Bacterial infection, immune responses, and autophagy in lutzomyia longipalpis sand flies

Doctor of PhilosophyDepartment of EntomologyMarcelo Ramalho-OrtigaoKun Yan ZhuMicrobial communities residing within the midgut of insect vectors play a critical role in the response to various zoonotic and human pathogens, and can directly alter the development and survival of the insects. Sand flies are the primary vector of Leishmania, the causative pathogen of leishmaniasis, a neglected tropical disease. Sand flies acquire many microbes from the soil where immature stages develop until emergence as adults. Gram-negative Pantoea agglomerans and gram-positive Bacillus subtilis are two bacteria commonly associated with sand fly populations. Here, I demonstrated that an EGFP- and a GFP-expressing version of these two bacteria localize to different compartments of the midgut; a phenomenon that is achieved, in part, to pH differences found across the length of the gut. Additionally, P. agglomerans is able to selectively induce midgut epithelial apoptosis while B. subtilis does not. This is accompanied by differential immune and homeostasis responses to both bacteria highlighted by immune pathway suppression via the Poor Immune Response upon Knock-in (Pirk) gene. These effects may actually be representative of a broader type of response to bacterial infection that might be present across several insect species. Finally, I demonstrated that during metamorphosis the sand fly relies, at least in part, upon the activation of multiple genes from the autophagy pathway to aid in generating adult tissues. More specifically, I demonstrate, using microscopy, the presence of ATG6 in the cytoplasm of developing midgut epithelial cells of the sand fly pupae

\u3ci\u3eVarroa destructor\u3c/i\u3e Mites Vector and Transmit Pathogenic Honey Bee Viruses Acquired From an Artificial Diet

The ectoparasitic mite Varroa destructoris one of the most destructive pests of the honey bee (Apis mellifera) and the primary biotic cause of colony collapse in many regions of the world. These mites inflict physical injury on their honey bee hosts from feeding on host hemolymph and fat body cells/cellular components, and serve as the vector for deadly honey bee viruses, including Deformed wing virus (DWV) and the related Varroa destructor virus-1 (VDV-1) (i.e., DWV-like viruses). Studies focused on elucidating the dynamics of Varroa-mediated vectoring and transmission of DWV-like viruses may be confounded by viruses present in ingested host tissues or the mites themselves. Here we describe a system that includes an artificial diet free of insect tissue-derived components for maintaining Varroa mites for in vitro experimentation. Using this system, together with the novel engineered cDNA clone-derived genetically tagged VDV-1 and wild-type DWV, we demonstrated for the first time that Varroa mites provided an artificial diet supplemented with engineered viruses for 36 hours could acquire and transmit sufficient numbers of virus particles to establish an infection in virus-naïve hosts. While the in vitro system described herein provides for only up to five days of mite survival, precluding study of the long-term impacts of viruses on mite health, the system allows for extensive insights into the dynamics of Varroa-mediated vectoring and transmission of honey bee viruses

Diversity and Global Distribution of Viruses of the Western Honey Bee, Apis mellifera

Funding: This research was permitted and funded by the COLOSS network (https://coloss.org/).Peer reviewedPublisher PD

COVID-19 symptoms at hospital admission vary with age and sex: results from the ISARIC prospective multinational observational study

Background: The ISARIC prospective multinational observational study is the largest cohort of hospitalized patients with COVID-19. We present relationships of age, sex, and nationality to presenting symptoms. Methods: International, prospective observational study of 60 109 hospitalized symptomatic patients with laboratory-confirmed COVID-19 recruited from 43 countries between 30 January and 3 August 2020. Logistic regression was performed to evaluate relationships of age and sex to published COVID-19 case definitions and the most commonly reported symptoms. Results: ‘Typical’ symptoms of fever (69%), cough (68%) and shortness of breath (66%) were the most commonly reported. 92% of patients experienced at least one of these. Prevalence of typical symptoms was greatest in 30- to 60-year-olds (respectively 80, 79, 69%; at least one 95%). They were reported less frequently in children (≤ 18 years: 69, 48, 23; 85%), older adults (≥ 70 years: 61, 62, 65; 90%), and women (66, 66, 64; 90%; vs. men 71, 70, 67; 93%, each P < 0.001). The most common atypical presentations under 60 years of age were nausea and vomiting and abdominal pain, and over 60 years was confusion. Regression models showed significant differences in symptoms with sex, age and country. Interpretation: This international collaboration has allowed us to report reliable symptom data from the largest cohort of patients admitted to hospital with COVID-19. Adults over 60 and children admitted to hospital with COVID-19 are less likely to present with typical symptoms. Nausea and vomiting are common atypical presentations under 30 years. Confusion is a frequent atypical presentation of COVID-19 in adults over 60 years. Women are less likely to experience typical symptoms than men

Deep River: Slavery, Empire, and Emancipation in the Upper Mississippi River Valley, 1730-1860

"Deep River" offers a continental perspective on human bondage and emancipation in mainland North America. It unearths the deep history of indigenous and African slavery in the upper Mississippi River Valley and traces its connections outward toward the Atlantic and Gulf coasts. "Deep River" argues for a new spatial frame for the history of slavery and freedom to understand how colonial experiences in the upper Mississippi River Valley shaped the trajectory of emancipation in the United States. It also offers new perspectives on the history of emancipation by exposing free and enslaved black agency to eradicate slavery from Illinois. "Deep River" moves past legal categories as an organizing framework for slave and free societies. It demonstrates that inheritable bondage long survived its legal abolition. Displacing laws as the engine of change, it argues the collaborations between free black migrants, fugitive slaves, and white anti-slavery activists drove the processes of emancipation forward. Free and fugitive migrants into Illinois settled in black freedom villages which afforded slaves limited access to capital, avenues toward finding attorneys, and support in legal proceedings. In this way, Illinois's movement to a free society sprang from domestic migrations and a longer colonial legacy of trade and settlement in the Mississippi Valley, not laws and statutes passed by the United States. By focusing on the ways in which black northern migration and litigation in local courts shaped emancipation in the state, "Deep River" illuminates how legal and political development in Illinois followed the paths that enslaved African Americans created

Analysis of EST’s encoding pea aphid Acyrthosiphon pisum C002 & the effect of armet transcript knockdown in Tribolium castaneum

Master of ScienceDepartment of BiochemistryGerald ReeckAphids mount a remarkable salivary secretion to overcome plant host defenses. Our group has previously reported a gene unique to aphids enriched in the salivary glands of the pea aphid A. pisum, C002, which is required for successful feeding on its host plant Vicia fava. Here I present an analysis of genetic variation within the available EST data for C002 in pea aphids. From 596 total ESTs, 332 are full-length, and segregate into 8 validated haplotypes based on the criteria I set in place to access the quality of EST data. Additionally, Armet, is a putative multi-functional gene implicated as a neurotrophic factor during development, and as a part of the unfolded protein response during stress. I employ RNA interference in the model organism T. castaneum to determine the effect of transcript knockdown during development from early in-star larval stages, through pupation, and its effect on adult emergence. I report that knockdown of Armet transcript significantly hinders the ability for beetles to emerge from the pupae

Bacterial Infection and Immune Responses in Lutzomyia longipalpis Sand Fly Larvae Midgut.

The midgut microbial community in insect vectors of disease is crucial for an effective immune response against infection with various human and animal pathogens. Depending on the aspects of their development, insects can acquire microbes present in soil, water, and plants. Sand flies are major vectors of leishmaniasis, and shown to harbor a wide variety of Gram-negative and Gram-positive bacteria. Sand fly larval stages acquire microorganisms from the soil, and the abundance and distribution of these microorganisms may vary depending on the sand fly species or the breeding site. Here, we assess the distribution of two bacteria commonly found within the gut of sand flies, Pantoea agglomerans and Bacillus subtilis. We demonstrate that these bacteria are able to differentially infect the larval digestive tract, and regulate the immune response in sand fly larvae. Moreover, bacterial distribution, and likely the ability to colonize the gut, is driven, at least in part, by a gradient of pH present in the gut

Midgut morphological changes and autophagy during metamorphosis in sand flies

During metamorphosis, holometabolous insects undergo significant remodeling of their midgut and become able to cope with changes in dietary requirements between larval and adult stages. At this stage, insects must be able to manage and recycle available food resources in order to develop fully into adults, especially when no nutrients are acquired from the environment. Autophagy has been previously suggested to play a crucial role during metamorphosis of the mosquito. Here, we investigate the overall morphological changes of the midgut of the sand fly during metamorphosis and assess the expression profiles of the autophagy-related genes ATG1, ATG6, and ATG8, which are associated with various steps of the autophagic process. Morphological changes in the midgut start during the fourth larval instar, with epithelial degeneration followed by remodeling via the differentiation of regenerative cells in pre-pupal and pupal stages. The changes in the midgut epithelium are paired with the up-regulation of ATG1, ATG6 and ATG8 during the larva-adult transition. Vein, a putative epidermal growth factor involved in regulating epithelial midgut regeneration, is also up-regulated. Autophagy has further been confirmed in sand flies via the presence of autophagosomes residing within the cytoplasmic compartment of the pupal stages. An understanding of the underlying mechanisms of this process should aid the future management of this neglected tropical vector

mRNA expression profiles of 3^rd instar <i>L</i>. <i>longipalpis</i> larvae post infection with <i>B</i>. <i>subtilis</i> and <i>P</i>. <i>agglomerans</i>.

<p><i>B</i>. <i>subtilis</i> (<i>Bs</i>) or <i>P</i>. <i>agglomerans</i> (<i>Pa</i>) bacteria were fed to larvae and the expression of selected transcripts relative to agar fed control larvae was assessed. Expression profiles were obtained for the effector molecules <i>Att</i> and <i>IMPer</i>, in addition to the epithelial growth factor <i>Vein</i> are shown in A. D, and G, Expression profiles for the JAK/STAT receptor <i>Domeless</i>, the transcription factor for immunodeficiency <i>IMD</i>, and the negative regulator of IMD pathway <i>Pirk</i> are shown in are shown in B, E and H. Expression profiles for the E3 Ubiquitin ligase associated with <i>IMD</i>, and the effector molecules dual oxidase <i>DUOX</i> and <i>Def1</i>are shown in C, F, and I. Following bacterial feeding, total RNA was obtained at 12 h (A, B, and C), at 24 h (D, E, and F), and at 36 h (G, H, and I) post infection (PI). All Ct values were normalized to the ribosomal protein S6 (RPS6). Error bars are represented as the standard deviation determined from three biological replicates each with n = 20 midguts. Significance was determined using student t-tests. * denotes P<0.05 and ** denotes P<0.01. Y-axis, fold change.</p