Name: captive maintenance and venom extraction of tityus serrulatus (brazilian yellow scorpion) for antivenom production
Uploaded: 2023-10-06T13:10:00.000+00:00
Duration: 5 min 27 s
Description: Watch this Scientific Journal Video about Optimizing Scorpion Venom Extraction for Antivenom Production at JoVE.com

Protocols involving invertebrate animals are exempt from Instituto Butantan's Committee on the Use and Care of Animals' approval. However, the maintenance of scorpions described here follows ethical parameters, and animal welfare is respected according to the needs of the species.
1. Housing
<ol>
	<li>Keep the scorpions in communal polypropylene containers as enclosures (height 35 cm, width 35.5 cm, length 72 cm) containing a maximum of 350 animals.</li>
	<li>Maintain the room where the animal enclosures are housed under a controlled temperature of 24 °C (±0.5 °C) in a light cycle of 10 h with lights on and 14 h with lights off.</li>
	<li>Line the enclosure floor (bottom) of each container with kraft paper fixed with masking tape on the edges on all sides so that the animals cannot get under the paper (Figure 3A).</li>
	<li>Use four cardboard trays (egg trays) as vertical substrates, stacked on one side of the container, with a rigid cardboard sheet placed between each tray (Figure 3B,C).</li>
	<li>Place a shallow polypropylene tray (water tray) on the opposite side of the enclosure, containing soaked cotton as a water source for the animals (Figure 3B).</li>
	<li>Keep the containers without lids to increase the airflow. Place a strip of approximately 10 cm of self-adhesive plastic tape on the top of the internal surface of the enclosure and on the upper corners to make the walls smooth enough to prevent the animals from escaping from the container (Figure 3A).</li>
</ol>
<img alt="Insect rearing setup using egg cartons in a plastic container for behavior observation experiment." class="xfigimg" src="/files/ftp_upload/65737/65737fig03.jpg" data-altupdatedat="1747736610000" data-alt="Figure 3"> 
Figure 3: Housing and enclosure preparation. (A) Arrows indicate the kraft paper on the bottom, fixed with masking tape on the edges. Dotted lines indicate the self-adhesive plastic tape placed on the top of the internal surface of the enclosure. (B) Vertical substrate (egg trays piled on one side of the container) and a shallow polypropylene tray (water tray) placed on the opposite side. (C) Vertical substrate detail: egg trays pilled with a rigid cardboard sheet in between two trays. (D) Cardboard sheets and egg trays positioned in layers, simulating the natural dark and humid habitat of T. serrulatus. (E) Animals accommodated in the substrate. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
2. Hygiene routine
NOTE: The hygiene routine is divided into two maintenance procedures: complete and partial maintenance. The first one is performed 2 days after feeding the animals or when the enclosure is selected for venom extraction. Partial maintenance is performed when the animals are not fed.
<ol>
	<li>Complete maintenance
	<ol>
		<li>Prepare a clean enclosure with new substrate (egg trays, cardboard, and kraft paper) and a clean water tray, as described in section 1.</li>
		<li>Utilizing long forceps, remove the water tray from the enclosure. If there are any scorpions on the water tray, remove them and place them in the clean enclosure.</li>
		<li>Repeat the same procedure described in step 2.1.2 with all egg trays and cardboard sheets, removing all the scorpions and placing them in the clean enclosure.</li>
		<li>After the removal of the trays, remove all the scorpions left on the floor of the enclosure and place them in the clean enclosure. Be sure to account for any carcasses and discard them properly. Collect all remaining live prey (insects) and remove them from the enclosure.</li>
		<li>Remove all the disposable substrates from the enclosure (egg trays, cardboard sheet, and kraft paper) and properly discard them. Send the reusable items (water tray and polypropylene container) for sanitation. 
		NOTE: The sanitation of the reusable items is performed with 0.5% hypochlorite combined with a neutral detergent. It is mandatory that the materials are thoroughly and abundantly rinsed with running water after using any sanitation product or solution.</li>
	</ol>
	</li>
	<li>Partial maintenance 
	NOTE: Partial maintenance consists of inspecting all the substrates (egg trays and kraft paper) with the aim of removing dead scorpion carcasses and other undesirable debris, but without discarding the substrates or changing the enclosure.
	<ol>
		<li>Perform the inspection using long forceps to gently lift each egg tray and remove the waste.</li>
		<li>If needed, replace one or more egg trays with a clean one.</li>
		<li>Completely replace the water tray with a clean one using long forceps. Transfer any scorpions on the water tray to the egg tray in another part of the enclosure.</li>
	</ol>
	</li>
</ol>
3. Feeding
<ol>
	<li>Feed the animals every 15 days according to the venom extraction schedule. Feed animals that will be milked 7 days prior and 7 days after the venom extraction.</li>
	<li>For the feeding, supply cockroaches (Phoetalia pallida) and crickets (Gryllus sp.) alternately.</li>
	<li>Calculate the amount of food according to the energy intake of the animals; offer 1 prey for every 3 or 5 scorpions in the enclosure.</li>
	<li>Count the calculated amount of prey items and place them alive inside the enclosure, leaving them for 2 days prior to the complete maintenance (section 2.1).</li>
	<li>When offering cockroaches, apply a calcium carbonate with alcohol solution around the top of the enclosures. The solution causes the cockroaches to slip and not be able to climb the side surface of the enclosure, thus preventing the insects from escaping.</li>
</ol>
4. Venom extraction
NOTE: The animals used in the venom extraction routine are adults or pre-adults. Due to the difficulty in determining the complete sexual maturity of the Brazilian yellow scorpion (adult phase), it was determined that animals with a total body length of 5 to 7 cm are eligible for the procedure.
<ol>
	<li>Milk each animal every 2-3 months.</li>
	<li>On the day of venom extraction, count all alive animals in the enclosure and transfer them to a glass container utilizing long forceps (Figure 4).</li>
	<li>Prepare and arrange all the materials and equipment to be used on the top of the airflow cabinet work surface. Plug the electrostimulator power cord into the outlet and turn the electrostimulator and the airflow cabinet on (Figure 2B).
	<ol>
		<li>Use the following electro stimulator parameters settings: potential (difference of the potential between the electrodes): 540 mV, stress (duration of the electric pulse): 2 ms, action (intermittency to moderate the potency used, which would reduce the heating of the telson organic tissue): 70%, cycle (adjust the cyclic repetition): 1 s, and intensity (electric current that circulates through the animal's muscle): 0.75 mA.</li>
	</ol>
	</li>
	<li>Manually restrain each animal in a way that the telson stands still, following the steps:
	<ol>
		<li>Hold the metassoma of the animal with straight forceps (Figure 5A).</li>
		<li>Immobilize the telson with curved forceps (Figure 5B).</li>
		<li>Restrain the telson with the hand (Figure 5C,D).</li>
	</ol>
	</li>
	<li>After restraint, place the telson in direct contact with the electrostimulator conductor, without the need to use a conductive solution. The electric shock causes an involuntary muscle contraction of the venom gland and consequent ejection of the venom.</li>
	<li>Collect the venom drops directly into a plastic microtube placed close to the electrostimulator conductor (Figure 6).</li>
	<li>Next, release the animal back into the glass container, following the steps:
	<ol>
		<li>Transfer the animal's telson from the hand to the curved forceps.</li>
		<li>Place the animal gently on the bottom of the glass container.</li>
	</ol>
	</li>
	<li>Perform steps 4.4-4.7 with each animal in the glass recipient.</li>
	<li>After all the animals in the glass recipient have been milked, place them back into the enclosure.</li>
	<li>Store the collected venom at -20 °C until the lyophilization process.</li>
</ol>
<img alt="Scorpions in glass container with tweezers, research setup for arachnid behavior study." class="xfigimg" src="/files/ftp_upload/65737/65737fig04.jpg" data-altupdatedat="1747736610000" data-alt="Figure 4"> 
Figure 4: Scorpions in the glass container. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Scorpion handling procedure using forceps, shown in step-by-step image sequence." class="xfigimg" src="/files/ftp_upload/65737/65737fig05.jpg" data-altupdatedat="1747736610000" data-alt="Figure 5"> 
Figure 5: Restraining procedure for venom extraction. (A) Metassoma restrained with straight forceps (black arrow). (B) Telson immobilized with curved forceps (black arrow). (C) Transference of the restrained telson from the forceps to the hand. (D) Telson restrained by the hand. It is recommended that the animals be handled with bare hands since when using gloves, the scorpions can grab the material with their claws, making it difficult to release them at the end of the procedure and considerably increasing the risk of accidents. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig05large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Insect venom extraction setup using pipette and tweezers for biochemical research." class="xfigimg" src="/files/ftp_upload/65737/65737fig06.jpg" data-altupdatedat="1747736610000" data-alt="Figure 6"> 
Figure 6: Venom extraction. A plastic microtube (white arrow) placed close to the electrostimulator (yellow arrow). The venom drops are collected directly into the tube maintained at room temperature during the procedure. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig06large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Rearing of Brazilian Yellow Scorpion for Venom Extraction

<ol>
	<li>Polis, G. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The biology of scorpions. , (1990).</li><li>Lacerda, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpion+envenomation+in+the+state+of+S&#227;o+Paulo,+Brazil:+Spatiotemporal+analysis+of+a+growing+public+health+concern.">Scorpion envenomation in the state of S&#227;o Paulo, Brazil: Spatiotemporal analysis of a growing public health concern.</a> PLoS One. 17 (4), 0266138 (2022).</li><li>Reckziegel, G. C., Pinto, V. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpionism+in+Brazil+in+the+years+2000+to+2012.">Scorpionism in Brazil in the years 2000 to 2012.</a> Journal of Venomous Animals and Toxins Including Tropical Diseases. 20, 46 (2014).</li><li>Guerra-Duarte, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpion+envenomation+in+Brazil:+Current+scenario+and+perspectives+for+containing+an+increasing+health+problem.">Scorpion envenomation in Brazil: Current scenario and perspectives for containing an increasing health problem.</a> PLoS Neglected Tropical Diseases. 17 (2), 0011069 (2023).</li><li>Pimenta, R. J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Selected+to+survive+and+kill:+Tityus+serrulatus,+the+Brazilian+yellow+scorpion.">Selected to survive and kill: Tityus serrulatus, the Brazilian yellow scorpion.</a> PLoS One. 14 (4), e0214075 (2019).</li><li>Lacerda, A. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+areas+vulnerable+to+scorpionism+and+its+association+with+environmental+factors+in+S&#227;o+Paulo,+Brazil.">Detection of areas vulnerable to scorpionism and its association with environmental factors in S&#227;o Paulo, Brazil.</a> Acta Tropica. 230, 106390 (2022).</li><li>Torrez, P. P. Q., Dourado, F. S., Bertani, R., Cupo, P., Fran&#231;a, F. O. d. e. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Scorpionism+in+Brazil:+exponential+growth+of+accidents+and+deaths+from+scorpion+stings.">Scorpionism in Brazil: exponential growth of accidents and deaths from scorpion stings.</a> Journal of the Brazilian Society of Tropical Medicine. 52, 20180350 (2019).</li><li>Amado, T. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Vulnerable+areas+to+accidents+with+scorpions+in+Brazil.">Vulnerable areas to accidents with scorpions in Brazil.</a> Tropical Medicine &amp; International Health. 26 (5), 13561 (2021).</li><li>Brasil, Minist&#233;rioda da Sa&#250;de, Funda&#231;&#227;o Nacional de Sa&#250;de. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Manual de diagn&#243;stico e tratamento de acidentes por animais pe&#231;onhentos. , (2001).</li><li>Candido, D. M., Lucas, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Maintenance+of+scorpions+of+the+genus+Tityus+Koch+(Scorpiones,+Buthidae)+for+venom+obtention+at+Instituto+Butantan,+S&#227;o+Paulo,+Brazil.">Maintenance of scorpions of the genus Tityus Koch (Scorpiones, Buthidae) for venom obtention at Instituto Butantan, S&#227;o Paulo, Brazil.</a> Journal of Venomous Animals and Toxins including Tropical Diseases. 10 (1), 000100007 (2004).</li><li>Lucas, S. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laborat&#243;rio+de+Artr&#243;podes+do+Instituto+Butantan+e+os+aracn&#237;deos+pe&#231;onhentos.">Laborat&#243;rio de Artr&#243;podes do Instituto Butantan e os aracn&#237;deos pe&#231;onhentos.</a> Hist&#243;ria Ci&#234;ncias Sa&#250;de Manguinhos. 10 (3), 000300011 (2003).</li><li>Braga-Pereira, G. 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The authors have no conflicts of interest to disclose.

The application of the methods described allows us to keep a large number of T. serrulatus individuals and gives us consistent predictability of the number of scorpions needed for annual venom production. This way, we are able to provide enough batches of venom in advance to supply the antivenom manufacturing process. At the same time, the development of pre-established schedules for maintenance, feeding, and venom extraction is an essential part of the activities and assists in complying with the described prot...

Scorpions are arthropods belonging to the class Arachnida, order Scorpiones, which comprises approximately 2,621 species1,2. These animals have a wide geographical range and are present on all continents except Antarctica1. Envenomation caused by scorpions results in the morbidity or death of thousands of people every year globally3. In 2019, it was estimated that there were more than 1.2 million accidents and 3,500 annual deaths caused by these animals. In Brazil, the number of cases has been increasing exponentially, reaching more than 100,000 cases per year since 20174,5,6. The uncontrolled urbanization observed in Brazil in the past decades, without adequate sewage treatment and regular collection and disposal of garbage, associated with environmental degradation and climate changes, has provided conditions for the proliferation of invasive scorpions, such as T. serrulatus, increasing the contact with humans and thus resulting in harmful accidents4,7,8. There are about 178 scorpion species in Brazil, but stings of medical importance are caused by the genus Tityus, with four species (T. serrulatus, T. bahiensis, T. stigmurus, and T. obscurus) being of medical concern, with Tityus serrulatus being responsible for the most severe cases and deaths7,9.
Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing approximately 70,000 vials of scorpion antivenom for the Brazilian health system. Briefly, the steps involved in antivenom manufacturing include the inoculation of venom-derived antigens in equines, the collection and purification of rich-antibodies plasma, resulting in 5 mL vials of scorpion antivenom. Each vial is capable of neutralizing, at least, 1 mg of scorpion venom per mL of antivenom. The Arthropods Bioterium is an integral part of the industrial center, responsible for supplying the source material for the scorpion antivenom.
The Arthropods Bioterium at Instituto Butantan was originally named the Laboratory of Arthropods and was established in 1967. However, it was only in 1995 that the Laboratory moved to an exclusive facility dedicated to housing venomous arthropods10,11. At that time, the scorpions were kept in a room of 12 m2 with no controlled temperature and distributed among approximately 13 enclosures, each housing a maximum of 300 animals10. Due to the increasing demand for venom over the years and the consequent need to increase the number of animals, along with improvements in the venom extraction process, a major change occurred in 2016. The Laboratory was integrated into the industrial center of Instituto Butantan and was renamed the Arthropods Bioterium. Additionally, in 2016, the Bioterium moved to a new purpose-built facility. The new scorpion room covers an area of 24 m2 and contains approximately 48 polypropylene enclosures, each housing up to 300 animals, as shown in Figure 1. This results in a total of 10,000 to 20,000 individuals being maintained under controlled captive conditions, with variations throughout the year. The scorpions are carefully managed under strict husbandry protocols to ensure their welfare while meeting the high production demands and adhering to good practices and ethical standards for animal care. For venom extraction, a specific room was designed and constructed, housing two airflow cabinets as shown in Figure 2A. These cabinets serve as safety measures to prevent technicians from inhaling venom particles during the extraction procedure. Personal protective equipment (PPE) is mandatory for the technicians, including synthetic aprons and FFP2 masks, to avoid any contamination and ensure their safety.
<img alt="Research storage room with bins on shelves, workspace for data analysis." class="xfigimg" src="/files/ftp_upload/65737/65737fig01v2.jpg" data-altupdatedat="1747736610000" data-alt="Figure 1"> 
Figure 1: General view of the scorpion's room of the Arthropods Bioterium. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Laboratory biosafety cabinets setup for contamination prevention and sterile environment control." class="xfigimg" src="/files/ftp_upload/65737/65737fig02.jpg" data-altupdatedat="1747736610000" data-alt="Figure 2"> 
Figure 2: Working area. (A) General view of the venom extraction room of the Arthropods Bioterium. (B) Airflow cabinet work surface. All the materials and equipment are prepared and arranged on the top of the airflow cabinet work surface. The black arrow indicates the electrostimulator device, and the red arrow indicates the forceps. <a href="https://www.jove.com/files/ftp_upload/65737/65737fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
About 90% of the T. serrulatus scorpions in the Arthropods Bioterium originate from different sites, collected by municipal health agents as part of a surveillance and control program for these animals. The scorpions are caught in urban areas and then sent to Instituto Butantan as part of a collaborative program. T. serrulatus scorpions are parthenogenetic, meaning that birth occurs as a consequence of the development of offspring from unfertilized eggs, without the presence of an individual of the opposite sex12. Due to this type of reproduction, some animals are born in captivity in our facility and are maintained until they reach the adult phase, following a similar husbandry protocol as described below. Once they reach the adult stage, they are added to the venom extraction routine. Upon arrival, the scorpions undergo initial screening, and those in good health conditions are kept in collective enclosures containing a maximum of 350 individuals, divided by their origin. For each enclosure, a control form is filled out daily, providing data on the date of feeding and the type of prey offered (cricket or cockroach), water supply, cleaning procedures, the number of dead animals, and the number of remaining live ones inside the enclosure.
In a pre-scheduled agenda, animals are subjected to electric stimulation, and the resulting venom of hundreds of individuals is then lyophilized, resulting in batches of freeze-dried venom. The standardized venom obtained from this process is destined for horse immunization as part of the manufacturing process, as well as to provide reference material for quality control of the active substance and the final product.
There are very few facilities in the world capable of keeping such a large number of scorpions and performing the volume of venom extractions in accordance with good manufacturing practices and ethical use of the animals, as done in the Arthropods Bioterium of Instituto Butantan. Therefore, our objective is to describe the scorpion maintenance protocols and venom extraction procedures used in T. serrulatus husbandry, which successfully provide the necessary amount of venom for the production of scorpion antivenom.

<table><tbody><tr><td>Carboard sheet</td><td>NA</td><td>NA</td><td></td></tr><tr><td>Egg tray</td><td>NA</td><td>NA</td><td>cardboard 36 places egg trays</td></tr><tr><td>Electro stimulator device</td><td>Instituto Butantan</td><td>ART-FV-01</td><td>Internally designed for the venom extraction&amp;nbsp;</td></tr><tr><td>Kraft paper</td><td>NA</td><td>NA</td><td>regular kraft paper</td></tr><tr><td>Making tape</td><td>NA</td><td>NA</td><td>White masking tape (24 mm x 50 m)</td></tr><tr><td>Polyproplene container</td><td>NA</td><td>NA</td><td>70 L Polypropylene box (H &amp;ndash; 35 cm, W &amp;ndash; 35.5 cm, L - 72 cm)</td></tr><tr><td>Polyproplene tray</td><td>NA</td><td>NA</td><td>Polypropylene tray (H &amp;ndash; 3.0 cm, W &amp;ndash; 18.5 cm, L - 30 cm)</td></tr><tr><td>Self-adhesive plastic</td><td>NA</td><td>NA</td><td></td></tr></tbody></table>

captive maintenance and venom extraction of tityus serrulatus (brazilian yellow scorpion) for antivenom production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) are responsible for approximately 150,000 envenoming cases per year in Brazil, of which 10% require antivenom treatment to reverse life-threatening venom effects. Therefore, thousands of T. serrulatus individuals are maintained under controlled captivity conditions for venom extraction, subsequently used in the production of the national supply of scorpion antivenom. Instituto Butantan is the main antivenom-manufacturing laboratory in Brazil, providing about 70,000 vials of scorpion antivenom for the Brazilian health system. Thus, the husbandry protocols and venom extraction methodologies are key points for the success of large-scale, standardized venom production. The objective of this article is to describe the captivity protocols of T. serrulatus husbandry, encompassing the husbandry routine and the venom extraction procedures, following good manufacturing practices, and ensuring animal welfare. These practices allow for the maintenance of up to 20,000 animals in captivity, with a routine of 3,000 to 5,000 scorpions milked monthly according to antivenom manufacturing demand, achieving an average of 90% of positive extraction.

Thirty standard operational procedures (SOPs) were developed for each procedure, ensuring the repeatability of the procedures among technicians and compliance with quality parameters. The average mortality rate after the extraction procedure is around 12%, which may be considered low, considering the electrostimulation routine and the captivity environment, where stress factors are successfully reduced by applying the procedures.
To keep the animals healthy, a tray covered with cotton and wate...

Watch this Scientific Journal Video about Optimizing Scorpion Venom Extraction for Antivenom Production at JoVE.com

Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production

Scorpion envenomation is a public health problem in several tropical and subtropical countries. Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow ...

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2.2K Views.  Instituto Butantan. Here, we present successful maintenance and venom extraction protocols for large-scale Tityus serrulatus Lutz and Mello, 1922 (Brazilian yellow scorpion) husbandry, with the aim of providing venom for subsequent scorpion antivenom production to meet the demand of the Brazilian health system.

Video: Author Spotlight: Optimizing Scorpion Venom Extraction for Antivenom Production 

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, and the majority of extant heteropterans retain this trophic strategy. Some heteropterans have transitioned to feeding on vertebrate blood (such as the kissing bugs, Triatominae; and bed bugs, Cimicidae) while others have reverted to feeding on plants (most Pentatomomorpha). However, with the exception of saliva used by kissing bugs to facilitate blood-feeding, little is known about heteropteran venoms compared to the venoms of spiders, scorpions and snakes.
One obstacle to the characterization of heteropteran venom toxins is the structure and function of the venom/labial glands, which are both morphologically complex and perform multiple biological roles (defense, prey capture, and extra-oral digestion). In this article, we describe three methods we have successfully used to collect heteropteran venoms. First, we present electrostimulation as a convenient way to collect venom that is often lethal when injected into prey animals, and which obviates contamination by glandular tissue. Second, we show that gentle harassment of animals is sufficient to produce venom extrusion from the proboscis and/or venom spitting in some groups of heteropterans. Third, we describe methods to harvest venom toxins by dissection of anaesthetized animals to obtain the venom glands. This method is complementary to other methods, as it may allow harvesting of toxins from taxa in which electrostimulation and harassment are ineffective. These protocols will enable researchers to harvest toxins from heteropteran insects for structure-function characterization and possible applications in medicine and agriculture.

Although many insects in the suborder Heteroptera (Insecta: Hemiptera) are venomous, their venom composition and the functions of their venom toxins are mostly unknown. This protocol describes methods to harvest heteropteran venoms for further characterization, using electrostimulation, harassment, and gland dissection.

Heteropteran insects such as assassin bugs (Reduviidae) and giant water bugs (Belostomatidae) descended from a common predaceous and venomous ancestor, ...

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Culture and Maintenance of Human Embryonic Stem Cells  

Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be  fed  every day by performing a complete medium change to replenish lost nutrients and to keep the cultures free of unwanted differentiation factors. Although a small amount of differentiation is normal and expected in stem cell cultures, the culture should be routinely cleaned up by manually removing, or "picking" differentiated areas. Identifying and removing excess differentiation from hES cell cultures are essential techniques in the maintenance of a healthy population of cells.

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Human embryonic stem (hES) cells must be monitored and cared for in order to maintain healthy, undifferentiated cultures. At minimum, the cultures must be ...

A Trap-Vaccinate-Release Protocol for Immunization of Skunks and Additional Rabies Vectors Against Rabies

Oral vaccination of wildlife against rabies via the distribution of vaccine-laden baits is used widely as a management tool in Europe and North America. Over the past several decades, successful programs have targeted important reservoirs, including coyotes, foxes, raccoon dogs, and raccoons, for prevention and control. However, other species (e.g., skunks) or certain habitats (e.g., urban) may be less amenable to oral vaccination, requiring consideration of additional strategies. Trap-vaccinate-release (TVR) is one alternate method that entails the use of live traps to capture targeted wildlife. Traps are distributed along grids in suitable, accessible habitats. Captured animals are identified by tagging, vaccinated parenterally with an inactivated commercial vaccine, sexed, aged, weighed, and released at the site of capture. Since 2001, cross-species transmission of rabies virus from bats to mesocarnivores, such as skunks, has been detected in Flagstaff, Arizona. During the past two decades, TVR has been used as a safe and effective method to prevent and control rabies during such events in this region.

Here, we present a protocol to demonstrate safe, trap-vaccinate-release (TVR) techniques for carnivores susceptible to rabies.

Oral vaccination of wildlife against rabies via the distribution of vaccine-laden baits is used widely as a management tool in Europe and North America. ...

Artificial Host Parasitization by Trichogramma dendrolimi and Venom Extraction

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, parasitoid wasps inject venom into their hosts to suppress their hosts&#39; immunity, modulate hosts&#39; development, metabolism, and even behavior. With over 600,000 estimated species, the diversity of parasitoid wasps surpasses that of other venomous animals, such as snakes, cone snails, and spiders. Parasitoid wasp venom is an underexplored source of bioactive molecules with potential applications in pest control and medicine. However, collecting parasitoid venom is challenging due to the inability to use direct or electrical stimulation and the difficulty in dissection because of their small size. Trichogramma is a genus of tiny (~0.5 mm) egg parasitoid wasps that are widely used for the biological control of lepidopteran pests in both agriculture and forests. Here, we report a method for extracting venom from T. dendrolimi using artificial hosts. These artificial hosts are created with polyethylene film and amino acid solutions and then inoculated with Trichogramma wasps for parasitism. The venom was subsequently collected and concentrated. This method enables the extraction of large amounts of Trichogramma venom while avoiding contamination from other tissues caused by dissection, a common issue in venom reservoir dissection protocols. This innovative approach facilitates the study of Trichogramma venom, paving the way for new research and potential applications.

Author Spotlight: Efficient Venom Extraction Method from Trichogramma Parasitoid Wasps 

Here, we present a protocol for extracting venom from Trichogramma dendrolimi using an artificial host created with polyethylene film and amino acid solution.

Parasitoid wasps are a diverse group of hymenopteran insects that serve as invaluable resources for pest biocontrol. To ensure successful parasitism, ...

Biochemistry

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and behavioural research. As a vertebrate, zebrafish share considerable genetic sequence similarity with humans and are being used as an animal model for various human disease conditions. The advantages of zebrafish in comparison to other common vertebrate models include high fecundity, low maintenance cost, transparent embryos, and rapid development. Due to the spur of interest in zebrafish research, the need to establish and maintain a productive zebrafish housing facility is also increasing. Although literature is available for the maintenance of a zebrafish laboratory, a concise video protocol is lacking. This video illustrates the protocol for regular housing, feeding, breeding and raising of zebrafish larvae. This process will help researchers to understand the natural behaviour and optimal conditions of zebrafish husbandry and hence troubleshoot experimental issues that originate from the fish husbandry conditions. This protocol will be of immense help to researchers planning to establish a zebrafish laboratory, and also to graduate students who are intending to use zebrafish as an animal model.

Regular Care and Maintenance of a Zebrafish Danio rerio Laboratory: An Introduction

This protocol outlines regular maintenance and care to maintain optimal conditions for zebrafish husbandry. The video illustrates the protocol for system maintenance, regular housing, feeding, breeding, and raising of zebrafish larvae.

This protocol describes regular care and maintenance of a zebrafish laboratory. Zebrafish are now gaining popularity in genetics, pharmacological and ...

Extraction of Tissue Antigens for Functional Assays

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune diseases and cancer2. Our aim is to investigate the antigens recognized by human T cells in the autoimmune disease type 1 diabetes 1,3,4,5. To analyze human T-cell responses against tissue where the antigens recognized by T cells are not identified we developed a method to extract protein antigens from human tissue in a format that is compatible with functional assays 6. Previously, T-cell responses to unpurified tissue extracts could not be measured because the extraction methods yield a lysate that contained detergents that were toxic to human peripheral blood mononuclear cells. Here we describe a protocol for extracting proteins from human tissues in a format that is not toxic to human T cells. The tissue is homogenized in a mixture of butan-1-ol, acetonitrile and water (BAW). The protein concentration in the tissue extract is measured and a known mass of protein is aliquoted into tubes. After extraction, the organic solvents are removed by lyophilization. Lyophilized tissue extracts can be stored until required. For use in assays of immune function, a suspension of immune cells, in appropriate culture media, can be added directly to the lyophilized extract. Cytokine production and proliferation by PBMC, in response to extracts prepared using this method, were readily measured. Hence, our method allows the rapid preparation of human tissue lysates that can be used as a source of antigens in the analysis of T-cell responses. We suggest that this method will facilitate the analysis of adaptive immune responses to tissues in transplantation, cancer and autoimmunity.

A simple protocol for preparing extracts of human tissue to be used as a source of antigens in functional T-cell assays is described. This method allows T-cell responses to tissue-derived antigens to be measured in vitro.

Many of the antigen targets of adaptive immune response, recognized by B and T cells, have not been defined 1. This is particularly true in autoimmune ...

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. However, purifying proteins is sometimes challenging since many proteins are expressed in an insoluble form. When working with difficult or multiple targets it is therefore recommended to use high throughput (HTP) protein expression screening on a small scale (1-4&#160;ml cultures) to quickly identify conditions for soluble expression. To cope with the various structural genomics programs of the lab, a quantitative (within a range of 0.1-100 mg/L culture of recombinant protein) and HTP protein expression screening protocol was implemented and validated on thousands of proteins. The protocols were automated with the use of a liquid handling robot but can also be performed manually without specialized equipment.
Disulfide-rich venom proteins are gaining increasing recognition for their potential as therapeutic drug leads. They can be highly potent and selective, but their complex disulfide bond networks make them challenging to produce. As a member of the FP7 European Venomics project (<a href="http://www.venomics.eu">www.venomics.eu</a>), our challenge is to develop successful production strategies with the aim of producing thousands of novel venom proteins for functional characterization. Aided by the redox properties of disulfide bond isomerase DsbC, we adapted our HTP production pipeline for the expression of oxidized, functional venom peptides in the E. coli cytoplasm. The protocols are also applicable to the production of diverse disulfide-rich proteins. Here we demonstrate our pipeline applied to the production of animal venom proteins. With the protocols described herein it is likely that soluble disulfide-rich proteins will be obtained in as little as a week. Even from a small scale, there is the potential to use the purified proteins for validating the oxidation state by mass spectrometry, for characterization in pilot studies, or for sensitive micro-assays.

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

A protocol for the quantitative, high throughput expression screening and analytical purification of fusion proteins from small-scale Escherichia coli cultures is described and applied to the expression of disulfide-rich animal venom protein targets.

Escherichia coli (E. coli) is the most widely used expression system for the production of recombinant proteins for structural and functional studies. ...

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and efficient expansion of large amounts of clinical-grade hPSCs are critical issues in hPSC-based therapies. Conventionally, hPSCs are propagated as colonies on both feeder and feeder-free culture systems. However, these methods have several major limitations, including low cell yields and generation of heterogeneously differentiated cells. To improve current hPSC culture methods, we have recently developed a new method, which is based on non-colony type monolayer (NCM) culture of dissociated single cells. Here, we present detailed NCM protocols based on the Rho-associated kinase (ROCK) inhibitor Y-27632. We also provide new information regarding NCM culture with different small molecules such as Y-39983 (ROCK I inhibitor), phenylbenzodioxane (ROCK II inhibitor), and thiazovivin (a novel ROCK inhibitor). We further extend our basic protocol to cultivate hPSCs on defined extracellular proteins such as the laminin isoform 521 (LN-521) without the use of ROCK inhibitors. Moreover, based on NCM, we have demonstrated efficient transfection or transduction of plasmid DNAs, lentiviral particles, and oligonucleotide-based microRNAs into hPSCs in order to genetically modify these cells for molecular analyses and drug discovery. The NCM-based methods overcome the major shortcomings of colony-type culture, and thus may be suitable for producing large amounts of homogeneous hPSCs for future clinical therapies, stem cell research, and drug discovery.

Here, we present human pluripotent stem cell (hPSC) culture protocols, based on non-colony type monolayer (NCM) growth of dissociated single cells. This new method, utilizing Rho-associated kinase inhibitors or the laminin isoform 521 (LN-521), is suitable for producing large amounts of homogeneous hPSCs, genetic manipulation, and drug discovery.

Human pluripotent stem cells (hPSCs) hold great promise for regenerative medicine and biopharmaceutical applications. Currently, optimal culture and ...

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses

Venoms are chemically complex secretions typically comprising numerous proteins and peptides with varied physiological activities. Functional characterization of venom proteins has important biomedical applications, including the identification of drug leads or probes for cellular receptors. Spiders are the most species rich clade of venomous organisms, but the venoms of only a few species are well-understood, in part due to the difficulty associated with collecting minute quantities of venom from small animals. This paper presents a protocol for the collection of venom from spiders using electrical stimulation, demonstrating the procedure on the Western black widow (Latrodectus hesperus). The collected venom is useful for varied downstream analyses including direct protein identification via mass spectrometry, functional assays, and stimulation of venom gene expression for transcriptomic studies. This technique has the advantage over protocols that isolate venom from whole gland homogenates, which do not separate genuine venom components from cellular proteins that are not secreted as part of the venom. Representative results demonstrate the detection of known venom peptides from the collected sample using mass spectrometry. The venom collection procedure is followed by a protocol for dissecting spider venom glands, with results demonstrating that this leads to the characterization of venom-expressed proteins and peptides at the sequence level.

This article provides a protocol for the extraction of venom from spiders using electrical stimulation in order to 1) conduct proteomic characterization, 2) stimulate venom gland gene expression, and 3) perform functional studies of venoms. This is followed by a description of venom gland microdissections for gene expression studies.

Captive Maintenance and Venom Extraction of Tityus serrulatus (Brazilian Yellow Scorpion) for Antivenom Production

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Chapters in this video

Harvesting Venom Toxins from Assassin Bugs and Other Heteropteran Insects

Culture and Maintenance of Human Embryonic Stem Cells

A Trap-Vaccinate-Release Protocol for Immunization of Skunks and Additional Rabies Vectors Against Rabies

Extracting Venom from the Parasitoid Wasp Trichogramma dendrolimi Using an Artificial Host

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Regular Care and Maintenance of a Zebrafish (Danio rerio) Laboratory: An Introduction

Extraction of Tissue Antigens for Functional Assays

High Throughput Quantitative Expression Screening and Purification Applied to Recombinant Disulfide-rich Venom Proteins Produced in E. coli

Alternative Cultures for Human Pluripotent Stem Cell Production, Maintenance, and Genetic Analysis

Extraction of Venom and Venom Gland Microdissections from Spiders for Proteomic and Transcriptomic Analyses