E. saccharina. Identification of field-collected stem borer specimens was done using classical taxonomic techniques, except for Busseola spp. where DNA barcoding was used. As field-collected larval material of Busseola died before reaching the adult stage, identification of species using adult morphology was not possible. 'Sequence divergence in the COl gene was used as a tool to identify the species of Busseola attacking Ethiopian sugarcane. Partial COl sequences from Ethiopian specimens were compared with sequences of already identified noctuid species from the East African region. Results of the sequence analysis indicated that the Busseola species complex in Ethiopian sugarcane comprised B. fusca and B. phaia. Sequence divergences between Ethiopian Busseola species was as high as 5.0 %, whereas divergences within species were less than 1% in both species identified. Several larval parasitoids, bacterial and fungal diseases of stem boring caterpillars were also recorded in Ethiopian sugarcane. Amongst these was Cotesia flavipes Cameron (Hymenoptera: Braconidae). This exotic parasitoid has been introduced into several African countries for the control of C. partellus in maize and sorghum, but had never been released in Ethiopia. To investigate the origin of C. flavipes in Ethiopian sugarcane, molecular analyses were conducted on Ethiopian specimens from sugarcane and specimens of C. flavipes from different countries of Africa released from the Kenyan laboratory colony, again using COl sequences. Results of the analysis revealed that the C. flavipes population that had established in sugarcane fields of Ethiopia was similar to the south east Asian populations released against C. partellus in maize in other parts of Africa, and different from other populations of this species, providing evidence that the Ethiopian C. flavipes is likely to be a descendant of the original Pakistani population that was released in different parts of Africa. The study reveals the importance of lepidopteran stem borers in sugarcane production in Ethiopia and highlights the role of molecular methods in species identification and determining phylogenetic relationships. More importantly, this study establishes the continental phylogeographical pattern of the indigenous moth, E. saccharina. The impact of geological events, geographic barriers and cropping systems on the evolution, distribution and abundance of stem borers are discussed. Future areas of research for understanding more about the phylogeographic relationships of E. saccharina and management of stem borers are discussed.

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