BIOLOGY AND MANAGEMENT OF ARTHROPOD PESTS OF PUBLIC AND ANIMAL HEALTH IMPORTANCE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: TERMINATED
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1003465
• Project No.: KS517
• Project Start Date: Jul 2, 2014
• Project End Date: Dec 10, 2018

Project Director
Zurek, LU, .

Recipient Organization
KANSAS STATE UNIV
(N/A)
MANHATTAN,KS 66506

Performing Department
Entomology

Non Technical Summary
Research in Zurek's group is focused on better understanding of the microbial-ecology of muscod flies and biting midges with aim to establish a platform for novel approaches for management of these insect pests and/or pathogens they transmit.Research in the Ortigao laboratory focuses on several aspects of the biology of insect-borne diseases. Our studies aim at identifying underlying details of pathogen development within vectors allowing for the development of novel disease and vector control strategies to mitigate the impact of such diseases in animals and humans.

Animal Health Component

20%

Research Effort Categories

Basic

100%

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
312	3110	1110	20%
315	3110	1110	40%
721	3110	1040	20%
712	3110	1040	20%

Knowledge Area
312 - External Parasites and Pests of Animals; 712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 315 - Animal Welfare/Well-Being and Protection; 721 - Insects and Other Pests Affecting Humans;

Subject Of Investigation
3110 - Insects;

Field Of Science
1040 - Molecular biology; 1110 - Parasitology;

Keywords

insect disease vectors

bacterial community

gut microbiology

food safety

peritrophic matrix

rna interference

sand flies

mosquitoes

biting midges

muscoid flies

Goals / Objectives
Role of muscoid flies in the ecology of Shiga-toxigenic Escherichia coli (STEC) (Zurek Lab)Significance of bacteria in oviposition, development, fitness, and vector competence of Culicoides sonorensis for EHDV. (Zurek Lab)Microbial ecology and epithelial immunity of house flies and stable flies (Zurek Lab)The insect peritrophic matrix type 1 permeability and effect on parasite development within vectors. (Ortigao Lab)RNA interference to investigate insect vector physiology. (Ortigao Lab)Paratransgenesis approaches against sand fly-transmitted leishmaniasis. (Ortigao Lab)Morphological and physiological analyses of mosquito tissues. (Ortigao Lab)

Project Methods
Role of muscoid flies in the ecology of Shiga-toxigenic Escherichia coli (STEC) The CDC (2010) estimates ~37,000 cases of STEC annually and ~0.57 cases per 100,000 population. One of the potential means of spread of these pathogens in the environment is by insects that develop in animal feces/manure (primarily house flies, Musca domestica L). Previously, a laboratory-based study demonstrated that E. coli O157 ingested by HF remain viable in the fly excreta and the flies were able to carry and disseminate E. coli for several days (Kobayashi et al. 1999). In another study, we have shown that house flies can transmit E. coli O157 to cattle and likely play a role in the ecology of this organism in the cattle environment (Ahmad et al., 2007). House flies and stable flies will be collected from dairy and beef cattle from ~12 states. Flies will be surface sterilized and homogenized individually in 1 ml PBS and screened for STEC by a) direct plating on modified Posse (mPosse) agar and cefixime tellurite supplemented sorbitol MacConkey (CT-SMAC) agar and b) enrichment in EC broth followed by immuno-magnetic separation (IMS) for seven different serotypes (O26, O111, O104, O103, O45, O121, O145). For each fly sample, from the mPosse, morphologically different colonies will be screened by serotype-specific 8-plex PCR. The prevalence and diversity of STEC will be assessed in flies and corresponding animal manure (collaboration with Drs. Renter and Cernicchiaro).Microbial ecology and vector competence of Culicoides sonorensis. C. sonorensis is an important vector of orbiviruses, including bluetongue virus (BTV) and epizootic hemorrhagic disease virus (EHDV) (Gibbs et al., 1994; Allison et al., 2010). No effective mitigation strategies exist for controlling Culicoides biting midges. Larval Culicoides develop in microbe-rich habitats and the adult gut is colonized with bacteria. However, how the microbial communities in the natural developmental substrate and in the digestive tract of C. sonorensis impact midge development, fitness, and ultimately vector competence for orbiviruses is unknown. Our project goals are to: 1) evaluate microbe-midge interactions in relation to midge life history and fitness and 2) assess the impact of the gut microbiota on the vector competence of C. sonorensis for BTV and EHDV. Our approach will be to characterize the microbial community of natural larval habitats, and gut of larvae, newly-emerged and wild-trapped adults using culture-dependent and culture-independent approaches. A better understanding of midge-bacterial interactions will result in the establishment of a new platform for development of alternative strategies for management of C. sonorensis and BTV and EHDV.Microbial ecology of muscoid flies. Muscoid flies, primarily house flies and stable flies are major pests of food animals and represent a major concern from food safety perspective. Stable flies (Stomoxys calcitrans L.), are blood-feeding insects with a great negative impact on livestock resulting in annual losses around $2 billion in the USA alone (Taylor et al., 2012). Stable fly and house fly larvae develop primarily in animal manure and live bacteria were shown to be essential for stable fly development (Zurek et al., 2000). We hypothesize that the microbial community of animal manure changes over time and plays an important role in stable fly fitness. We plan to assess the microbial community changes in horse and cattle manure over time (16S rDNA pyrosequencing) and correlate that with fly ovipostion, larval development, and fitness of adult flies by conducting oviposition and developmental laboratory bioassays. In addition, we will investigate epithelial immune responses (qPCR) of house flies and stable flies to various bacterial species in order to better understand the fly-microbial interactions that will potentially lead to novel strategies for muscoid fly control.Peritrophic matrix (PM) type 1 permeability and effect on Leishmania development. Sand flies are vectors of several important pathogens including, bacteria, viruses, and parasites of the genus Leishmania. During a complex cycle within the sand fly midgut, Leishmania must escape from the endoperitrophic space and bind to the midgut epithelia to prevent excretion. Our goal is to test the effect of three antibodies targeting PpChit1 and PpPer1 & 3, shown to the involved in PM scaffolding, maturation, and degradation. Following feeding of sand flies on these antibodies, we will measure PM porosity and permeability using fluorescently labeled beads. Decreased PM permeability will likely cause a greater number of beads to remain within the (endo)peritrophic space, including some of the smaller ones (70K to 150K beads). In contrast, if the PM permeability increases, larger size beads (and possibly more beads), such as those in the range of 250-to-500K will likely be found in the ectoperitrophic space. This will be suggestive that a thicker PM would allow a smaller proportion of Leishmania parasites to escape. The goal is to assess if targeting such PM-molecules in sand flies is a suitable approach towards transmission blocking vaccines against canine visceral leishmaniasis. We will also assess the effects on the structure and scaffolding of the PM by various microscopic techniques. For the investigation on the effects on PM permeability, we intend to feed adult sand flies with the three different mouse derived antibodies against PpPer1, PpPer3, PpChit1 in conjunction with FITC-dextran beads (Sigma). Midguts from sand flies will be dissected 24-96 hrs post blood meal. Details on each procedure can be found in various publications by our group in the last 5 years (Martins et al 211 and 2012; Pascini et al. 2011; Leodido et al 2013)RNA interference to investigate insect vector physiology: We have developed an efficient RNAi platform to investigate various aspects of sand fly-Leishmania interaction. We have shown that RNAi knock-down (KD) of PpChit1 led to > 95% in reduction of PpChit1 protein in the midgut of P. papatasi and a 65% reduction in Leishmania load (Coutinho-Abreu et al. 2010). In contrast, PpPer1peritrophin KD led to an increase in parasite load (Coutinho-Abreu et al. 2013). We are using this RNAi platform to assess the role of various genes both on Leishmania as well as innate response of sand flies.Sand fly midgut microbiota: We are assessing the effects of bacteria on sand fly development, innate response, and Leishmania development. We are also assessing whether selected bacteria can be used to negatively interfere with sand fly fitness and/or behavior (Hurwitz et al. 2011, Hurwitz et al. 2012).We have identified several bacteria present within the sand fly midgut and correlated that to larval food source (Peterkova-Koci et al. 2012). These studies shall continue for the duration of this project (i.e., for the next five years) (Coutinho-Abreu and Ramalho-Ortigao 2010, Coutinho-Abreu et al. 2010, Ramalho-Ortigao and Coutinho-Abreu 2012)investigating changes in the fat body cells following starvation, feeding (sugar and blood), and infection with Dengue viruses (Pascini et al. 2011, Pascini et al. 2012). We are also investigating the basic architecture of organs such as the mosquito heart, which may provide insights into the specific function this organ, contributing to our understanding on how pathogens are able to circulate (or be re-circulated) through the mosquito hemolymph. The approaches used are based on microscopical analyzes and will continue for the next five years (Martins and Ramalho-Ortigão 2012, Martins et al 2011).

Progress 07/02/14 to 12/10/18

Outputs
Target Audience:Fellow scientists, policy makers, veterinarians, food-safety agencies, animal industry, pest control agencies, farmers and ranchers, funding agencies, dog owners, preventive medicine, public health organizations Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Understanding factors that affect Culicoides larval development is critical in suppressing adult midge populations that transmit economically important pathogens to ruminants such as bluetongue virus and epizootic hemorrhagic disease virus. In this study, development of Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae) was investigated in sterilized mud mixed with varying concentrations (3.2, 6.4, 12.6, 25.0, 50.0, and 100.0%) of dairy cattle and white tailed deer manures. In addition, C. sonorensis development was also evaluated in manure (25.0% concentration) of six other farm animals (beef cattle, sheep, goat, pig, horse, and chicken). First instar larvae (~100/ treatment) were added to each substrate, and adult emergence and development times were monitored for 90 d. In substrates with dairy cattle manure, significantly more adults emerged (≥76.7%) and development time was shorter (≤25.5 d) from 25.0% manure substrate than from lower or higher manure concentrations (≤41.3% emerged; ≥31.2 d). Comparatively, white-tailed deer and chicken manures supported C. sonorensis development poorly with low emergence rates (deer ≤ 13.0%; chicken = 0%) and longer development time (deer ≥ 29.0 d). Mud enriched with manure of beef cattle, sheep, goat, pig, and horse generally supported C. sonorensis development, although adult emergence and development times varied widely between species. These results suggest that manure of several farm animals, except for white-tailed deer and chicken, can contribute substantially to C. sonorensis development in the field. Therefore, the potential of animals other than cattle in sustaining local populations of C. sonorensis cannot be overlooked when designing management strategies.

Publications

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:Fellow scientists, policy makers, veterinarians, food-safety agencies, animal industry, pest control agencies, farmers and ranchers, funding agencies, dog owners, preventive medicine, public health organizations Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?Project being terminated early due to Dr. Zurek's resignation.

Impacts
What was accomplished under these goals? Understanding factors that affect Culicoides larval development is critical in suppressing adult midge populations that transmit economically important pathogens to ruminants such as bluetongue virus and epizootic hemorrhagic disease virus. In this study, development of Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae) was investigated in sterilized mud mixed with varying concentrations (3.2, 6.4, 12.6, 25.0, 50.0, and 100.0%) of dairy cattle and white tailed deer manures. In addition, C. sonorensis development was also evaluated in manure (25.0% concentration) of six other farm animals (beef cattle, sheep, goat, pig, horse, and chicken). First instar larvae (~100/ treatment) were added to each substrate, and adult emergence and development times were monitored for 90 d. In substrates with dairy cattle manure, significantly more adults emerged (≥76.7%) and development time was shorter (≤25.5 d) from 25.0% manure substrate than from lower or higher manure concentrations (≤41.3% emerged; ≥31.2 d). Comparatively, white-tailed deer and chicken manures supported C. sonorensis development poorly with low emergence rates (deer ≤ 13.0%; chicken = 0%) and longer development time (deer ≥ 29.0 d). Mud enriched with manure of beef cattle, sheep, goat, pig, and horse generally supported C. sonorensis development, although adult emergence and development times varied widely between species. These results suggest that manure of several farm animals, except for white-tailed deer and chicken, can contribute substantially to C. sonorensis development in the field. Therefore, the potential of animals other than cattle in sustaining local populations of C. sonorensis cannot be overlooked when designing management strategies.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Erram D. and L. Zurek (2018). Larval development of Culicoides sonorensis (Diptera: Ceratopogonidae) in mud supplemented with manure of various farm animals. Journal of Medical Entomology DOI: 10.1093/jme/tjx197.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Louradour I., C.C. Monteiro, E. Inbar, K. Ghosh, R. Merkhofer, P. Lawyer, A. Paun, M. Smelkinson, N. Secundino, M. Lewis, D. Erram, L. Zurek and D. Sacks (2017). The midgut microbiota plays an essential role in sand fly vector competence for Leishmania major. Cellular Microbiology e12755. DOI: 10.1111/cmi.12755.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Kim D., P. Maldonado-Ruiz, L. Zurek, and Y. Park (2017). Water absorption through salivary gland type I acini in the blacklegged tick, Ixodes scapularis. PeerJ 5:e3984. DOI:10.7717/ peerj.3984.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Channaiah LH, S. Bhadriraju, and L. Zurek (2018). Molecular characterization of antibiotic resistant and potentially virulent enterococci isolated from swine farms and feed mills. Journal of Stored Products Research 77:189-196. DOI:10.1016/j.jspr.2018 .04.007

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:Farmers, ranchers, vector biologists, veterinarians, entomologists, ecologists, microbial ecologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?This project was published in a peer reviewd journal: Journal of Medical Entomology. What do you plan to do during the next reporting period to accomplish the goals?We are planning to characterize a new bacterial taxon that we isolated from larvae of C. sonorensis.

Impacts
What was accomplished under these goals? Understanding factors that affect Culicoides larval development is critical in suppressing adult midge populations that transmit economically important pathogens to ruminants such as bluetongue virus and epizootic hemorrhagic disease virus. In this study, development of Culicoides sonorensis Wirth and Jones (Diptera: Ceratopogonidae) was investigated in sterilized mud mixed with varying concentrations (3.2, 6.4, 12.6, 25.0, 50.0, and 100.0%) of dairy cattle and white tailed deer manures. In addition, C. sonorensis development was also evaluated in manure (25.0% concentration) of six other farm animals (beef cattle, sheep, goat, pig, horse, and chicken). First instar larvae (~100/ treatment) were added to each substrate, and adult emergence and development times were monitored for 90 d. In substrates with dairy cattle manure, significantly more adults emerged (≥76.7%) and development time was shorter (≤25.5 d) from 25.0% manure substrate than from lower or higher manure concentrations (≤41.3% emerged; ≥31.2 d). Comparatively, white-tailed deer and chicken manures supported C. sonorensis development poorly with low emergence rates (deer ≤ 13.0%; chicken = 0%) and longer development time (deer ≥ 29.0 d). Mud enriched with manure of beef cattle, sheep, goat, pig, and horse generally supported C. sonorensis development, although adult emergence and development times varied widely between species. These results suggest that manure of several farm animals, except for white-tailed deer and chicken, can contribute substantially to C. sonorensis development in the field. Therefore, the potential of animals other than cattle in sustaining local populations of C. sonorensis cannot be overlooked when designing management strategies.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Erram D. and L. Zurek (2018). Larval development of Culicoides sonorensis (Diptera: Ceratopogonidae) in mud supplemented with manure of various farm animals. Journal of Medical Entomology DOI: 10.1093/jme/tjx197.

Progress 10/01/15 to 09/30/16

Outputs
Target Audience:Farmers, ranchers, vector biologists, veterinarians, entomologists, ecologists, microbial ecologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Nothing Reported How have the results been disseminated to communities of interest?This study was presented at the IV International Conference on Bluetongue and Related Orbiviruses. November 5?7, 2015 ? Rome, Italy. What do you plan to do during the next reporting period to accomplish the goals?We are planning to conduct a new study assessing the biosolid products from different wastewater treatement facilities for their attractivness to house flies and their potential to serve as a habiait for house fly laraval development.

Impacts
What was accomplished under these goals? Although the biting midge Culicoides sonorensis is an important vector of orbiviruses causing significant disease in domestic and wild ruminants in the US, little is known about factors contributing to midge vector competence. In other vectors such as mosquitoes, interactions among the immune response, microbiota, and ingested pathogens within the vector gut directly impact pathogen survival and therefore vectoring potential. We recently described components of the humoral immune response in the reference transcriptome for adult female C. sonorensis and analysed their temporal expression profiles across several dietary states (unfed, blood, or sugar fed). Genes for immune effectors, such as antimicrobial peptides, were in particular highly induced. Since blood feeding also stimulated proliferation and diversification of bacterial populations colonising the gut of female midges, we infer that changes in immune gene expression were a result of fluctuations in gut microbiota. Thus, diet can indirectly (via microbiota) impact gut immune status and therefore should be carefully considered in subsequent studies assessing vector competence in biting midges.

Publications

• Type: Journal Articles Status: Published Year Published: 2015 Citation: Wada-Katsumata A., L. Zurek, G. Nalyanya, W.L. Roelofs, A. Zhang, and C. Schal. (2015). Gut bacteria mediate aggregation in the German cockroach. Proceedings of the National Academy of Sciences 112: 15678-15683
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Nayduch D., D. Erram, M.B. Lee, L. Zurek and C.A. Saski (2015). Impact of the blood meal on humoral immunity and microbiota in the gut of female Culicoides sonorensis. Veterinaria Italiana 51: 385-392.

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Farmers, ranchers, vector biologists, veterinarians, entomologists, ecologists, microbial ecologists Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One Ph.D. student and one undergardauate student were trained under this objective. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals?We plan to make a great progress in nall projects under these objectives.

Impacts
What was accomplished under these goals? Current knowledge of vector ecology and control tactics for North American Culicoides species, and delineate research recommendations aimed to fill knowledge gaps were outlined.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Pfannenstiel R.S., B.A. Mullens, M.G. Ruder, L. Zurek, L.W. Cohnstaedt, and D.Nayduch (2015). Management of North American Culicoides biting midges: Current knowledge and research needs. Vector-Borne and Zoonotic Diseases 15: 374-384.

Progress 07/02/14 to 09/30/14

Outputs
Target Audience: Nothing Reported Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? 5 graduate students, 2 postdoctoral research associates and 5 undergraduet students are working on the projects under these major goals. How have the results been disseminated to communities of interest? Nothing Reported What do you plan to do during the next reporting period to accomplish the goals? We plan to make a great progress in nall projects under these objectives.

Impacts
What was accomplished under these goals? All projects under the major goals have started.

Publications