The greenbottle blowflies, Lucilia sericata (Meigen, 1826) and Lucilia cuprina (Wiedemann, 1830) (Diptera: Calliphoridae) are very difficult to distinguish on the basis of their external morphology. The literature suggests that these two species may be interbreeding. Sequencing two nuclear (28S rRNA and Period) and one mitochondrial (COI) gene indicated that there has been an ancient hybridization event and that mtDNA of L. sericata has become fixed in a lineage of L. cuprina through mtDNA introgression, possibly involving Wolbachia infection. This has implications for identifications of these species based on mtDNA alone. No study has shown explicitly that hybrids of L. sericata and L. cuprina can be identified morphologically. Morphological characters used to identify L. sericata and L. cuprina were scored and tested using specimens of both species and known hybrids. Discriminant function analysis of the characters successfully separated the specimens into three unambiguous groups – L. sericata, L. cuprina and hybrids. This is the first evidence that hybrids of these two species can be identified from physical characteristics.Lucilia sericata and L. cuprina have medical, veterinary and forensic importance. Knowing their distribution in South Africa would allow more effective management and utilisation of these flies. Their predicted geographic distributions in South Africa were modelled using maximum entropy analysis of selected climatic variables. The most important environmental variables in modelling their distributions were magnitude of monthly rainfall and the magnitude of the monthly maximum temperature for L. sericata, and the seasonal variation in monthly mean humidity and magnitude of monthly rainfall for L. cuprina. Both species have a widespread distribution in South Africa and one therefore cannot identify specimens of these flies by locality of capture alone.Luciliinae is a diverse and geographically widespread subfamily containing four genera - Hemipyrellia, Lucilia, Dyscritomyia and Hypopygiopsis – that all contain parasitic species ranging from saprophages to obligate parasites. The phylogenetic relationships between these genera are unclear. The 28S rRNA, COI and Period genes of 14 species of Lucilia and Hemipyrellia were partially sequenced and analysed together with 11sequences from GenBank and the Barcode of Life Database (BOLD). Lucilia sericata and L. cuprina were shown to be sister-species. Three cases of paraphylly were identified within Lucilia that affects identification of these species using mtDNA alone. Hemipyrellia consistently caused Lucilia to be paraphyletic when it was included in analyses, so Hemipyrellia should be synonymized with Lucilia. The relationships of Dyscritomyia and Hypopygiopsis to Lucilia are unclear and further studies are required. No geographic pattern was found within the different forms of parasitism within this group, but the different degrees of parasitism were phylogenetically clustered.

Abstract not available

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

THESIS ABSTRACT Current control strategies for livestock ectoparasites are limited by problems associated with chemical resistance and residues. Fungal biopesticides could provide an alternative control without these problems. However, a strategic approach is needed to first evaluate the suitability of selected fungal isolates for fungal biopesticide development. Two ectoparasites of significance to cattle and sheep are the cattle tick Rhipicephalus (Boophilus) microplus (Canestrini) and the Australian sheep blowfly Lucilia cuprina (Wiedmann). The fungus Metarhizium anisopliae (Metsch.) Sorokin) was evaluated for its potential to control these livestock ectoparasites. The growth characteristics of 30 isolates of M. anisopliae were investigated. Radial growth measurements were used to identify vigorous isolates that grew well at 30C and were capable of growing at 35C. A qualitative assessment of sporulation capacity further refined the candidate isolate group. A possible nutritive role of oil in the formulation was also investigated. However, there was no clear support for the theory that oil as a formulation additive could boost the germination and growth of the fungal conidia in vitro. Quantal response bioassays were conducted with cattle ticks and sheep blowflies using a range of conidial doses of three different isolates of M. anisopliae and different methods of inoculation. Ticks were either dosed with 2 µl or immersed in the conidial doses. Blowflies were either dosed with 2 µl of the conidial doses or fed conidia mixed with sugar. Probit analyses were carried out on the mortality data to compare the virulence of these isolates to ticks and blowflies and look for indications of different virulence mechanisms employed by M. anisopliae isolates when invading these hosts. One isolate (ARIM16) showed high virulence to both hosts killing 95 % of ticks after two days and 88 (±2) % of blowflies after four days. Strikingly different mortality patterns indicated quite different virulence mechanisms operating when M. anisopliae invades ticks or blowflies. The mortality pattern seen with ticks suggested that the number of conidia adhering per unit area of the cuticle was more important for rapid tick death than the total number of conidia contacting the entire tick surface. Blowflies fed conidia mixed with food died rapidly after an initial lag phase regardless of dose. Microscopic investigations were carried out to resolve the basis of the virulence patterns observed. The spatial and temporal aspects of the invasion of ticks and blowflies by M. anisopliae isolate ARIM16 were investigated with different types of microscopy. The scanning electron microscope and stereo light microscope were used to record surface changes and events and the compound light microscope revealed internal changes. Two distinctly different patterns of invasion were found in ticks and blowflies. Fungal conidia germinated on the surface of ticks then hyphae simultaneously penetrated into the tick body and grew across the tick surface. There was extensive fungal degradation of the tick cuticle with a preference for the outer endocuticle. While large numbers of conidia adhered to the surface of blowflies, no conidia were recorded germinating on external surfaces. One germinating conidium was seen in the entrance to the buccal cavity. Investigations of the fly interior revealed a higher density of hyphal bodies in the haemolymph surrounding the buccal cavity than in haemolymph from regions of the upper thorax. This pattern suggested that fungal invasion of the blowfly is through the buccal cavity. Plentiful extracellular mucilage was seen around the hyphae on ticks, and crystals of calcium oxalate were seen amongst the hyphae on the surface of ticks and in the haemolymph of blowflies killed by M. anisopliae isolate ARIM16. It was considered that cattle ticks are more suited for control with fungal biopesticides than adult blowflies. Three field trials were conducted over twelve months to assess the pathogenicity of M. anisopliae to parasitic stages of R. microplus on dairy heifers under different environmental conditions. Two isolates were selected based on their high optimal growth temperature (30oC), good conidial production characteristics and ability to kill adult engorged ticks in the laboratory in minimum time. Conidia were formulated in an oil emulsion and applied using a motor driven spray unit. Surface temperatures of selected animals were monitored, as were the ambient temperature and relative humidity. Unengorged ticks sampled from each animal immediately after treatment were incubated under laboratory conditions to assess the efficacy of the formulation and application. Egg production by engorged ticks collected in the first 3 days after treatment was monitored. Side counts of standard adult female ticks were conducted daily, before and after treatment to assess the performance of the fungus against all tick stages on the animals. At each trial the formulation caused 100% mortality in unengorged ticks that were removed from cattle and cultured under laboratory conditions. A significant reduction in egg production was recorded for engorged ticks collected in the three days post treatment. In the field, the fungal formulation had an inconsistent effect on ticks, which might be due to the influence of environmental temperature and humidity.

Zugl.: Hohenheim, Univ., Diss., 2008. / Enthält u.a. vier Abstracts.

<div>Forensic entomology is a subdiscipline of entomology that involves the use of insect behavior and developmental data to aid in criminal investigations. Genetic data has become increasingly important to the field as there has been a push for DNA-based species identification methods of forensically relevant insects. Genetic data can also elucidate population structure and relatedness of these insects, and such knowledge can contribute to the development of more specific datasets for insects in different regions. The first study presented here investigated the phylogenetics of sister species <i>Lucilia cuprina</i> and <i>Lucilia sericata</i> to identify possible subspecies divisions and issues with DNA-based identifications in the United States. The initial aim of this study was to identify genetic differences between specimens of <i>L. cuprina</i> that preferred live versus carrion flesh. Flies collected from Indiana, USA and South Africa were sequenced and analyzed. Upon sequencing of the genes <i>COI, Period,</i> and <i>28s,</i> our results indicated that <i>L. cuprina</i> from Indiana possess a unique combination of nuclear and mitochondrial haplotypes that suggest a unique lineage, possibly indicating modern hybridization with <i>L. sericata. </i>The inability of both nuclear and mitochondrial genes to distinguish between <i>L. cuprina</i> and <i>L. sericata</i> raises questions about the capabilities of DNA-based species identifications within this genus. Additionally, the inability of these genes to distinguish between specimens that preferred live versus carrion flesh highlights a need for continued research of these behavioral differences. The second study presented here investigated the population structure and relatedness of house flies in the American southwest in relation to a civil lawsuit where neighbors of a poultry farm alleged that flies were emanating from the farm to their homes. <i>Musca domestica</i> (house fly) specimens were collected from the chicken farm and from locations in varying directions and distances from the farm. Amplified fragment length polymorphism (AFLP) analysis was performed and the data were used in a number of analyses. Population reallocation simulations generally indicated that samples from different locations were not genetically different enough from other locations to allocate to their true origin population over others. Kinship analysis showed differences in samples collected in a later season that indicate a genetic bottleneck over time. Population structure analysis indicated the presence of two intermixing genetic populations in the dataset. AMOVA revealed that the majority of genetic variation laid within, rather than among, populations. A Mantel test revealed no significant correlation between genetic and geographic distances. These results indicate that the <i>M. domestica</i> population in this region of southwestern America is large and intermixing, with no clear genetic distinctions between specimens collected at the poultry farm versus the surrounding locations. In regard to the civil lawsuit, it was not possible to conclude that the flies did not emanate from the poultry farm. In a broader perspective, these data can be utilized to develop pest management strategies in this region. Overall, the data from both studies presented here will be useful to forensic investigations, development of more specific and detailed data and identification techniques, and pest control measures.</div>