Phylogeographic analysis of the New World Screwworm fly, Cochliomyia hominivorax

Orientador: Ana Maria Lima de Azeredo-EspinTese (doutorado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: A mosca-da-bicheira, Cochliomyia hominivorax, é um ectoparasito obrigatório dos animais de sangue quente endêmico das Américas. As miíases causadas por esta praga geram severos prejuízos econômicos na produção pecuária da América do Sul e da região do Caribe, devido à mortalidade e redução na produtividade dos animais infestados. Nesta tese, abordamos alguns aspectos da distribuição da variação de seqüências do DNA mitocondrial de C. hominivorax em duas escalas geográficas e os eventos históricos que podem explicar os padrões observados. As principais contribuições foram compiladas em três artigos científicos. No primeiro artigo, (artigo 1) investigamos o padrão geral de estruturação geográfica da variabilidade genética em 38 localidades ao longo de toda a distribuição da espécie. As análises das seqüências parciais da região controle (CR) e dos genes COI e COII mitocondriais revelaram que a diversidade genética da espécie está estruturada em quatro grupos regionais principais, denominados Cuba (CG), República Dominicana (DRG), região norte e região sul da Amazônia (NAG e SAG, respectivamente). Foi sugerido que o padrão observado foi principalmente formado por eventos históricos, como a colonização das ilhas do Caribe, vicariância na região Amazônica e expansão das populações desde o último máximo glacial. O segundo artigo (artigo 2) apresenta um teste da hipótese de perturbação-vicariância na região Amazônica, que foi realizado através de simulações de coalescência e modelagem ecológica da espécie. Os resultados indicaram que os ciclos climáticos do Pleistoceno promoveram a expansão, fragmentação e mistura das populações na região Amazônica em diferentes momentos do passado. Detectamos que o primeiro momento de divergência dos grupos de populações analisadas (SAG, NAG e amostras da América Central e do Norte) é mais antigo que o último período glacial e deve ter acontecido no interglacial Yarmouth (~250.000 anos atrás). Porém, a demografia histórica da espécie e as relações filogenéticas das seqüências mitocondriais sugerem um padrão mais complexo, incluindo reticulação, para a evolução e divergência das populações na região Amazônica. No terceiro artigo (artigo 3), apresentamos o primeiro estudo da variabilidade genética de C. hominivorax em escala local, em que se analisaram indivíduos de 25 sítios de coleta distribuídos numa área de 90 km por 60 km próxima às cidades de Quaraí e Artigas, na fronteira entre Brasil e Uruguai. Os resultados sugerem que os indivíduos pertencem a uma única população com alto grau de polimorfismo, indicando que esta é uma população estável capaz de sobreviver às condições adversas do inverno local e que flutua demograficamente de acordo com o clima da região. Porém, há evidências de que estas amostras não estão em equilíbrio e, portanto, a ausência de estruturação baseada nas estimativas tradicionais deve ser interpretada com cuidado. Como esta é uma região importante para a atividade pecuária do Brasil e Uruguai, estes resultados são de interesse para um possível plano de controle na região. No entanto, serão necessárias análises de amostras seriadas ao longo de um ano, assim como de diferentes anos na mesma escala geográfica, para melhor entender o padrão de fluxo gênico na regiãoAbstract: The New World screwworm fly, Cochliomyia hominivorax, is an obligate ectoparasite of warm blooded vertebrates, endemic of the Americas. Myiasis caused by this pest fly generate great profit looses to the livestock sector in South America and the Caribbean region, due to mortality and productivity reduction of infested animals. In this thesis, we approach some aspects of mitochondrial DNA sequences variation of C. hominivorax in two geographic scales, and the historical events that could explain the observed patterns. The main contributions were compiled in three research articles. In the first (article 1), we investigate the general pattern of genetic variability distribution in 38 localities encompassing the species geographic range. The analysis of partial sequences of the mitochondrial control region (CR), COI and COII genes revealed four main regional groups of genetic diversity, denominated Cuba (CG), Dominican Republic (DRG), north region (NAG) and south region (SAG) of the Amazon basin. Results suggest that current distribution of C. hominivorax genetic diversity was mainly shaped for historical events, i.e. Caribbean islands colonization, vicariance in the Amazon region and population expansion since the last glacial maximum. The second article (article 2) presents a hypotheses test of vicariance-perturbation in the Amazon region, through coalescent simulations and ecological niche modeling of the species. The results indicate that Pleistocene climatic cycles promote expansion, fragmentation and mixture of Amazon region populations during the past. We detected that the divergence among groups of analyzed populations (SAG, NAG and samples from North and Central America) occurred before the last glacial maximum and may have happened during the interglacial Yarmouth (~250.000 years ago). However, the species demographic history and the phylogenetic relationships of the mitochondrial sequences suggest a more complex pattern, including reticulation, for the evolution and divergence of the Amazonian region populations. Finally, in the third article (article 3) we present the first study of the genetic variability of C. hominivorax in a local scale, in which individuals of 25 localities in a 90 km for 60 km area in the proximity of Artigas city and Quaraí city, in the border between Brazil and Uruguay, were analyzed. The results suggest that the individuals are from a unique population, with high level of polymorphism, indicating that this is a stable population which is capable of survive the adverse local winter conditions and demographically fluctuate in accordance to the region climate. However, there are evidences that these samples are not in equilibrium hence the absence of structure interpretation based on traditional estimates must be done carefully. As this is an important region for livestock industry of Brazil and Uruguay, these results are interesting for a possible control program in the region. However, studies of serially collected samples throughout the year and in different years at the same geographic scale will be necessary to better understand the gene flow pattern in the regionDoutoradoGenetica Animal e EvoluçãoDoutor em Genetica e Biologia Molecula

Revisão das interações ecológicas da mosca Cochliomyia hominivorax e avaliação dos possíveis impactos ecológicos de sua erradicação no Uruguai

Controlling a pest species raises questions about the harmful effects its eradication could cause in ecosystems. Currently, in Uruguay are being analyzed strategies to eradicate the New World Screwworm (NWS), Cochliomyia hominivorax (Diptera: Calliphoridae), an obligatory ectoparasite that causes myiasis in warm-blooded vertebrates, including humans. The loss of a species implies a risk of secondary extinctions and cascading effects that depend on the ecological function of the species and the capacity of the rest of the species with which it interacts to compensate for its loss. To assess the ecological impact of the NWS eradication, we analyzed its ecological redundancy, its effect as an indirect regulatory agent for other species, and its participation in critical ecosystem functions. We conclude that the NWS presents high functional redundancy in most ecological interactions. It does not play an essential role as a regulatory agent of other species and does not play an important role in critical ecosystem functions. Without ignoring the information gaps that certainly exist about the ecological interactions network of the NWS in Uruguay, we estimate that its eradication is unlikely to cause secondary extinctions or cascading effects in the networks it integrates. In order to timely detect possible impacts during the course of the program, it is recommended to monitor the ecosystems using indicators related to direct and indirect partners in the ecological interactions of the NWS.El control de una especie plaga genera interrogantes sobre los efectos nocivos que su erradicación podría causar en los ecosistemas. Actualmente, en Uruguay se analizan estrategias para erradicar la mosca de la bichera (MB), Cochliomyia hominivorax (Diptera: Calliphoridae), un ectoparásito obligatorio que causa miasis en vertebrados de sangre caliente, incluidos los humanos. La pérdida de una especie supone un riesgo de extinciones secundarias y efectos en cascada que dependen de la función ecológica de la especie que se pierde y la capacidad del resto de las especies con las que interactúa para compensar su pérdida. Para evaluar el impacto ecológico de la erradicación de la MB analizamos su redundancia ecológica, su efecto como agente regulador indirecto de otras especies y su participación en funciones críticas de los ecosistemas. Concluimos que la MB presenta alta redundancia funcional en la mayoría de sus interacciones ecológicas, no juega un papel importante como agente regulador de otras especies y no juega un papel importante en funciones ecosistémicas críticas. Sin desconocer los vacíos de información que ciertamente existen sobre la red de interacciones ecológicas de la MB en Uruguay, estimamos que es poco probable que su erradicación cause extinciones secundarias o efectos en cascada en las redes de interacciones ecológicas que integra. A fin de detectar oportunamente eventuales impactos durante el curso del programa, se recomienda monitorear los ecosistemas utilizando indicadores relacionados con socios directos e indirectos en las interacciones ecológicas de la MB.O controle de uma espécie praga levanta questões sobre os efeitos nocivos que a erradicação pode causar nos ecossistemas. Atualmente, no Uruguai estão sendo analisadas estratégias de controle para erradicar a mosca da bicheira (MB), Cochliomyia hominivorax (Diptera: Calliphoridae), um ectoparasita obrigatório que causa miíase em vertebrados de sangue quente, incluindo o homem. A perda de uma espécie implica um risco de extinções secundárias e efeitos em cascata que dependem da função ecológica da espécie que se perde e da capacidade do resto das espécies com as quais interage para compensar a sua perda. Para avaliar o impacto ecológico da erradicação da MB, analisamos sua redundância ecológica, seu efeito como agente regulador indireto para outras espécies e sua participação em funções críticas do ecossistema. Concluímos que a MB apresenta alta redundância funcional na maioria de suas interações ecológicas, não desempenha um papel importante como agente regulador de outras espécies e não desempenha um papel importante em funções críticas do ecossistema. Sem ignorar as lacunas de informação que certamente existem sobre a rede de interações ecológicas do NWS no Uruguai, estimamos que é improvável que sua erradicação cause extinções secundárias ou efeitos em cascata nas redes de interações ecológicas que integra. Para detectar oportunamente possíveis impactos durante o curso do programa, recomenda-se monitorar os ecossistemas usando indicadores relacionados a parceiros diretos e indiretos nas interações ecológicas da MB

A new class of glycomimetic drugs to prevent free fatty acid-induced endothelial dysfunction

Background: Carbohydrates play a major role in cell signaling in many biological processes. We have developed a set of glycomimetic drugs that mimic the structure of carbohydrates and represent a novel source of therapeutics for endothelial dysfunction, a key initiating factor in cardiovascular complications. Purpose: Our objective was to determine the protective effects of small molecule glycomimetics against free fatty acid­induced endothelial dysfunction, focusing on nitric oxide (NO) and oxidative stress pathways. Methods: Four glycomimetics were synthesized by the stepwise transformation of 2,5­dihydroxybenzoic acid to a range of 2,5­substituted benzoic acid derivatives, incorporating the key sulfate groups to mimic the interactions of heparan sulfate. Endothelial function was assessed using acetylcholine­induced, endotheliumdependent relaxation in mouse thoracic aortic rings using wire myography. Human umbilical vein endothelial cell (HUVEC) behavior was evaluated in the presence or absence of the free fatty acid, palmitate, with or without glycomimetics (1µM). DAF­2 and H2DCF­DA assays were used to determine nitric oxide (NO) and reactive oxygen species (ROS) production, respectively. Lipid peroxidation colorimetric and antioxidant enzyme activity assays were also carried out. RT­PCR and western blotting were utilized to measure Akt, eNOS, Nrf­2, NQO­1 and HO­1 expression. Results: Ex vivo endothelium­dependent relaxation was significantly improved by the glycomimetics under palmitate­induced oxidative stress. In vitro studies showed that the glycomimetics protected HUVECs against the palmitate­induced oxidative stress and enhanced NO production. We demonstrate that the protective effects of pre­incubation with glycomimetics occurred via upregulation of Akt/eNOS signaling, activation of the Nrf2/ARE pathway, and suppression of ROS­induced lipid peroxidation. Conclusion: We have developed a novel set of small molecule glycomimetics that protect against free fatty acidinduced endothelial dysfunction and thus, represent a new category of therapeutic drugs to target endothelial damage, the first line of defense against cardiovascular disease

The genetic structure of an invasive pest, the Asian citrus psyllid Diaphorina citri (Hemiptera: Liviidae).

The Asian citrus psyllid Diaphorina citri is currently the major threat to the citrus industry as it is the vector of Candidatus Liberibacter, the causal agent of huanglongbing disease (HLB). D. citri is native to Asia and now colonizes the Americas. Although it has been known in some countries for a long time, invasion routes remain undetermined. There are no efficient control methods for the HLB despite the intensive management tools currently in use. We investigated the genetic variability and structure of populations of D. citri to aid in the decision making processes toward sustainable management of this species/disease. We employed different methods to quantify and compare the genetic diversity and structure of D. citri populations among 36 localities in Brazil, using an almost complete sequence of the cytochrome oxidase I (COI) gene. Our analyses led to the identification of two geographically and genetically structured groups. The indices of molecular diversity pointed to a recent population expansion, and we discuss the role of multiple invasion events in this scenario. We also argue that such genetic diversity and population structure may have implications for the best management strategies to be adopted for controlling this psyllid and/or the disease it vectors in Brazil

A relic of the past: Current genetic patterns of the palaeoendemic tree Nothofagus macrocarpa were shaped by climatic oscillations in central Chile

• Background and Aims The Mediterranean-type forest of central Chile is considered a 'biodiversity hotspot' and a relic of a wider ancient distribution produced by past climatic oscillations. Nothofagus macrocarpa, commonly known as 'roble de Santiago', is a threatened palaeoendemic of this forest, poorly represented in the protected area system. This tree has been repeatedly misidentified as the sister species N. obliqua, which has affected its recognition and protection. Only a few populations of N. macrocarpa remain within a matrix of intensive land use that has been affected by recent forest fires. We tested the hypothesis that current populations of N. macrocarpa are a relic state of a previously widespread range, with the aim of contributing to its identification, its biogeographical history and the design of conservation measures using genetic information. • Methods We analysed remnant N. macrocarpa forests using nuclear (nDNA) and chloroplast DNA (cpDNA) sequences, conducted phylogenetic and phylogeographical analyses to reconstruct its biogeographical history, and assessed microsatellites [simple sequence repeats (SSRs)] to determine contemporary patters of diversity within and among all remnant populations. We also examined the degree of past, current and potential future isolation of N. macrocarpa populations using ecological niche models (ENMs). • Key Results The species N. macrocarpa was confirmed by nDNA sequences, as previously suggested by chromosomal analysis. Small isolated populations of N. macrocarpa exhibited moderate to high genetic diversity according to SSRs. cpDNA analysis revealed a marked past latitudinal geographical structure, whereas analysis of SSRs did not find such current structure. ENM analyses revealed local expansion-contraction of the N. macrocarpa range during warmer periods, particularly in the northern and central ranges where basal-most cpDNA haplotypes were detected, and recent expansion to the south of the distribution. • Conclusions Genetic patterns confirm that N. macrocarpa is a distinct species and suggest a marked latitudinal relic structure in at least two evolutionarily significant units, despite contemporary among-population gene flow. This information must be considered when choosing individuals (seeds and/or propagules) for restoration purposes, to avoid the admixture of divergent genetic stocks.Fil: Mathiasen, Paula. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; ArgentinaFil: Venegas González, Alejandro. Universidad Mayor; ChileFil: Fresia, Pablo. Instituto Nacional de Investigacion Agropecuaria; . Instituto Pasteur de Montevideo; UruguayFil: Premoli Il'grande, Andrea Cecilia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Patagonia Norte. Instituto de Investigaciones en Biodiversidad y Medioambiente. Universidad Nacional del Comahue. Centro Regional Universidad Bariloche. Instituto de Investigaciones en Biodiversidad y Medioambiente; Argentin

Comparative transcriptome analysis of lufenuron-resistant and susceptible strains of Spodoptera frugiperda (Lepidoptera: Noctuidae)

Abstract\ud \ud Background\ud The evolution of insecticide resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) has resulted in large economic losses and disturbances to the environment and agroecosystems. Resistance to lufenuron, a chitin biosynthesis inhibitor insecticide, was recently documented in Brazilian populations of S. frugiperda. Thus, we utilized large-scale cDNA sequencing (RNA-Seq analysis) to compare the pattern of gene expression between lufenuron-resistant (LUF-R) and susceptible (LUF-S) S. larvae in an attempt to identify the molecular basis behind the resistance mechanism(s) of S. frugiperda to this insecticide.\ud \ud \ud Results\ud A transcriptome was assembled using approximately 19.6 million 100 bp-long single-end reads, which generated 18,506 transcripts with a N50 of 996 bp. A search against the NCBI non-redundant database generated 51.1 % (9,457) functionally annotated transcripts. A large portion of the alignments were homologous to insects, with the majority (45 %) being similar to sequences of Bombyx mori (Lepidoptera: Bombycidae). Moreover, 10 % of the alignments were similar to sequences of various species of Spodoptera (Lepidoptera: Noctuidae), with 3 % of them being similar to sequences of S. frugiperda. A comparative analysis of the gene expression between LUF-R and LUF-S S. frugiperda larvae identified 940 differentially expressed transcripts (p ≤ 0.05, t-test; fold change ≥ 4). Six of them were associated with cuticle metabolism. Of those, four were overexpressed in LUF-R larvae. The machinery involved with the detoxification process was represented by 35 differentially expressed transcripts; 24 of them belonging to P450 monooxygenases, four to glutathione-S-transferases, six to carboxylases and one to sulfotransferases. RNA-Seq analysis was validated for a number of selected candidate transcripts by using quantitative real time PCR (qPCR).\ud \ud \ud Conclusions\ud The gene expression profile of LUF-R larvae of S. frugiperda differs from LUF-S larvae. In general, gene expression is much higher in resistant larvae when compared to the susceptible ones, particularly for those genes involved with pathways for xenobiotic detoxification, mainly represented by P450 monooxygenases transcripts. Our data indicate that enzymes involved with the detoxification process, and mostly the P450, are one of the resistance mechanisms employed by the LUF-R S. frugiperda larvae against lufenuron.This work was part of research conducted in partial fulfillment of the M.Sc.\ud Degree in Entomology by ARBN at Escola Superior de Agricultura “Luiz de\ud Queiroz” (ESALQ/USP). We thank National Council for the Improvement of\ud Higher Education (CAPES) for the M.Sc. scholarship to ARBN and\ud postdoctoral fellowship to PF. We also thank National Council for Scientific\ud and Technological Development (CNPq) for granting research fellowships to\ud FLC (Process 312094/2013-2) and CO (Process 312086/2013-0)

Haplotype network of populations of <i>Diaphorina citri</i> from Brazil based on partial sequences of the COI gene (996 bp), built by using the TCS program.

<p>Each circle represents a haplotype and circles are gradually colored depending on the frequency haplotypes were observed, from one occurrence (light yellow) to more than 40 occurrences (dark red).</p

Mismatch curves of <i>Diaphorina citri</i> from the whole sample (a), and from group I (b) and II (c) independently.

<p>Mismatch curves of <i>Diaphorina citri</i> from the whole sample (a), and from group I (b) and II (c) independently.</p

The Phylogeographic History of the New World Screwworm Fly, Inferred by Approximate Bayesian Computation Analysis

<div><p>Insect pest phylogeography might be shaped both by biogeographic events and by human influence. Here, we conducted an approximate Bayesian computation (ABC) analysis to investigate the phylogeography of the New World screwworm fly, <i>Cochliomyia hominivorax</i>, with the aim of understanding its population history and its order and time of divergence. Our ABC analysis supports that populations spread from North to South in the Americas, in at least two different moments. The first split occurred between the North/Central American and South American populations in the end of the Last Glacial Maximum (15,300-19,000 YBP). The second split occurred between the North and South Amazonian populations in the transition between the Pleistocene and the Holocene eras (9,100-11,000 YBP). The species also experienced population expansion. Phylogenetic analysis likewise suggests this north to south colonization and Maxent models suggest an increase in the number of suitable areas in South America from the past to present. We found that the phylogeographic patterns observed in <i>C. hominivorax</i> cannot be explained only by climatic oscillations and can be connected to host population histories. Interestingly we found these patterns are very coincident with general patterns of ancient human movements in the Americas, suggesting that humans might have played a crucial role in shaping the distribution and population structure of this insect pest. This work presents the first hypothesis test regarding the processes that shaped the current phylogeographic structure of <i>C. hominivorax</i> and represents an alternate perspective on investigating the problem of insect pests.</p> </div

Analysis of molecular variance (AMOVA) for <i>Diaphorina citri</i> samples using <i>COI</i> sequences.

<p>Analysis of molecular variance (AMOVA) for <i>Diaphorina citri</i> samples using <i>COI</i> sequences.</p