Entomology Commons^™

The Effects Of Urbanization On Bee Communities Depends On Floral Resource Availability And Bee Functional Traits., Caleb J Wilson, Mary A Jamieson

Entomology Faculty Publications

Wild bees are important pollinators in many ecosystems threatened by anthropogenic disturbance. Urban development can reduce and degrade natural habitat for bees and other pollinators. However, some researchers suggest that cities could also provide refuge for bees, given that agricultural intensification may pose a greater risk. In this study, we surveyed bee communities at 15 farms and gardens across an urban-rural gradient in southeastern Michigan, USA to evaluate the effect of urbanization on bees. We examined how floral resources, bee functional traits, temperature, farm size, and the spatial scale of analysis influence bee response to urbanization. We found that urbanization …

Altering Social Cue Perception Impacts Honey Bee Aggression With Minimal Impacts On Aggression-Related Brain Gene Expression, James W. Harrison, Joseph H. Palmer, Clare C. Rittschof

Entomology Faculty Publications

Gene expression changes resulting from social interactions may give rise to long term behavioral change, or simply reflect the activity of neural circuitry associated with behavioral expression. In honey bees, social cues broadly modulate aggressive behavior and brain gene expression. Previous studies suggest that expression changes are limited to contexts in which social cues give rise to stable, relatively long-term changes in behavior. Here we use a traditional beekeeping approach that inhibits aggression, smoke exposure, to deprive individuals of aggression-inducing olfactory cues and evaluate whether behavioral changes occur in absence of expression variation in a set of four biomarker genes …

Hippodamia Convergens Overwintering Feeding, Nathan Mercer

Lady Beetle Research Data

Impact of food supplements on overwintering Hippodamia convergens energy reserves, survival and spring reproduction.

Development Of Cs-Tpp-Dsrna Nanoparticles To Enhance Rnai Efficiency In The Yellow Fever Mosquito, Aedes Aegypti, Ramesh Kumar Dhandapani, Dhandapani Gurusamy, Jeffrey L. Howell, Subba Reddy Palli

Entomology Faculty Publications

Mosquito-borne diseases are a major threat to human health and are responsible for millions of deaths globally each year. Vector control is one of the most important approaches used in reducing the incidence of these diseases. However, increasing mosquito resistance to chemical insecticides presents challenges to this approach. Therefore, new strategies are necessary to develop the next generation vector control methods. Because of the target specificity of dsRNA, RNAi-based control measures are an attractive alternative to current insecticides used to control disease vectors. In this study, Chitosan (CS) was cross-linked to sodium tripolyphosphate (TPP) to produce nano-sized polyelectrolyte complexes with …

Downregulation Of Orco And 5-Htt Alters Nestmate Discrimination In The Subterranean Termite Ondontotermes Formosanus (Shiraki), Pengdong Sun, Shuxin Yu, Austin Merchant, Chaoliang Lei, Xuguo Zhou, Qiuying Huang

Entomology Faculty Publications

Nestmate discrimination allows social insects to recognize nestmates from non-nestmates using colony-specific chemosensory cues, which typically evoke aggressive behavior toward non-nestmates. Functional analysis of genes associated with nestmate discrimination has been primarily focused on inter-colonial discrimination in Hymenopterans, and parallel studies in termites, however, are grossly lacking. To fill this gap, we investigated the role of two genes, Orco and 5-HTT, associated with chemosensation and neurotransmission respectively, in nestmate discrimination in a highly eusocial subterranean termite, Odontotermes formosanus (Shiraki). We hypothesized that knocking down of these genes will compromise the nestmate recognition and lead to the antagonistic behavior. To …

Geographic Biases In Bee Research Limits Understanding Of Species Distribution And Response To Anthropogenic Disturbance, Mary A. Jamieson, Adrian L. Carper, Caleb J. Wilson, Virginia L. Scott, Jason Gibbs

Entomology Faculty Publications

Habitat loss and degradation due to agricultural intensification and urbanization are key threats facing wild pollinators, especially bees. However, data on the distribution and abundance of most of the world's 20,000+ bee species is lacking, making it difficult to assess the effects of anthropogenic disturbance through time. Moreover, there are geographic biases in the study of bees creating gaps in our understanding of species distributions and regional patterns of diversity. Research efforts are often focused around cities or field stations associated with universities and other research institutions. In this perspectives paper, we provide examples of geographic bias in knowledge regarding …

Bacterially Expressed Dsrna Causes Gene Silencing In Eab, Lynne Rieske-Kinney

Entomology Research Data

RNA interference (RNAi) is a naturally occurring, biological process in which RNA molecules inhibit gene expression or translation by neutralizing target mRNA molecules. Recent advances in our understanding of the RNAi mechanism have allowed its development as an emerging tool to protect against insect pests. The major challenge for efficient pest management using RNAi as a molecular bio-pesticide in the field is development of convenient and efficient methods for production, and practical delivery of stable dsRNAs to the target insect. Our goal is to assess the potential for deploying bacterially produced RNAi as a bio-pesticide against an invasive forest pest, …

Rna Interference And Validation Of Reference Genes For Gene Expression Analyses Using Qpcr In Southern Pine Beetle, Dendroctonus Frontalis, Bethany R. Kyre, Thais B. Rodrigues, Lynne K. Rieske

Entomology Faculty Publications

RNA interference (RNAi) is a highly specific gene-silencing mechanism that can cause rapid insect mortality when essential genes are targeted. RNAi is being developed as a tool for integrated pest management of some crop pests. Here we focus on an aggressive forest pest that kills extensive tracts of pine forests, the southern pine beetle (SPB), Dendroctonus frontalis. We sought to identify reference genes for quantitative real-time PCR (qPCR) and validate RNAi responses in SPB by mortality and gene silencing analysis. Using an adult beetle feeding bioassay for oral ingestion of dsRNA, we measured the expression and demonstrated knockdown of …

Molecular Evolutionary Trends And Feeding Ecology Diversification In The Hemiptera, Anchored By The Milkweed Bug Genome, Kristen A. Panfilio, Iris M. Vargas Jentzsch, Joshua B. Benoit, Deniz Erezyilmaz, Yuichiro Suzuki, Stefano Colella, Hugh M. Robertson, Monica F. Poelchau, Robert M. Waterhouse, Panagiotis Ioannidis, Matthew T. Weirauch, Daniel S. T. Hughes, Shwetha C. Murali, John H. Werren, Chris G. C. Jacobs, Elizabeth J. Duncan, David Armisén, Barbara M. I. Vreede, Patrice Baa-Puyoulet, Chloé S. Berger, Chun-Che Chang, Hsu Chao, Mei-Ju M. Chen, Yen-Ta Chen, Christopher P. Childers, Ariel D. Chipman, Andrew G. Cridge, Antonin J. J. Crumière, Peter K. Dearden, Elise M. Didion, Subba Reddy Palli, Jayendra Nath Shukla

Entomology Faculty Publications

Background: The Hemiptera (aphids, cicadas, and true bugs) are a key insect order, with high diversity for feeding ecology and excellent experimental tractability for molecular genetics. Building upon recent sequencing of hemipteran pests such as phloem-feeding aphids and blood-feeding bed bugs, we present the genome sequence and comparative analyses centered on the milkweed bug Oncopeltus fasciatus, a seed feeder of the family Lygaeidae.

Results: The 926-Mb Oncopeltus genome is well represented by the current assembly and official gene set. We use our genomic and RNA-seq data not only to characterize the protein-coding gene repertoire and perform isoform-specific RNAi, but …

Photocatalytic Degradation Of Profenofos And Triazophos Residues In The Chinese Cabbage, Brassica Chinensis, Using Ce-Doped Tio2, Xiangying Liu, You Zhan, Zhongqin Zhang, Lang Pan, Lifeng Hui, Kailin Liu, Xuguo Zhou, Lianyang Bai

Entomology Faculty Publications

Pesticides have revolutionized the modern day of agriculture and substantially reduced crop losses. Synthetic pesticides pose a potential risk to the ecosystem and to the non-target organisms due to their persistency and bioaccumulation in the environment. In recent years, a light-mediated advanced oxidation processes (AOPs) has been adopted to resolve pesticide residue issues in the field. Among the current available semiconductors, titanium dioxide (TiO₂) is one of the most promising photocatalysts. In this study, we investigated the photocatalytic degradation of profenofos and triazophos residues in Chinese cabbage, Brassica chinensis, using a Cerium-doped nano semiconductor TiO₂ (TiO …

A Determining Factor For Insect Feeding Preference In The Silkworm, Bombyx Mori, Zhong-Jie Zhang, Shuai-Shuai Zhang, Bao-Long Niu, Dong-Feng Ji, Xiao-Jing Liu, Mu-Wang Li, Hua Bai, Subba Reddy Palli, Chen-Zhu Wang, An-Jiang Tan

Entomology Faculty Publications

Feeding preference is critical for insect adaptation and survival. However, little is known regarding the determination of insect feeding preference, and the genetic basis is poorly understood. As a model lepidopteran insect with economic importance, the domesticated silkworm, Bombyx mori, is a well-known monophagous insect that predominantly feeds on fresh mulberry leaves. This species-specific feeding preference provides an excellent model for investigation of host-plant selection of insects, although the molecular mechanism underlying this phenomenon remains unknown. Here, we describe the gene GR66, which encodes a putative bitter gustatory receptor (GR) that is responsible for the mulberry-specific feeding preference …

Synopsis Of New World Sigalphinae (Hymenoptera, Braconidae) With The Description Of Two New Species And A Key To Genera, Michael J. Sharkey, Angélica Maria Penteado-Dias, M. Alex Smith, Winnie Hallwachs, Daniel Janzen

Entomology Faculty Publications

We describe and illustrate Paphanus paloi sp. n., first generic record for Brazil, and Minanga patriciamadrigalae, first generic record for Costa Rica. We present illustrated keys for the New World genera of Sigalphinae, and the New World species of Paphanus and Minanga. Minanga patriciamadrigalae sp. n. was reared from caterpillars of Chloropteryx nordicariaDHJ01 (Geometridae).

Predation On Stink Bugs (Hemiptera: Pentatomidae) In Cotton And Soybean Agroecosystems [Predator Gut Contents], Kacie J. Athey, John R. Ruberson, Dawn Olson, James D. Harwood

Entomology Research Data

Stink bugs (Hemiptera: Pentatomidae) are significant pests of cotton and soybeans in the southeastern United States with annual control costs exceeding $14 million in these crops. Three of the most prominent stink bug pests are the southern green (Nezara viridula), brown (Euschistus servus) and green (Chinavia hilaris) stink bugs. To determine trophic linkages between generalist arthropod predators and these pests, species-specific 16S molecular markers were designed and used to detect the presence of prey DNA in predator gut-contents. Over 2700 predators were collected over two growing seasons in cotton and soybean in southern Georgia …

Predation On Stink Bugs (Hemiptera: Pentatomidae) In Cotton And Soybean Agroecosystems [Predator Capture Numbers], Kacie J. Athey, John R. Ruberson, Dawn Olson, James D. Harwood

Entomology Research Data

Stink bugs (Hemiptera: Pentatomidae) are significant pests of cotton and soybeans in the southeastern United States with annual control costs exceeding $14 million in these crops. Three of the most prominent stink bug pests are the southern green (Nezara viridula), brown (Euschistus servus) and green (Chinavia hilaris) stink bugs. To determine trophic linkages between generalist arthropod predators and these pests, species-specific 16S molecular markers were designed and used to detect the presence of prey DNA in predator gut-contents. Over 2700 predators were collected over two growing seasons in cotton and soybean in southern Georgia …

Rna Sequencing, Selection Of Reference Genes And Demonstration Of Feeding Rnai In Thrips Tabaci (Lind.) (Thysanoptera: Thripidae), Satnam Singh, Mridula Gupta, Suneet Pandher, Gurmeet Kaur, Neha Goel, Pankaj Rathore, Subba Reddy Palli

Entomology Faculty Publications

Background: Thrips tabaci is a severe pest of onion and cotton. Due to lack of information on its genome or transcriptome, not much is known about this insect at the molecular level. To initiate molecular studies in this insect, RNA was sequenced; de novo transcriptome assembly and analysis were performed. The RNAseq data was used to identify reference and RNAi pathway genes in this insect. Additionally, feeding RNAi was demonstrated in T. tabaci for the first time.

Results: From the assembled transcriptome, 27,836 coding sequence (CDS) with an average size of 1236 bp per CDS were identified. About 85.4% of …

Trade-Offs Shape Carotenoid-Based Color Variation In Redheaded Pine Sawfly (Neodiprion Lecontei) Larvae, Maranda Gaines

Lewis Honors College Capstone Collection

Carotenoids serve various ecological roles in animals including coloration, immune responses, and vision. Carotenoid-derived coloration is greatly emphasized in the literature, particularly relating to mate choice and aposematic warning. However, the trade-offs between the color and non-color functions of carotenoids are not thoroughly explored. In the redheaded pine sawfly (Neodiprion lecontei), some larval populations have yellow pigmentation, using carotenoids derived from their diets for aposematic warning coloration. Other larval populations are white in color, having genetically lost the ability to produce the yellow pigment. Because carotenoids are essential to life functions in both the yellow and white populations, we aim …

Biology And Management Of The Soybean Stem Borer, Dectes Texanus Leconte, In Kentucky, Izabela Gomes

Theses and Dissertations--Entomology

Dectes texanus LeConte (Coleoptera: Cerambycidae) is a longhorn beetle species endemic to eastern United States. Originally described as a pest of weeds from the family Asteraceae, D. texanus has expanded its host range and is found infesting the stems of soybeans, Glycine max (L.) through the southwestern and middle United States. Female D. texanus chews a hole in the epidermis of a petiole and oviposits on it. Then, the D. texanus larva depletes all the pith of the stem making a tunnel down to the base of the plant and girdle the stem about 5 cm above the …

Nanomaterials For Double-Stranded Rna Delivery, Stuart Lichtenberg

Theses and Dissertations--Plant and Soil Sciences

RNA interference has enormous potential as a potent, specific, and environmentally friendly alternative to small molecule pesticides for crop protection. The use of exogenous double-stranded RNA offers flexibility in targeting and use in crops in which transgenic manipulation is not an option. The combination of RNAi with nanotechnology offers further advantages that are not available with dsRNA alone. In this work, I have evaluated several different combinations of nanomaterials and polymers for use in RNAi-based pest control systems. First, I have characterized the use of chitosan/dsRNA polyplex nanoparticles for gene knockdown using the model nematode Caenorhabditis elegans. Though chitosan/dsRNA …

Quantifying Bee Assemblages And Attractiveness Of Flowering Woody Landscape Plants For Urban Pollinator Conservation, Bernadette M. Mach, Daniel A. Potter

Entomology Faculty Publications

Urban and suburban landscapes can be refuges for biodiversity of bees and other pollinators. Public awareness of declining pollinator populations has increased interest in growing plants that provide floral resources for bees. Various publications and websites list “bee-friendly” plants, but such lists are rarely based on empirical data, nor do they emphasize flowering trees and shrubs, which are a major component of urban landscapes. We quantified bee visitation to 72 species of flowering woody landscape plants across 373 urban and suburban sites in Kentucky and southern Ohio, USA, sampling and identifying the bee assemblages associated with 45 of the most …

Epigenetic Modifications Acetylation And Deacetylation Play Important Roles In Juvenile Hormone Action, Amit Roy, Subba Reddy Palli

Entomology Faculty Publications

Background: Epigenetic modifications including DNA methylation and post-translational modifications of histones are known to regulate gene expression. Antagonistic activities of histone acetyltransferases (HATs) and histone deacetylases (HDACs) mediate transcriptional reprogramming during insect development as shown in Drosophila melanogaster and other insects. Juvenile hormones (JH) play vital roles in the regulation of growth, development, metamorphosis, reproduction and other physiological processes. However, our current understanding of epigenetic regulation of JH action is still limited. Hence, we studied the role of CREB binding protein (CBP, contains HAT domain) and Trichostatin A (TSA, HDAC inhibitor) on JH action.

Results: Exposure of Tribolium castaneum cells …

Patterns Of Authorship In Ecology And Evolution: First, Last, And Corresponding Authorship Vary With Gender And Geography, Charles W. Fox, Josiah P. Ritchey, C. E. Timothy Paine

Entomology Faculty Publications

The position of an author on the byline of a paper affects the inferences readers make about their contributions to the research. We examine gender differences in authorship in the ecology literature using two datasets: submissions to six journals between 2010 and 2015 (regardless of whether they were accepted), and manuscripts published by 151 journals between 2009 and 2015. Women were less likely to be last (i.e., “senior”) authors (averaging ~23% across journals, years, and datasets) and sole authors (~24%), but more likely to be first author (~38%), relative to their overall frequency of authorship (~31%). However, the proportion of …

Development Of A Diagnostic Marker For Phlebotomus Papatasi To Initiate A Potential Vector Surveillance Program In North America, Austin Merchant, Tian Yu, Jizhe Shi, Xuguo Zhou

Entomology Faculty Publications

Phlebotomus papatasi, an Old World sand fly species, is primarily responsible for the transmission of leishmaniasis, a highly infectious and potentially lethal disease. International travel, especially military rotations, between domestic locations and P. papatasi-prevalent regions in the Middle East poses an imminent threat to the public health of US citizens. Because of its small size and cryptic morphology, identification of P. papatasi is challenging and labor-intensive. Here, we developed a ribosomal DNA-polymerase chain reaction (PCR)-based diagnostic assay that is capable of detecting P. papatasi genomic DNA from mixed samples containing multiple sand flies native to the Americas. Serial …

Continental-Scale Suppression Of An Invasive Pest By A Host-Specific Parasitoid Underlines Both Environmental And Economic Benefits Of Arthropod Biological Control, Kris A. G. Wyckhuys, Prapit Wongtiem, Aunu Rauf, Anchana Thancharoen, George E. Heimpel, Nhung T. T. Le, Muhammad Zainal Fanani, Geoff M. Gurr, Jonathan G. Lundgren, Dharani D. Burra, Leo K. Palao, Glenn Hyman, Ignazio Graziosi, Vi X. Le, Matthew J. W. Cock, Teja Tscharntke, Steve D. Wratten, Liem V. Nguyen, Minsheng You, Yanhui Lu, Johannes W Ketelaar, Georg Goergen, Peter Neuenschwander

Entomology Faculty Publications

Biological control, a globally-important ecosystem service, can provide long-term and broad-scale suppression of invasive pests, weeds and pathogens in natural, urban and agricultural environments. Following (few) historic cases that led to sizeable environmental up-sets, the discipline of arthropod biological control has—over the past decades—evolved and matured. Now, by deliberately taking into account the ecological risks associated with the planned introduction of insect natural enemies, immense environmental and societal benefits can be gained. In this study, we document and analyze a successful case of biological control against the cassava mealybug, Phenacoccus manihoti (Hemiptera: Pseudococcidae) which invaded Southeast Asia in 2008, where …

Identification Of Target Genes For Rnai-Mediated Control Of The Twospotted Spider Mite, June-Sun Yoon, Dipak K. Sahoo, Indu B. Maiti, Subba Reddy Palli

Entomology Faculty Publications

RNA interference (RNAi) is being developed for the management of pests that destroy crops. The twospotted Spider Mite (TSSM), Tetranychus urticae is a worldwide pest due to its unique physiological and behavioral characteristics including extraordinary ability to detoxify a wide range of pesticides and feed on many host plants. In this study, we conducted experiments to identify target genes that could be used for the development of RNAi-based methods to control TSSM. Leaf disc feeding assays revealed that knockdown in the expression genes coding for proteins involved in the biosynthesis and action of juvenile hormone (JH) and action of ecdysteroids …

Better Than Deet Repellent Compounds Derived From Coconut Oil, Junwei J. Zhu, Steven C. Cermak, James A. Kenar, Gary Brewer, Kenneth F. Haynes, Dave Boxler, Paul D. Baker, Desen Wang, Changlu Wang, Andrew Y. Li, Rui-De Xue, Yuan Shen, Fei Wang, Natasha M. Agramonte, Ulrich R. Bernier, Jaires G. De Oliveira Filho, Ligia M. F. Borges, Kristina Friesen, David B. Taylor

Entomology Faculty Publications

Hematophagous arthropods are capable of transmitting human and animal pathogens worldwide. Vector-borne diseases account for 17% of all infectious diseases resulting in 700,000 human deaths annually. Repellents are a primary tool for reducing the impact of biting arthropods on humans and animals. N,N-Diethyl-meta-toluamide (DEET), the most effective and long-lasting repellent currently available commercially, has long been considered the gold standard in insect repellents, but with reported human health issues, particularly for infants and pregnant women. In the present study, we report fatty acids derived from coconut oil which are novel, inexpensive and highly efficacious repellant compounds. These coconut …

Transcriptional Response Of Honey Bee (Apis Mellifera) To Differential Nutritional Status And Nosema Infection, Farida Azzouz-Olden, Arthur G. Hunt, Gloria Degrandi-Hoffman

Plant and Soil Sciences Faculty Publications

Background: Bees are confronting several environmental challenges, including the intermingled effects of malnutrition and disease. Intuitively, pollen is the healthiest nutritional choice, however, commercial substitutes, such as Bee-Pro and MegaBee, are widely used. Herein we examined how feeding natural and artificial diets shapes transcription in the abdomen of the honey bee, and how transcription shifts in combination with Nosema parasitism.

Results: Gene ontology enrichment revealed that, compared with poor diet (carbohydrates [C]), bees fed pollen (P > C), Bee-Pro (B > C), and MegaBee (M > C) showed a broad upregulation of metabolic processes, especially lipids; however, pollen feeding promoted more functions, and …

Japanese Beetles’ Feeding On Milkweed Flowers May Compromise Efforts To Restore Monarch Butterfly Habitat, Adam M. Baker, Daniel A. Potter

Entomology Faculty Publications

The eastern North American migratory population of monarch butterflies (Danaus plexippus) is in serious decline. Habitat restoration, including adding millions of host plants to compensate for loss of milkweed in US cropland, is a key part of the international conservation strategy to return this iconic butterfly to sustainable status. We report here that Popillia japonica, a polyphagous, invasive beetle, aggregates and feeds on flowers of Asclepias syriaca, the monarch’s most important larval food plant, reducing fruiting and seed set by >90% and extensively damaging milkweed umbels in the field. The beetle’s ongoing incursion into the monarch’s …

Double-Stranded Rna Binding Protein, Staufen, Is Required For The Initiation Of Rnai In Coleopteran Insects, June-Sun Yoon, Kanakachari Mogilicherla, Dhandapani Gurusamy, Xien Chen, Shankar C. R. R. Chereddy, Subba R. Palli

Entomology Faculty Publications

RNA interference (RNAi) is being used to develop methods to control pests and disease vectors. RNAi is robust and systemic in coleopteran insects but is quite variable in other insects. The determinants of efficient RNAi in coleopterans, as well as its potential mechanisms of resistance, are not known. RNAi screen identified a double-stranded RNA binding protein (StaufenC) as a major player in RNAi. StaufenC homologs have been identified in only coleopteran insects. Experiments in two coleopteran insects, Leptinotarsa decemlineata and Tribolium castaneum, showed the requirement of StaufenC for RNAi, especially for processing of double-stranded RNA (dsRNA) to small interfering …

Revision Of Zosteragathis Sharkey Of Thailand (Hymonoptera, Braconidae, Agathidinae, Agathidini), Michael J. Sharkey, Eric G. Chapman

Entomology Faculty Publications

Based on cladistic analyses recently conducted by Sharkey and Chapman, the genus Zosteragathis Sharkey is revised. Twenty-two species are reported from Thailand, three previously described species, Z. samensis, Z. contrasta and Z. nuichuaensis, and 19 new species, i.e., Z. chaiyaphumensis, Z. eukos, Z. hinensis, Z. hongensis, Z. inthanonensis, Z. krachanensis, Z. lampangensis, Z. lampooensis, Z. luangensis, Z. ngamensis, Z. perknos, Z. petchaburiensis, Z. phahompokensis, Z. phuphanensis, Z. sakaeratensis, Z. sakonensis, Z. samensis, Z. surinensis, Z. taemensis, Z. tonensis. …

Quantifying Plant Soluble Protein And Digestible Carbohydrate Content, Using Corn (Zea Mays) As An Exemplar, Carrie A. Deans, Gregory A. Sword, Paul A. Lenhart, Eric Burkness, William D. Hutchison, Spencer T. Behmer

Entomology Faculty Publications

Elemental data are commonly used to infer plant quality as a resource to herbivores. However, the ubiquity of carbon in biomolecules, the presence of nitrogen-containing plant defensive compounds, and variation in species-specific correlations between nitrogen and plant protein content all limit the accuracy of these inferences. Additionally, research focused on plant and/or herbivore physiology require a level of accuracy that is not achieved using generalized correlations. The methods presented here offer researchers a clear and rapid protocol for directly measuring plant soluble proteins and digestible carbohydrates, the two plant macronutrients most closely tied to animal physiological performance. The protocols combine …