Name: a versatile model of hard tick infestation on laboratory rabbits
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Description: Watch this Scientific Journal Video about A Versatile Model of Hard Tick Infestation on Laboratory Rabbits at JoVE.com

We acknowledge the technical assistance of Evelyne Le Naour, French National Institute of Agricultural Research (INRA), Alain Bernier (INRA) and Oc&#233;ane Le Bidel (ANSES). Consuelo Almaz&#225;n was supported by a fellowship from the Laboratory of Excellence, Integrative Biology of Emerging Infectious Diseases (LabEx IBEID), Pasteur Institute. The rabbits and sheep were purchased by ANSES. Part of this work was funded by National Institutes of Health grant RO1AI090062 to Y. Park. Dr. Jeffrey L. Blair is acknowledged for reviewing the manuscript.

NOTE: In this study, rabbits were maintained in standard cages with food and water offered ad libitum at the French Agency for Food, Environmental and Occupational Health &#38; Safety (ANSES) accredited animal facilities in Maisons-Alfort, France. Animals were monitored twice daily by two experienced technicians for any abnormal skin reactions, health problems or complications. The experimental room was secured by framing the interior of the doorway with double-sided tape to avoid accidental escape of ticks. The...

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The most important step in this entire protocol is to glue the capsule firmly to the shaved skin. For this reason, constant pressure for at least 3 minutes is critical, especially at the corners. When inoculating the ticks into the capsule, it is important to apply them deep into the opposite corner from the open one to avoid tick escape during the sealing. When planning the experiments, make sure that all the capsules are covered by the jacket to avoid damage by chewing or scratching. Make sure that the neck region of t...

The hard ticks (Ixodidae) are well known as slow feeding arthropods and can be attached on a host for several days, or weeks, depending on the species and developmental stage1. These obligatory hematophagous arthropods are vectors of a wide variety of infectious agents, such as bacteria, protozoa, and viruses, and thus present a significant risk to humans and animal health1. When studying tick biology or evaluating new control methods, the establishment of an effective tick feeding system is crucial in order to effectively design the experiments and accomplish the goal(s) of the study. Recently, several artificial tick feeding systems (avoiding the use of live animals) have been developed2,3,4 and they should be used whenever possible. However, these systems have not been able to completely replace tick feeding on live animals, and they are not suitable substitutes for many physiologic conditions required for scientific studies. Therefore, in some cases, the use of experimental animal hosts is crucial to guarantee the relevance of experimental results.
Laboratory New Zealand rabbits have shown to be the most suitable and accessible hosts for several ixodid tick species5,6,7,8,9. Two common strategies of tick feeding on rabbits have been frequently used: a) feeding on rabbit ears covered with cotton cloth or socks6,7, and b) feeding in cotton bags9, nylon bottles10 or neoprene chambers11&#160;glued to the rabbit&#39;s back. The feeding on rabbit&#39;s ears is not an elegant system, because ticks (especially early stages, larvae or nymphs) may crawl and attach deep in the ear canal, which is uncomfortable for the animal and makes the monitoring of tick feeding and/or the recovery of engorged ticks difficult. This system is also limited to only two tick groups on ears fully covered by socks protected by Elizabethan collars,representing a significant discomfort for the animal. Other systems9,10,11 are definitely more advanced and well suited for hard tick colony maintenance. However, they are limited in the number of experimental groups placed on the rabbit as well as in the modifiable sizes/shapes of the feeding chambers. In addition, these protocols often require hobbling the rear rabbit legs to avoid scratching and the use of Elizabethan collars to prevent grooming.
Herein we propose a simple, non-laborious and very effective method to feed multiple groups of hard ticks in closed chambers glued to the rabbit back covered by a jacket, eliminating the need for Elizabethan collars or hobbling during the experiment. Specifically, our system uses elastic capsules made from an ethylene-vinyl acetate (EVA) foam sheet covered by mosquito mesh and glued to the shaved rabbit back with fast solidifying (3 min) non-irritating latex glue. This technique allows the attachment of multiple capsules of desired size or shape, and few weeks after the experiment the rabbits are fully recovered. The system is suitable mainly for the nymphal and adult hard tick stages, but with a little modification it can be used for larvae feeding as well. The EVA foam-based methods for hard tick feeding may be adapted to other types of vertebrate hosts, for example sheep (which is shown as one of the alternatives in this paper).

<table border="0" cellpadding="0" cellspacing="0" style="width:1157px;" width="1158">
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		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">New Zealand Rabbits (2.5-3.5 kg)</td>
			<td style="width:197px;">Charles River&#160;</td>
			<td style="width:399px;">Strain Code 571</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Rambouillet sheep</td>
			<td style="width:197px;">Local provider-tick free farm</td>
			<td>Female 6 months old</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">EVA foam 5 mm thick&#160;</td>
			<td style="width:197px;">Cosplay Shop</td>
			<td style="width:399px;">EVA-PE451kg (950mm x 450mm)</td>
			<td>10 mm PE45kg foam from the Cosplay Shop may be used for the large adult tick species</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Foam Sheet 9&#34; X 12&#34; 6 mm-White</td>
			<td style="width:197px;">Amazon</td>
			<td style="width:399px;">FOAMSHT6-20</td>
			<td>6 mm-EVA foam ca be ordered via Amazon as an alternative to the foam from Cosplay Shop</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Full length rabbit jackets&#160;</td>
			<td style="width:197px;">Harvard Apparatus, Inc.&#160;</td>
			<td style="width:399px;">620077- medium, 6270078 - large&#160;</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Non-toxic latex glue&#160;</td>
			<td style="width:197px;">Tear mender&#160;</td>
			<td style="width:399px;">Fabric &#38; Leather Adhesive</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Tubular cotton orthopedic stockinette</td>
			<td style="width:197px;">BSN Medical</td>
			<td style="width:399px;">9076 (12-15 cm wide)</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Mosquito mesh&#160;</td>
			<td style="width:197px;">Loisirs Creatifs</td>
			<td style="width:399px;">Very fine filter nylon mesh fabric</td>
			<td>Any mosquito mesh, or curtain material with the mesh size less than 50 microns is suitable.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:295px;">Leukoplast&#160;</td>
			<td style="width:197px;">BSN medical S.A.S</td>
			<td style="width:399px;">LF 72361-02</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:295px;">Adhesive hook-and-loop tape</td>
			<td style="width:197px;">AIEX store</td>
			<td style="width:399px;">AIEX 39.37 Feet/12m Hook and Loop Self Adhesive Tape Roll, 20 mm width, white colour</td>
			<td>Fullfiled by Amazon</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fast drying glue &#160;</td>
			<td>Fixtout&#160;</td>
			<td>Superglue</td>
			<td></td>
		</tr>
	</tbody>
</table>

a versatile model of hard tick infestation on laboratory rabbits

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the laboratory. In ticks, all developmental stages (except egg) are hematophagous, and acquiring a blood-meal when attached to their vertebrate hosts is essential for the successful completion of their life cycle. Here we demonstrate a simple method that uses easily openable capsules for feeding of hard ticks on rabbits. The advantages of the proposed method include its simplicity, short duration and most importantly versatile adjustment to the needs of specific experimental requirements. The method makes possible the use of multiple chambers (of various sizes) on the same animal, which permits feeding of multiple stages or different experimental groups while reducing the overall animal requirement. The non-irritating and easily accessible materials used minimizes discomfort to the animals, which can be easily recovered from an experiment and offered for adoption or reused if the ethical protocol allows it.

Here we propose for the first time a detailed step-by step method of hard tick feeding in EVA foam capsules applied to a shaved rabbit&#39;s back, covered by a jacket (Figure&#160;1 and&#160;Figure 2). This protocol is suitable for various types of experiments when different tick groups on the same host are required and can be also used for mass rearing of hard ticks. The tick feeding success in the laboratory mo...

Watch this Scientific Journal Video about A Versatile Model of Hard Tick Infestation on Laboratory Rabbits at JoVE.com

A Versatile Model of Hard Tick Infestation on Laboratory Rabbits

We have developed a simple and versatile system to feed hard ticks on laboratory rabbits. Our non-laborious protocol uses easily accessible materials and can be adjusted depending on the requirements of the various experimental settings. The method allows comfortable monitoring and/or sampling of ticks during the entire feeding period.

The use of live animals in tick research is crucial for a variety of experimental purposes including the maintenance of hard tick colonies in the ...

This method is suitable for maintaining hard colonies, studying tick biology or host-vector pathogen interactions or evaluating different control measures of these medically important attributes. The main advantages of these techniques are its simplicity and short duration. and most importantly ability to be modified, according to the experimental needs of the study.Although this method can be used for feeding the different life stages of hard ticks on laboratory rabbits. It can also be adapted to other host systems such as sheep. Begin by cutting an appropriately-sized capsule frame from a sheet of EVA Foam rounding the outer corners to minimize accidental detachment during gluing.Cut four eight millimeter wide strips of self-adhesive hook tape and attach the strips to the sides of the frame. Cut the same size strips from the self-adhesive loop tape and bind them to the hook tape on the frame. Next, cut a piece of pine mosquito mesh big enough to cover the frame and stick the mesh to the self-adhesive loop strips, trimming any overhanging material as necessary.When the capsule is ready, manually restrain the rabbit and shave the experimental regions on the back and sides of the animal. Apply fast-drying non-irritating latex glue to the entire surface of the prepared capsule and after one minute, gently pressed the capsule to the skin. The most critical step of the protocol is the field attachment of the capsule to the skin.Therefore, constant pressure of the capsule onto the rabbit skin especially at the corners for at least three minutes is required. After three minutes, slightly lift the capsule to visually confirm its attachment using a spatula to apply glue to any non-attached parts. When the capsule is secured, apply protective tape to the rear paws to prevent jacket damage and place the front legs through the front jacket openings tightening the neck of the jacket to one finger width between the animal and the jacket.To initiate the tick infestation, first add the ticks into the plunger end of a plastic cotton plug to syringe with a cut-off tip. Next, lift the mesh from one corner of the capsule and place the syringe deep into the capsule through the opening. Insert the plunger all the way into the syringe to infest the animal with the ticks.Then slowly twist the plunger toward the skin to remove any remaining ticks while simultaneously pulling the plunger out of the capsule. When all the ticks have been delivered, refasten the hook and loop tape. Close the jacket by zipping the backside of it and insert the rear legs of the rabbit into the rear elastic enclosures.Then return the rabbit to its cage. At the appropriate experimental time point, transfer the rabbit to the bench and unzip the jacket. Gently restraining the rabbit, open the capsule and use forceps to collect the fully engorged adults and a tick twister to collect the partially fed adults.Engorged larvae and nymphs can be collected by brushing them into the dish. Then re-close the capsule if experimentally appropriate. At the end of the experiment, remove the mosquito mesh completely from the capsule without removing the jacket.Three to four weeks later, gently trim one of the corners to try to remove the capsule. Once the capsule has been removed, remove the jacket and allow the rabbit to recover in its cage. If the protocol and experiments allow, the recovered rabbit can be reused or offered for adoption.This protocol is suitable for various types of experiments including mass rearing of multiple tick groups on the same host. The EVA Foam capsule system has been proven to be highly successful when feeding different developmental stages of various hard tick species. It may also be adapted for other type of laboratory hosts such as sheep.In addition, this versatile method facilitates the creation of a variety of different experimental settings based on the modifiable number, shape and composition of the capsules on the host. The procedure also allows a complete recovery of the rabbit hosts after the experiments. This protocol is not laborious and no intensive training is required to become familiar with the technique.In addition, a minimal amount of materials, steps and stress to the experimental animals is required.

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Laser-Induced Chronic Ocular Hypertension Model on SD Rats

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular hypertension is one of the well established animal models. This video demonstrates how to induce ocular hypertension by Argon laser photocoagulation in rat.

Glaucoma is one of the major causes of blindness in the world. Elevated intraocular pressure is a major risk factor. Laser photocoagulation induced ocular ...

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System 

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers. Genetic studies in mammalian systems have been limited due to the lack of readily available tools including defined mutant genetic cell lines, regulatory expression systems, and appropriate selectable markers. To circumvent these difficulties, model genetic systems in lower eukaryotes have become an attractive choice for the study of functionally conserved DNA repair proteins and pathways. We have developed a model yeast system to study the poorly defined genetic functions of the Werner syndrome helicase-nuclease (WRN) in nucleic acid metabolism. Cellular phenotypes associated with defined genetic mutant backgrounds can be investigated to clarify the cellular and molecular functions of WRN through its catalytic activities and protein interactions. The human WRN gene and associated variants, cloned into DNA plasmids for expression in yeast, can be placed under the control of a regulatory plasmid element. The expression construct can then be transformed into the appropriate yeast mutant background, and genetic function assayed by a variety of methodologies. Using this approach, we determined that WRN, like its related RecQ family members BLM and Sgs1, operates in a Top3-dependent pathway that is likely to be important for genomic stability. This is described in our recent publication [1] at <a href="http://www.impactaging.com">www.impactaging.com</a>. Detailed methods of specific assays for genetic complementation studies in yeast are provided in this paper.

Genetic Studies of Human DNA Repair Proteins Using Yeast as a Model System

Genetic studies in yeast can be employed to investigate the molecular and cellular functions of human genes in cellular DNA metabolism. Methods are described for the genetic characterization of the human WRN gene product defective in the premature aging disorder Werner syndrome in functionally conserved pathways using yeast as a tractable model system.

Understanding the roles of human DNA repair proteins in genetic pathways is a formidable challenge to many researchers.  Genetic studies in mammalian ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Endotracheal Intubation of Rabbits Using a Polypropylene Guide Catheter

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the avoidance of airway obstruction, prevention of gastric tympany, and to allow ventilatory support. Due to the difficulty of intubation, alternative methods such as the use of laryngeal mask airways or laryngeal tubes have been explored. However, these methods do not result in direct access to the trachea and thus may present a risk for development of complications. In addition, lack of direct intubation of the trachea can result in personnel exposure to waste anesthetic gases. Numerous methods for endotracheal intubation have been described, including blind placement, use of a fiberoptic laryngoscope or endoscope, and cricoid placement. Despite these numerous publications, many still struggle to achieve success. Here we provide a detailed description of an intubation technique that can be taught with minimal training with a short time to proficiency. Briefly, after administration of injectable anesthesia and proper positioning of the rabbit, a polypropylene catheter is placed into the trachea by direct visualization using a laryngoscope. The catheter is then used as a guide to direct the endotracheal tube into the trachea. This method allows for intubation without the need for expensive equipment and can be performed by a single individual without the need for an assistant. In conclusion, this technique can be easily taught and performed at very little cost in any clinical or research setting.

Endotracheal intubation in rabbits is challenging due to their unusual anatomy. Here we present a technique for direct intubation of the trachea using a polypropylene catheter as a guide. This method utilizes relatively inexpensive supplies, requires minimal training, and can be easily performed in any clinical setting.

Endotracheal intubation in rabbits can be challenging due to their unusual anatomy. Achieving a patent airway during anesthesia is critical for the ...

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The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is regulated spatially and temporally. The use of a fluorescent gene under the control of an immunity-related promoter in combination with an automated fluorescence microscopy is a simple way to understand spatiotemporal regulation of plant immunity. In contrast to the root tissues that have been used for a number of various intravital fluorescent imaging experiments, there exist few fluorescent live-imaging examples for the leaf tissues that encounter an array of airborne microbial infections. Therefore, we developed a simple method to mount leaves of Arabidopsis thaliana plants for live-cell imaging over an extended period of time. We used transgenic Arabidopsis plants expressing the yellow fluorescent protein (YFP) genefused to the nuclear localization signal (NLS) under the control of the promoter of a defense-related marker gene, Pathogenesis-Related 1 (PR1). We infiltrated a transgenic leaf with Pseudomonas syringae pv. tomato DC3000 (avrRpt2) strain (Pst_a2) and performed in vivo time-lapse imaging of the YFP signal for a total of 40 h using an automated fluorescence stereomicroscope. This method can be utilized not only for studies on plant immune responses but also for analyses of various developmental events and environmental responses occurring in leaf tissues.

A Versatile Method for Mounting Arabidopsis Leaves for Intravital Time-lapse Imaging

We report a simple and versatile method for performing fluorescent live-imaging of Arabidopsis thaliana leaves over an extended period of time. We use a transgenic Arabidopsis plant expressing a fluorescent reporter gene under the control of an immunity-related promoter as an example for gaining spatiotemporal understanding of plant immune responses.

The plant immune response associated with a genome-wide transcriptional reprogramming is initiated at the site of infection. Thus, the immune response is ...

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A)

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer progression. How the chromosomal location and function of a centromere (i.e., centromere identity) are determined and participate in accurate chromosome segregation is a fundamental question. CENP-A is proposed to be the non-DNA indicator (epigenetic mark) of centromere identity, and CENP-A ubiquitylation is required for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability.
Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-CENP-A K124R mutant suggesting that ubiquitylation at a different lysine is induced because of the EYFP tagging in the CENP-A K124R mutant protein. Lysine 306 (K306) ubiquitylation in EYFP-CENP-A K124R was successfully identified, which corresponds to lysine 56 (K56) in CENP-A through mass spectrometry analysis. A caveat is discussed in the use of GFP/EYFP or the tagging of high molecular weight protein as a tool to analyze the function of a protein. Current technical limit is also discussed for the detection of ubiquitylated bands, identification of site-specific ubiquitylation(s), and visualization of ubiquitylation in living cells or a specific single cell during the whole cell cycle.
The method of mass spectrometry analysis presented here can be applied to human CENP-A protein with different tags and other centromere-kinetochore proteins. These combinatory methods consisting of several assays/analyses could be recommended for researchers who are interested in identifying functional roles of ubiquitylation.

Mass Spectrometry Analysis to Identify Ubiquitylation of EYFP-tagged CENP-A EYFP-CENP-A

CENP-A ubiquitylation is an important requirement for CENP-A deposition at the centromere, inherited through dimerization between cell division, and indispensable to cell viability. Here we describe mass spectrometry analysis to identify ubiquitylation of EYFP-tagged CENP-A (EYFP-CENP-A) protein.

Studying the structure and the dynamics of kinetochores and centromeres is important in understanding chromosomal instability (CIN) and cancer ...

A Capsule-Based Model for Immature Hard Tick Stages Infestation on Laboratory Mice

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of mouse models in tick research is critical for understanding their biology and tick-host-pathogen interactions. Here we demonstrate a non-laborious technique for the feeding of immature stages of hard ticks on laboratory mice. The benefit of the method is its simplicity, short duration, and the ability to monitor or collect ticks at different time points of an experiment. In addition, the technique allows attachment of two individual capsules on the same mouse, which is beneficial for a variety of experiments where two different groups of ticks are required to feed on the same animal. The non-irritating and flexible capsule is made from easily accessible materials and minimizes the discomfort of the experimental animals. Furthermore, euthanasia is not necessary, mice recover completely after the experiment and are available for re-use.

In this study, a feeding system for nymphal and larval stages of hard tick was developed using a capsule attached to laboratory mouse. The feeding capsule is made from flexible materials and remains firmly attached to the mouse for at least one week and allows comfortable monitoring of tick feeding.

Ticks are obligatory blood feeding parasites at all stages of development (except eggs) and are recognized as vectors of various pathogens. The use of ...

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human food. Outside its native range, culturing G. bimaculatus is feasible due to its dietary plasticity, rapid reproductive cycle, lack of diapause requirement, tolerance for high-density rearing, and robustness against pathogens. Thus, G. bimaculatus can be&#160;a versatile model for studies of insect physiology, behavior, embryology, or genetics.
Cultural parameters, such as stocking density, within-cage refugia, photoperiod, temperature, relative humidity, and diet, all impact cricket growth, behavior, and gene expression and should be standardized. In the burgeoning literature on farming insects for human consumption, these crickets are frequently employed to evaluate candidate feed admixtures derived from crop residues, food-processing byproducts, and other low-cost waste streams.
To support ongoing experiments evaluating G. bimaculatus growth performance and nutritional quality in response to variable feed substrates, a comprehensive set of standard protocols for breeding, upkeep, handling, measurement, and euthanasia in the laboratory was developed and is presented here. An industry-standard cricket feed has proven nutritionally adequate and functionally appropriate for the long-term maintenance of cricket breeding stocks, as well as for use as an experimental control feed. Rearing these crickets at a density of 0.005 crickets/cm3 in screen-topped 29.3 L polyethylene cages at an average temperature of 27 &#176;C on a 12 light (L)/12 dark (D) photoperiod, with moistened coconut coir serving both as hydration source and oviposition medium has successfully sustained healthy crickets over a 2-year span. Following these methods, crickets in a controlled experiment yielded an average mass of 0.724 g 0.190 g at harvest, with 89% survivorship and 68.2% sexual maturation between stocking (22 days) and harvest (65 days).

Maintaining Laboratory Cultures of Gryllus bimaculatus, a Versatile Orthopteran Model for Insect Agriculture and Invertebrate Physiology

This paper outlines basic methods to standardize important factors such as density, feed availability, hydration source, and environmental controls for the long-term rearing of laboratory cultures of the edible cricket, Gryllus bimaculatus.

Gryllus bimaculatus (De Geer)&#160;is a large-bodied cricket distributed throughout Africa and Southern Eurasia where it is often wild-harvested as human ...

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many effectors with pharmaceutical properties that can diminish host immune responses and enhance pathogen transmission. One group of such effectors are microRNAs (miRNAs). miRNAs are short non-coding sequences that regulate host gene expression at the tick-host interface and within the organs of the tick. These small RNAs are transported in the tick saliva via extracellular vesicles (EVs), which serve inter-and intracellular communication. Vesicles containing miRNAs have been identified in the saliva of ticks. However, little is known about the roles and profiles of the miRNAs in tick salivary vesicles and glands. Furthermore, the study of vesicles and miRNAs in tick saliva requires tedious procedures to collect tick saliva. This protocol aims to develop and validate a method for isolating miRNAs from purified extracellular vesicles produced by ex vivo organ cultures. The materials and methodology needed to extract miRNAs from extracellular vesicles and tick salivary glands are described herein.

Isolation of microRNAs from Tick Ex Vivo Salivary Gland Cultures and Extracellular Vesicles

The present protocol describes the isolation of microRNAs from tick salivary glands and purified extracellular vesicles. This is a universal procedure that combines commonly used reagents and supplies. The method also allows the use of a small number of ticks, resulting in quality microRNAs that can be readily sequenced.

Ticks are important ectoparasites that can vector multiple pathogens. The salivary glands of ticks are essential for feeding as their saliva contains many ...

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite the growing burden of tick-borne diseases, research on ticks has lagged behind insect disease vectors due to challenges in applying genetic transformation tools for functional studies to the unique biology of ticks. Genetic interventions have been gaining attention to reduce mosquito-borne diseases. However, the development of such interventions requires stable germline transformation by injecting embryos. Such an embryo injection technique is lacking for chelicerates, including ticks. Several factors, such as an external thick wax layer on tick embryos, hard chorion, and high intra-oval pressure, are some&#160;obstacles that previously prevented embryo injection protocol development in ticks. The present work has overcome these obstacles, and an embryo injection technique for the black-legged tick, Ixodes scapularis, is described here.&#160;This technique can be used to deliver components, such as CRISPR/Cas9, for stable germline transformations.

Embryo Injection Technique for Gene Editing in the Black-Legged Tick, Ixodes scapularis

The present protocol describes a method for injecting tick embryos. Embryo injection is the preferred technique for genetic manipulation to generate transgenic lines.

Ticks can transmit various viral, bacterial, and protozoan pathogens and are therefore considered vectors of medical and veterinary importance. Despite ...