Host-microbiome-pathogen interactions in cockroaches

Traditionally, philosophy uses the discrimination of self and non-self to define individuality with the immune system performing this discrimination. In the evolutionary field of biology this distinction is not that simple. Nowadays it is becoming more and more apparent that individuals can no longer be considered as ‘lone isolated islands’ in the ‘environmental sea’. All kinds of eukaryotic taxa harbour their own microbiota consisting of bacteria, archaea, fungi, protozoa and viruses and they are tolerated by the host’s immune system because of their manifold beneficial functions on, for example, host nutrition, detoxification, development, fecundity or pathogen protection. However, not only the beneficial microbiome, but also the host’s nutrition can strongly affect its physiology and its ability to combat pathogen infections. Microbiome and host form a unit – the holobiome. Notably, even though we gained insights on either the function of the microbiome or of the nutrition on host immune defence in diverse separate studies we still poorly understand how they act together in particular organisms. An insect model system to study these interactions are cockroaches. This is because, 1) they are omnivorous generalists, which makes them easily accessible for nutritional studies; 2) they harbour a diverse microbiota which can be manipulated through sterilization methods; and 3) they feature effective strategies to combat pathogens since they are frequently exposed to a rich antigenic environment due to their lifestyle. First, in Chapter I I investigated the nutritional dependencies of immunity in the cockroach system by performing food choice experiments using the cockroach species Blatta orientalis upon exposure to the opportunistic Gram-negative bacterial insect pathogen Pseudomonas entomophila. I could show that depending on the strength of infection B. orientalis males reduce their overall nutrient intake and increase the protein to carbohydrate ratio being consumed. Interestingly, these behavioural shifts do not boost the insect’s immunity as indicated by the examination of the hemolymph’s antimicrobial activity, the abundance of immune proteins in the hemolypmph or the general host survival. This lack of benefits for the host highlights a possible decoupling of dietary macronutrient regulation from immunity in these invasive animals with the possibility that anorexia, in general, might be a more powerful tool if diet quality is highly unpredictable for generalist species. In Chapter II I evaluated two different approaches for the development of a germ-free Blatella germanica cockroach breeding system which forms the basis of any study dealing with the function of the cockroach microbiome. While one of these methods uses peracetic acid, the other one uses a combination of peracetic acid and sodium hypochlorite to surface-sterilize cockroach oothecas to deprive the hatchlings from their natural microbiota. These treatments should leave them only with their obligate symbiont Blattabacterium sp., which supplies essential vitamins and is required for the development into fecundant adults. I tested the success of those techniques by plating adult individuals on Lysogeny-broth-agar and by using state of art 16S metabarcoding. It turned out that both methods performed quite poorly leading to individuals which can be considered as germ-free in 40 % of all cases. I therefore developed our own method by combining sequential ootheca surface sterilizations with peracetic acid and sodium hypochlorite followed by a treatment of freshly hatched nymphs with the antibiotics rifampicin and gentamicin which significantly improved its effectiveness resulting in germ-free adult cockroaches to 99 % of all cases. In addition, I used our germ-free cockroach system for an early study on the impact of the absence of an intact microbiome on developmental time. I could show that B. germanica cockroaches deprived of their natural microbiota needed approximately 35 days longer from the day of hatching to the day molting into adults than their conventional counterparts, which already grants a small glimpse on the strong impacts of the microbiome on the host physiology and its overall performance. In Chapter III I analysed the transcriptome of germ-free and conventional B. germanica males and followed their survival upon P. entomophila systemic infection to gain further insights on the influence of the cockroach microbiome on host traits. The basis of our gene identification were two published genomes either the one by Harrison et al. (2018) or the one by He (2018). Depending on the reference genome used for the analyses small differences existed. When the Harrison et al. genome was used 25451 putative genes were identified and 184 of those including 19 immune-related genes were significantly different expressed between conventional and germ-free cockroaches. When the He genome was used 111778 putative genes were identified and 1082 of those including 30 immune-related genes were significantly different expressed between conventional and germ-free cockroaches. Immune-related genes which were significantly expressed between germ-free and conventional cockroaches identified with both reference genomes included hemolymph lps-binding protein related genes which were mostly upregulated in germ-free individuals because of their role in trapping the Blattabacterium sp. endosymbiont and tenecin-1 genes, a transferrin, a caspase-1, a alpha-1-macroglobulin, a lysozym c-1 as well as a catalase found to be upregulated in conventional individuals. All the latter ones contribute to the recognition and the suppression of microbial life to maintain a stable host microbiota. This regulation of gene expression by the microbiome might also assist the host in combating infections as indicated by the significantly higher survival of conventional cockroaches infected with P. entomophila. In conclusion I was able to show, that host biology is heavily shaped by microbial life. Those microbes can be either invading pathogens or commensal or beneficial microbiota. In all cases they radically alter host behaviour, development and/or physiology. While pathogens only harm their hosts, the microbiome promotes host phenotypes like development or immune competence. Therefore, pathogens do not only interact with their hosts but also with its microbiota. Since this fact became only apparent within the last few years more research is needed to reveal all its aspects. A stable foundation for such future work is paved by my recently established germ-free B. germanica breeding system. In this framework it will be particularly important and likewise exciting to perform refaunation experiments with single microbial taxa followed by infections with different pathogens and further transcriptomic analyses to uncover their special tasks in this broad network of interactions

Protein Assembly for a Functional Fibrous Product

Natural protein-based materials are exploring their new applications from the traditional uses. Structural proteins provide scaffolds, whereas functional proteins carry essential biological activities through millions of biochemical reactions. The idea of implementing functionalities into natural structures could provide manufactured protein products with a better fit for human desire. The development of synthetic biology and molecular assembly methods illustrates possibilities for the production of functional structures in-situ, which provides better connections between the functional partners with the structural scaffolds. Protein fibres are one of the natural structures which possess a unique shape and superior mechanical properties. Apart from the natural protein fibres, many disease-related peptides self-assembled into amyloid filaments and fibrous structures. Natural globular proteins could also form fibres through the directed assembly by changing their storage conditions or through fusion. Elongated polyglutamine peptides cause many neurodegenerative diseases as they assemble. In this thesis, a polyglutamine peptide (Q77) was fused with functional partners to direct the protein assembly in vitro. The role of the polyglutamine was studied during assembly and after the formation of a self-supportive fibrous product. The extensibility of traditionally size-limited fibrous materials formed by disease-related peptides was tested experimentally for the first time. The resultant fibrous product with embedded functionalities mimics the structure of silk, but the mechanical behaviour of collagen. Two structurally distinct proteins were chosen as the functional partners for Q77: a monomeric red fluorescent protein (mcRFP), which is relatively small in size and possesses a b-barrel structure, and firefly luciferase (Luc), which is a larger protein with a fragile structure consisting of two mobile domains. Both proteins have been widely used as reporters for intracellular activities with either fluorescence or bioluminescent signal. In this work, the functionalities of both proteins were investigated after Q77 fusion and after assembly towards respective fibrous products. The structural variation of these recombinant proteins resulted in the changes of their functionalities. Finally, a self-supportive fibrous ATP sensor was achieved for the first time with this dual functional protein product.Cambridge Trust, China Scholarship Counci

NEBLINE, April 2018

CONTENTS Feature: Can You Pass This Food Safety Quiz? Food & Health Farm & Acreage Pests & Wildlife Horticulture Early Childhood 4-H & Youth and other extension news and events Special Pullout Section: Weed Awarenes

Fluorescent-based nanosensors for selective detection of a wide range of biological macromolecules: A comprehensive review

Thanks to their unique attributes, such as good sensitivity, selectivity, high surface-to-volume ratio, and versatile optical and electronic properties, fluorescent-based bioprobes have been used to create highly sensitive nano -biosensors to detect various biological and chemical agents. These sensors are superior to other analytical instrumentation techniques like gas chromatography, high-performance liquid chromatography, and capillary electrophoresis for being biodegradable, eco-friendly, and more economical, operational, and cost-effective. Moreover, several reports have also highlighted their application in the early detection of biomarkers associ-ated with drug-induced organ damage such as liver, kidney, or lungs. In the present work, we comprehensively overviewed the electrochemical sensors that employ nanomaterials (nanoparticles/colloids or quantum dots, carbon dots, or nanoscaled metal-organic frameworks, etc.) to detect a variety of biological macromolecules based on fluorescent emission spectra. In addition, the most important mechanisms and methods to sense amino acids, protein, peptides, enzymes, carbohydrates, neurotransmitters, nucleic acids, vitamins, ions, metals, and electrolytes, blood gases, drugs (i.e., anti-inflammatory agents and antibiotics), toxins, alkaloids, antioxidants, cancer biomarkers, urinary metabolites (i.e., urea, uric acid, and creatinine), and pathogenic microorganisms were outlined and compared in terms of their selectivity and sensitivity. Altogether, the small dimensions and capability of these nanosensors for sensitive, label-free, real-time sensing of chemical, biological, and pharma-ceutical agents could be used in array-based screening and in-vitro or in-vivo diagnostics. Although fluorescent nanoprobes are widely applied in determining biological macromolecules, unfortunately, they present many challenges and limitations. Efforts must be made to minimize such limitations in utilizing such nanobiosensors with an emphasis on their commercial developments. We believe that the current review can foster the wider incorporation of nanomedicine and will be of particular interest to researchers working on fluorescence tech-nology, material chemistry, coordination polymers, and related research areas

Horizontal Transmission of Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) in Soybean Fields Infested with Helicoverpa zea (Boddie)

Helicoverpa armigera Nucleopolyhedrovirus (HearNPV) is a commercially available viral biopesticide that targets Heliothines, including Helicoverpa zea (Boddie) (Lepidoptera: Noctuidae), the most damaging pest of soybean (Glycine max (L.) Merrill) in the Mid-South. Previous formulations of HearNPV have been well studied; however, no research has been published on current formulations. The first objective of this thesis was to assess the rate of horizontal transmission of HearNPV in a soybean field infested with H. zea when HearNPV was applied as a bio-insecticide, and to identify arthropods that are important obligate carriers in dissemination. HearNPV spread 200 feet in 3 fields, and was present between 13 and 21 days post application. Ceresa festina (Hemiptera: Membracidae), H. zea larvae, and Geocoris spp. (Hemiptera: Geocoridae) were identified as important carriers, while spiders (Araneae) were determined to be suppressors of HearNPV. The second objective was to identify arthropods in contact with infected larvae and determine their importance in dissemination. HearNPV was found as far away as 200 feet by 3 days; however, only 2 samples were positive past 3 days. Several previously undocumented carriers were observed, including several families of Diptera and Lygus lineolaris (Hemiptera: Miridae). The third objective was to determine the ability of HearNPV to kill each H. zea instar, and a second infestation. HearNPV was successful in controlling 1st-3rd instars in 5 days. The second generation was controlled in 3.5 days. If applied to a soybean field as an insecticide when 1st-3rd instar H. zea populations are present, HearNPV should be able to spread 200 feet, utilizing several over-sprayed arthropods such as Ceresa festina, and remain in the canopy from 3 to 21 days. Five days after the application, several arthropods can disseminate HearNPV from liquefied to healthy larvae for 3 days before the majority of active HearNPV leaves the canopy. If another generation of healthy larvae infest during this time period, the epizootic event should continue, thus repeating this cycle

Transgenic and histological approaches to investigating the development of the stalk-eyed fly, Teleopsis dalmanni

Stalk-eyed flies of the family Diopsidae have their eyes laterally displaced on the end of head extensions called “eye-stalks”. Diopsids vary in their degree of sexual dimorphism for eyespan (distance between the eyes). In some dimorphic species it has been well established that females preferentially mate with males possessing exaggerated eyespan. With over 150 members, the family Diopsidae is an ideal model system for analysing the evolution and development of exaggerated sexual traits. Progress towards understanding the mechanisms underlying the development of exaggerated eyespan has been significantly hampered by the lack of modern molecular genetic technology in stalk-eyed flies. I have developed a transgenic protocol in Teleopsis dalmanni, a highly dimorphic diopsid species. I selected and tested embryo microinjection procedures. I used excision assays to compare the activity of three potential transposable element vector systems. Minos and piggyBac demonstrated suitable activity in T. dalmanni embryos but mariner did not. Using Minos and the transgene construct Px3-eGFP, I successfully achieved stable germline transformation in T. dalmanni. A number of transgenic lines were created. In one, a single copy of the insertion was seen to segregate with the X chromosome. I used a histological approach to investigate the relative contributions of cell size and cell number to variation in eyespan. I compared estimates of cell size in the eye-stalks of newly eclosed flies among fully fed and nutritionally stressed individuals. For comparison, I assessed cell size in a non-sexually dimorphic organ, the wing. I found that variation in eyespan was explained, at least in part, by variation in cell size. No inherent difference in eye-stalk cell size was detected between the sexes. The implications of the cell size findings and of the future studies made possible by transgenic technology are discussed

Division of Research and Economic Development Annual Report for FY2008

Annual report for the Division of Research and Economic Development of the University of Rhode Island for the year 2007-2008. Includes statistics of project proposals, expenditures, URI Foundation Awards, previous annual report summaries and awards received by individual academic and administrative departments