Orientadores: Ana Maria Lima de Azeredo Espin, Ana Carolina Martins Junqueira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-25T08:05:59Z (GMT). No. of bitstreams: 1 Paulo_DanielFernando_M.pdf: 4089154 bytes, checksum: f604296d2e31ee6a5dd7e3aa8f517f69 (MD5) Previous issue date: 2014 / Resumo: MicroRNAs (miRNAs) são pequenos RNAs não codantes que agem como moduladores pós-transcricionais da expressão gênica em todos os eucariotos investigados até o momento. Em animais, a complementariedade imperfeita de bases entre o miRNA e o sítio alvo do RNA mensageiro (mRNA) inibi sua tradução, tornando-os genes chaves no controle da expressão gênica. A identificação de miRNAs pode fornecer uma melhor compreensão de diversos processos biológicos e evolutivos das diferentes espécies. A família Calliphoridae é um grupo que compreende dípteros causadores de miíases, incluindo as espécies Cochliomyia hominivorax (mosca da bicheira) e Cochliomyia macellaria (mosca varejeira). A mosca da bicheira é uma das principais pragas na região Neotropical. Na fase larval, esta espécie causa infestações e alimenta-se de tecidos vivos de vertebrados de sangue quente, acarretando severas perdas na indústria pecuária. Diferentemente, a mosca varejeira, apresenta um hábito saprófago, se alimentando e reproduzindo em carcaças e tecidos em decomposição, ressaltando sua importância para a entomologia forense e para a saúde pública. Por serem filogeneticamente próximas e possuírem diferentes hábitos alimentares e reprodutivos, estas espécies representam modelos para estudos sobre as bases moleculares do parasitismo em Calliphoridae. Para identificar e caracterizar os miRNAs destas duas espécies, o transcriptoma de pequenos RNAs de adultos (macho e fêmea) e larva (terceiro instar) foram sequenciados em plataforma de nova geração MiSeq-Illumina. Os 6.2 milhões de reads gerados foram mapeados contra o genoma de Drosophila melanogaster e o banco de dados miRBase. Foram identificado 84 miRNAs evolutivamente conservados, dos quais 80 foram encontrados em C. hominivorax e 78 em C. macellaria. Também foi investigada a presença dos precursores em forma de grampo (pre-miRNAs) nos dados genômicos e transcriptômicos disponíveis para estas espécies. Foram preditos 10 pre-miRNAs conservados e outros 5 que não apresentaram similaridade com nenhum miRNA já descrito para outras espécies de animais. A caracterização evolutiva dos miRNAs identificados mostrou que essas sequências são altamente conservadas desde Nephrozoa (641 MA), na base de bilatéria, até Brachycera (195 MA). Substituições nucleotídicas observadas foram enviesadas na região 3¿-final com raras mutações na região seed. Análises preliminares de expressão revelaram 79 miRNAs diferentemente expressos entre as espécies e os estágios de desenvolvimento investigados. Os resultados deste trabalho irão contribuir para uma melhor compreensão sobre os hábitos de parasitismo nas espécies C. hominivorax e C. macellaria, com perspectivas para estudos evolutivos e funcionais na família e no controle de insetos-praga / Abstract: MicroRNAs (miRNAs) are small non-coding RNAs that act as post-transcriptional modulators of gene expression in all eukaryotes investigated so far. The imperfect complementarity between miRNA and the target site of messenger RNA (mRNA) inhibits their translation in animals, being key genes for the control of expression in cells. The identification of miRNAs can provide a better understanding of biological processes and evolution of traits in different species. The family Calliphoridae is a group of myiasis-causing flies with different feeding habits, which includes the species Cochliomyia hominivorax (screwworm fly) and Cochliomyia macellaria (secondary screwworm). The screwworm fly is one of the major pests in the Neotropical region. Their larvae infest and feed on live tissues of warm-blooded vertebrates, resulting in severe losses for livestock industry. Differently, the close-related secondary screwworm shows a saprophagous habit, feeding and breeding on carcasses and dead tissues, being crucial for forensic entomology and public health. Because of their close evolutionary relationship and contrasting feeding habits, they represent worthy models to study the molecular basis of parasitism and feeding specialization in the family Calliphoridae. To characterize the miRNAs from both species, the small-RNA transcriptomes of adults (male and female) and larvae (third instar) were sequenced using Illumina-MiSeq next generation sequencing platform. The 6.2 million reads generated were mapped against the Drosophila melanogaster genome and screened in miRBase. We identified 84 evolutionary conserved miRNAs which 80 was founded in C. hominivorax and 78 in C. macellaria. We also investigated the presence of hairpin precursors (pre-miRNAs) in the available genomic and transcriptomic data of these species, and predicted 10 conserved pre-miRNAs and others 5 that show no similarity with previously described animal miRNAs. The evolutionary characterization of identified miRNAs showed that their sequences were highly conserved since the Nephrozoa ancestor (641 MYA) in the basis of Bilaterian clade, until Brachycera ancestor (195 MYA), with nucleotide substitutions biased to 3¿-end portion of the miRNAs with rare substitutions in the seed region. The preliminary expression profile revealed 79 differentially expressed miRNAs between species, gender and life stages, given by hierarchical clustering and statistically significant change fold analysis. The results presented here will provide new information about the genetic background of parasitic habits in C. hominivorax and C. macellaria, with prospects to functional and evolutionary studies in Calliphoridae and pest control / Mestrado / Genetica Animal e Evolução / Mestre em Genética e Biologia Molecular

Cochliomyia macellaria (Fabricius) has great importance in forensics and is commonly utilized in estimating the period of insect activity (PIA) on a corpse due to its rapid colonization time of fresh remains, active oviposition during daylight, and its abundance throughout the southern United States. The purpose of this study is to determine what effects tissue and temperature will have on C. macellaria. The purpose of my secondary studies on attraction to larval resource is to gain insight into the behavior of adult blow flies in order to determine what factors drive colonization of remains. Forensic entomology can use development data of calliphorid flies to estimate their PIA on human remains. C. macellaria has major implications in medico-legal entomology as well as in veterinary entomology and are often found on human remains in Texas during the warm months of the year. Furthermore, C.macellaria is a secondary myiasis producer; this means that its larvae may be found infesting living animal or human muscle tissue and potentially could be involved in veterinary forensic cases. This study will help provide better insight on the behavior of C. macellaria in Texas as it relates to tissue type and temperature. In this experiment larvae were reared at three separate temperatures (21°C, 24°C, and 27°C) on either equine or porcine striated muscle tissues. Eggs were inoculated onto each tissue and monitored every hour for hatch. Once hatch occurred, observations were shifted to every twelve hours. Three larvae were sampled during each observation period. Sampled larvae were weighed and length was recorded. Additionally, stage of development was determined. Pupae were collected and time to adult emergence recorded. Furthermore, life-history traits, such as emergence pattern and adult longevity, were recorded. This study is the first in Texas and second in the United States to examine the development of C. macellaria and could provide significant information for cases of myiasis and neglect of both humans and animals. In contrast, rearing flies on beef liver or pork chops, which tends to be a standard procedure often employed may not be applicable to myiasis cases of equines. Data from this study could provide greater insight to developmental differences of forensically important blow flies on striated muscle from different vertebrate species. Since porcine tissue was used in the Florida study, it was also utilized in our study in order to allow a comparison. To date there is only one data set on the development of C. macellaria in the United States. Fly populations in different climates are suspected to have different development rates. This study will compare data from central Texas to data generated in Florida. If variation of development rates is demonstrated for different populations, then forensic entomologists should use data that are more conducive to their given geographic area. My secondary objective first looked at whether larval resource had an attraction effect on the subsequent adult C. macellaria flies. One cohort of larvae was reared on bovine testicles, while another was reared on bovine liver. The subsequent adults were then tested in a dual-choice olfactometer which possessed testicles at one end and liver at the other end. The second set of experiments also involved using a dual-choice olfactometer; however, all larvae were reared on bovine liver and fresh liver was placed at one end of the olfactometer, while fresh liver with conspecific larvae was placed at the end of the other. The purpose of this experiment was to assess whether adult C. macellaria flies were more attracted to the presence of conspecific larvae. These experiments will give us more insignt into adult blow fly behavior and help us to understand what factors drive colonization of remains.

Indiana University-Purdue University Indianapolis (IUPUI) / Development rate is a quantitative trait that displays significant variation within many species, including Cochliomyia macellaria Meigen (Diptera: Calliphoridae). Calliphorids are a family of dipterans known as blow flies and are commonly used in forensic entomology to estimate the minimum postmortem interval (PMIMIN), given some assumptions are made. In order to dissect the genetic underpinnings of development rate variation in this species, artificial selection for fast and slow development with population-based resequencing was used. The objective of this study is to isolate and characterize genomic regions that are correlated to development rate variation in blow flies. The first approach used known regulatory development genes from Drosophila melanogaster Meigen (Diptera: Drosophilidae) and isolated variants that were associated with development time changes in artificially selected fast and slow development C. macellaria strains. Three variants located in Ras and Eip74EF were associated with fast or slow development in selection strains with a significant change in allele frequency. The second approach involved the comparison of pooled artificially selected fast and slow C. macellaria genomes to investigate the genetic basis of development rate variation. When comparing the fast and slow genomes, 699 sequences were identified that contained 7290 variants with consistent changes in allele frequency. The variants indicated that the genomic regions that are associated with development rate were associated with developmental processes, including regulation of RNA polymerase II activity, and transporter activity, such as protein dimerization. Of the 699 sequences, 69 sequences were related to Achaete-scute complex and 14 were related to Cyp12A7. The identification of the genomic regions that associated with development rates from this study provides an important resource for future studies in identifying potential genetic markers to increase the effectiveness of PMIMIN estimates. By using significantly associated variants as a priori candidates for future studies, the data increases the understanding of natural development variation in blow flies.

This investigation explored the environmental and physiological factors affecting adult blow fly (Diptera: Calliphoridae) arrival and attendance at pig (Sus scrofa domesticus L.) carcasses in Brazos Co, TX in the summer and winter, and validated a new technique for estimating the pre-colonization interval. It also examined how the offspring of said blow flies compensate for adverse developmental conditions such as starvation or the presence of older competitors by determining the function of minimum viable weight, critical weight, and the terminal growth period in Cochliomyia macellaria (F.) (Diptera: Calliphoridae). Adult blow fly carcass attendance is poorly explained by temperature, wind speed, ambient light intensity, or body size for either winter or summer-active species. Time of day explained approximately 10% in carcass size variation for all four of the most common species. For summer flies, the degree of ovarian development changed significantly from 96%/98% fully developed on day 1 postmortem to 7%/2% fully developed on day 2 postmortem for C. macellaria and Chrysomya rufifacies (Macquart) iv respectively. Using the binomial distribution, the minimum postmortem interval was correctly estimated for 4/6 validation tests. Minimum viable weight for C. macellaria was found to be ~ 0.02 g, and was stable under conditions of starvation and simulated competition. Under starvation conditions, time to pupariation was not altered, whereas under simulated competition, growth rate was increased and terminal growth period shortened. Starved flies under simulated competition entered the pupal state ~12 h faster than starved flies without competition, but required ~12 longer to complete development. These effects should be considered when estimating post-colonization intervals.