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.

Espécies muito próximas da família Calliphoridae apresentam hábitos alimentares muito distintos, como alimentação em tecido de um hospedeiro vivo (parasitismo obrigatório) e alimentação em matéria orgânica em decomposição (necro-saprofagia). As origens evolu-tivas do hábito de parasitismo nesta família ainda são desconhecidas. No entanto, o que a torna ideal para o estudo da evolução do hábito alimentar é o aparecimento do parasitis-mo obrigatório em pelo menos três ocasiões independentes em sua história. Neste traba-lho, foram utilizados métodos para entender a evolução do parasitismo obrigatório assim como os genes envolvidos em três diferentes hábitos alimentares. O primeiro passo foi a inferência do hábito ancestral de Calliphoridae. Com o mapeamento de caracteres na filo-genia da família, o hábito ancestral mais provável seria a necro-saprofagia e o parasitismo obrigatório teria surgido posteriormente (independentemente do parasitismo facultati-vo). Ensaios de escolha com fêmeas permitiram a classificação precisa das espécies quan-to ao hábito alimentar. Ensaios com as larvas mostraram que tanto espécies necro-saprófagas como parasitas facultativas se alimentam tanto de carne em decomposição como carne fresca. Por outro lado, a espécie parasita obrigatória Co. hominivorax, apresentou um comportamento aversivo pela carne em decomposição e se desenvolveu somente na carne fresca. Esses resultados permitiram a formulação da hipótese de que o parasitismo tenha surgido a partir de uma mudança da atração das fêmeas pelos substra-tos para oviposição, seguida da especialização da larva parasita. A busca dos genes envol-vidos nos diferentes hábitos foi realizada por meio da análise de expressão gênica dife-rencial em dados de RNA-seq gerados para seis califorídeos. No total, foram encontrados 230 potenciais genes candidatos para investigação futura. Além disso, o padrão geral observado indicou que variações tanto nas regiões regulatórias como codificadoras, sofrem a ação predominante de seleção purificadora / Closely related species of the Calliphoridae family have contrasting feeding habits, such as feeding on living tissues of a host (obligate parasitism) and feeding on decaying organ-ic matter (necro-saprophagy). The evolutionary origins of parasitism in Calliphoridae are still unknown. However, what makes this family ideal for the study of the evolution of feeding habits is the appearance of obligate parasitism in at least three independent oc-casions. In this study, we used methods to understand the evolution of parasitism, as well as the genes involved in in three different feeding habits. First, we inferred the ancestral habit of Calliphoridae. By using stochastic character mapping along the phylogeny of the family, the most likely ancestral habit was revealed as necro-saprophagy. Obligate para-sitism could have evolved later (with an independent evolution of the facultative parasit-ism). Two-choice essays with females allowed the precise classification of the species regarding their feeding habits. Essays with larvae showed that both necro-saprophagous and facultative parasites feed on decaying flesh and fresh meat. On the other hand, the obligate parasite, Co. hominivorax, showed an aversive behavior to decaying meat and able to develop only in fresh meat. These results led to the formulation of the hy-pothesis that parasitism arose from a shift in the attraction of the female attraction to new oviposition sites, followed by the specialization of the parasitic larvae. The search for the genes involved in the different feeding habits was performed through an analysis of differential gene expression using RNA-seq data generated for six califorids. Within a da-taset containing more than 1000 candidate genes, 230 genes potential candidate genes were found for future research. In addition, the general pattern observed indicated that both regulatory and coding regions have predominantly undergone the action of purifying selection

Abstract not available

<p>This thesis attempts to improve our understanding of the role of population size for the process of speciation. First, the effect of population size on speciation is studied using several meta-analyses of published laboratory experiments. Second, the effect of population size on behaviour is studied using a laboratory population of the blowfly <i>Lucilia sericata</i>. Third, the effect of population size on morphological and genetic variation is studied using wings and microsatellites from wild populations of <i>L. illustris</i> as well as experimentally bottlenecked populations of <i>L. sericata</i>. The meta-analyses showed that the result of many previous laboratory experiments on sympatric and parapatric speciation may have been biased by too small population sizes. Reduced interbreeding was less likely to develop in small populations where the selection against hybridisation often seemed to have been opposed by inbreeding depression or loss of genetic variation. In allopatric speciation experiments, no general consistent effect of population size was observed. There was no support for speciation through founder events. In fact, significant assortative mating was only found in vicariance experiments where derived populations was tested against each other. Population size influenced reproductive behaviour in <i>L. sericata</i>. There was a positive effect of increasing number of males on egg-laying but only as long as the female was in the company of at least one other female. Female mate choice and a positive effect of number of eggs on larval survival are suggested to be the underlying factors. No historic bottlenecks could be detected in the fly populations, but strong genetic indications suggest a fine grained genetic population structure of wild <i>Lucilia</i> flies. Bottlenecks had unpredictable effects on wing morphology as well as on genetic variation and fitness in a laboratory stock of <i>L. sericata</i>. Thus a bottlenecked population will not necessarily have a higher chance of evolving morphological novelties than one which has not undergone a bottleneck. However, among many bottlenecked populations there is a good chance that in at least one of them the conditions will be conducive to morphological change and evolution. In this statistical sense, thus, strong population fluctuations may enhance the probability of speciation events.</p>

This thesis attempts to improve our understanding of the role of population size for the process of speciation. First, the effect of population size on speciation is studied using several meta-analyses of published laboratory experiments. Second, the effect of population size on behaviour is studied using a laboratory population of the blowfly Lucilia sericata. Third, the effect of population size on morphological and genetic variation is studied using wings and microsatellites from wild populations of L. illustris as well as experimentally bottlenecked populations of L. sericata. The meta-analyses showed that the result of many previous laboratory experiments on sympatric and parapatric speciation may have been biased by too small population sizes. Reduced interbreeding was less likely to develop in small populations where the selection against hybridisation often seemed to have been opposed by inbreeding depression or loss of genetic variation. In allopatric speciation experiments, no general consistent effect of population size was observed. There was no support for speciation through founder events. In fact, significant assortative mating was only found in vicariance experiments where derived populations was tested against each other. Population size influenced reproductive behaviour in L. sericata. There was a positive effect of increasing number of males on egg-laying but only as long as the female was in the company of at least one other female. Female mate choice and a positive effect of number of eggs on larval survival are suggested to be the underlying factors. No historic bottlenecks could be detected in the fly populations, but strong genetic indications suggest a fine grained genetic population structure of wild Lucilia flies. Bottlenecks had unpredictable effects on wing morphology as well as on genetic variation and fitness in a laboratory stock of L. sericata. Thus a bottlenecked population will not necessarily have a higher chance of evolving morphological novelties than one which has not undergone a bottleneck. However, among many bottlenecked populations there is a good chance that in at least one of them the conditions will be conducive to morphological change and evolution. In this statistical sense, thus, strong population fluctuations may enhance the probability of speciation events.

Thesis (M.S.)--Oregon State University, 2006. / Printout. Includes bibliographical references. Also available via the World Wide Web.

Blowflies (Diptera: Calliphoridae) are main colonizers of decomposing remains and any information on the influences of their lifecycle of growth and development are important to forensic entomologists when estimating postmortem interval during a medicolegal death investigation. Pupal survival of blowfly species, Lucilia sericata were studied at different developmental stages following pupation, white (0-12 hrs), young (24 hrs), medium (5 days), old (9 days) were submerged at various immersion intervals ranging from 1 hr to 120 hr (5 days) in different water types (fresh, salt, polluted, and drinking water). Control group (N= 100) were white pupae that were not submerged were also observed in order to compare the survival and eclosion rate. Two trials of the submergence process were conducted to observe the pupae survival. Data were analyzed using multiple logistic regressions in a 3-way interaction to determine if significant differences were seen between the water, time in water, and pupal stage. Dissolved inorganic nitrogen (DIN) of NH4+, NO2-, NO3-, and oP or DIP were analyzed to examine to determine if a correlation between the water types could be seen. DIN analyses exhibited similar NO2- + NO3- and NO3- in polluted and fresh water. The results show that as the pupae developmental age progressed, the pupae generally survived longer immersion intervals. The survival of white pupae decreased with the increase of submergence for all four water types, exhibiting a 100%mortality rate with pupae submerged longer than 24-36 hours. While pupae immersed in polluted water had similar survival rates in the white stage, the survival rate declined as the developmental age increased for pupae immersed in polluted water. Pupae immersed in salt and polluted water had earlier eclosion times when compared to other water groups, emerging at 36 hours from when the control group first emerged at day 12. Understanding the survival rate of submerged Lucilia sericata (Diperta: Calliphoridae) pupae will be potentially useful for criminal investigations by providing a better understanding of the survival of pupae submerged in different aquatic environments.

Made available in DSpace on 2014-08-20T14:31:32Z (GMT). No. of bitstreams: 1 dissertacao_sabrina_pires_capa.pdf: 403567 bytes, checksum: 5752dd89517a54f40cd0632e1db1c1c6 (MD5) Previous issue date: 2007-03-30 / The blowflies are the first insects to compose the faunistic succession of human cadavers, thus helping to determine the postmortem interval (PMI). However, failing to consider larval post-feeding dispersal can compromise medical-criminal inquiries. In order to investigate the relationship between post- feeding Lucilia sericata larvae and their weight, radial dispersion and depth, an artificial arena was built simulating the natural environment. We observed a difference in mean pupal weight between males (30.28mg) and females (32.35mg). We also observed that the majority of pupae were recovered a distance of 33 to 45cm from the center of the arena, and at a depth of 6 to 8cm from the surface. However, we observed no differences in radial dispersion and depth of pupation between males, females or unviable individuals. Correlation analysis between weight and distance (r = 0.246) and between weight and depth (r = 0.321) was directly proportional, suggesting that heavier pupae dispersed farther and deeper than lighter pupae. The experiment also allowed us to conclude that a circular arena permitted larval dispersion all directions. / Os dípteros califorídeos são os primeiros indivíduos a compor a sucessão faunística dos cadáveres humanos auxiliando assim na estimativa do intervalo pós- morte. Porém, a não consideração da dispersão pós-alimentar de larvas pode comprometer as investigações médico-criminais. Diante disto uma arena circular, simulando o ambiente natural, foi montada em laboratório a fim de verificar-se a dispersão larval radial pós-alimentar de Lucilia sericata, tentando assim estabelecer relações entre as variáveis peso, distância e profundidade de enterramento na arena. Os resultados demonstraram que a maioria das pupas foram recuperadas a uma profundidade compreendida entre 6cm e 8cm e a uma distância entre 33cm e 45cm do centro da arena. Não houve diferenças significativas quanto a propagação e profundidade de enterramento de machos, fêmeas e indivíduos inviáveis. Com relação ao peso, verificou-se que a média das fêmeas (x = 32,35mg) foi superior a dos machos (x = 30,28mg). A análise de correlação entre peso e distância percorrida (r = 0,246) e entre peso e profundidade (r = 0,321) foi diretamente proporcional, ou seja pupas mais pesadas propagaram e se aprofundaram mais. O experimento permitiu ainda concluir que uma arena circular possibilita o deslocamento das larvas em todas as direções.

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.