The authors would like to thank Hort Pome, Hort Stone, and the Technology and Human Resources for Industry Programme (THRIP: TP14062571871) for funding the project.
ORCID: 
Letodi L. Mathulwe http://orcid.org/0000-0002-5118-3578
Antoinette P. Malan http://orcid.org/0000-0002-9257-0312
Nomakholwa F. Stokwe http://orcid.org/0000-0003-2869-5652

1. Source of fungal strains
<ol>
	<li>Use South African isolated fungal strains of both M. pinghaense 5 HEID (GenBank Accession number: MT367414/MT895630) and M. robertsii 6EIKEN (MT378171/MT380849), collected from apple orchards in the Western Cape province, South Africa.</li>
	<li>Grow cultures of each EPF isolate on 60 g of Sabouraud dextrose agar medium, supplemented with 1 g of yeast extract (SDAY) and 10 &#181;L of Streptomycin. 
	NOTE: Incubate EPF cultures at a controlled temperature of &#177; 25 &#176;C in the dark.</li>
</ol>
2. Metarhizium pinghaense&#160;&#160;and M. robertsii conidial suspension inoculation
<ol>
	<li>Preparation of the solid substrates
	<ol>
		<li>Use two agricultural products, namely flaked oats and flaked barley, as growth mediums for the two EPF isolates and autoclave bags (305 mm &#215; 660 mm) as fermentation bags.</li>
		<li>Use a small polyvinylchloride waste pipe (1000 mm &#215; 50 mm) to create a neck for the fermentation bag at the open end of the autoclave bag and use autoclave tape to secure the pipe to the bag.</li>
		<li>Close the pipe with a sterile cotton wool plug to allow sufficient gas exchange during fermentation.</li>
		<li>Weigh dry grains (200 g) of both the flaked oats and flaked barley and place them in the fermentation bags, and to each bag, add 100 mL of distilled water and thoroughly mix the contents of the bags.</li>
		<li>Leave the wet grains to rest for 15-30 min to absorb moisture before autoclaving and sterilization. Prepare six bags for each grain type, and to prevent contamination, place the bags in other autoclave bags, and autoclave the substrates at 121 &#176;C for 55 min.</li>
	</ol>
	</li>
	<li>Preparation of conidial suspension inoculum
	<ol>
		<li>Harvest 2-3-week-old fungal conidia from fungal cultures of both M. pinghaense and M. robertsii by scraping, using a sterile surgical blade.</li>
		<li>Suspend the collected fungal conidia in 20 mL of sterile distilled water, supplemented with 0.05% Tween 20, and vortex-mix the conidial suspensions for 2 min.</li>
		<li>Prepare 20 mL of conidial suspensions, with a concentration of 1 &#215; 107 conidia/mL, and inoculate the flaked oat and flaked barley solid substrates, respectively. 
		NOTE: Use a hemocytometer to determine conidial concentrations.</li>
	</ol>
	</li>
	<li>Inoculation of the solid substrates
	<ol>
		<li>Open each bag by removing the cotton wool neck plugs, and add the prepared 20 mL conidial suspension to the cooled autoclaved substrate.</li>
		<li>Close the bags again, using the neck plugs, and massage the contents of the bag to allow the fungal inoculum to become evenly mixed with the grain substrate.</li>
		<li>Incubate the fermentation bags at a controlled temperature of &#177; 25 &#176;C, and ensure sufficient gaseous exchange between the culture and the environment. 
		NOTE: This procedure must be conducted under a laminar flow cabinet.</li>
	</ol>
	</li>
	<li>Fermentation phase
	<ol>
		<li>Manually massage the grain substrates in the fermentation bags 2 days after the inoculation and incubation, when visible mycelial growth occurs and the substrate has begun to be clumped by the growing fungus. 
		NOTE: This is done to mix the inoculated granules to allow for mycelial branching to take place during the first early stages of the vegetative growth of the fungi.</li>
		<li>Use a kitchen rolling pin to manipulate the substrate bed to avoid the physical heterogeneity of the grain substrate beds and the bed thickness of the substrate mass. 
		NOTE: The process promotes fungal metabolism, which optimizes fungal spore production, and maximizes the surface area, promoting conidial yield18.</li>
		<li>Leave the fermentation process to continue for up to 4-5 weeks, and check the fermentation bags every 2 days for the presence of any white vegetative overgrowth that can develop during the fermentation process, which can greatly affect the fungal conidia yield. 
		NOTE: Immediately terminate fermentation in the fermentation bags containing any white vegetative overgrowth, and dry the fungal cultures18.</li>
	</ol>
	</li>
</ol>
3. Blastospore inoculation
<ol>
	<li>Blastospore production and inoculation
	<ol>
		<li>Prepare a liquid culture medium, containing 1 L of distilled water, 30 g of glucose, 20 g of yeast extract, 4 g of potassium phosphate diabasic (K2HPO4), 15 mL of corn steep liquor, and 10 &#181;g/mL of the antibiotic Streptomycin, for both the M. pinghaense and the M. robertsii.</li>
		<li>First, heat the distilled water, switch off before reaching boiling point, and add each of the ingredients, except for Streptomycin, to the hot water in the pot. Bring the medium to a gentle boil for 3-4 min, and constantly stir the medium to allow for the proper mixing of the ingredients and prevent the settling of some of the ingredients at the bottom of the pot.</li>
		<li>Pour a total of 100 mL of the medium into nine different 250-mL flasks and place a cotton wool plug on each flask, and cover the cotton wool with aluminum foil as a stopper (Figure 1A).</li>
		<li>Autoclave the medium in the flasks for 55 min at 121 &#176;C. Following the autoclaving, allow the medium in the flasks to cool and add 10 mg/mL of Streptomycin to the medium in each flask (Figure 1B).</li>
		<li>Collect two to three bacterial loops of fungal conidia from 2-3-week-old fungal culture plates for both the EPF isolates, M. pinghaense and M. robertsii, and transfer to each 100 mL liquid media in the 250-mL flasks, under sterile conditions, and seal the flasks.</li>
		<li>Incubate the liquid culture flasks at &#177; 25 &#176;C, on an orbital shaker at 140 rpm for 3 days, and cease the incubation once the cultures show signs of high turbidity with fungal blastospore growth (Figure 1C).</li>
		<li>To detect any possible bacterial contamination from the cultures, draw a 100 &#181;L sample from each liquid culture flask after 24 h during the incubation and plate on three SDA plates per isolate. Incubate the plates for 48 h, at a controlled temperature of &#177; 25 &#176;C.</li>
	</ol>
	</li>
	<li>Preparation of the solid substrate
	<ol>
		<li>Use parboiled long-grain white rice as a solid substrate medium for the blastospores of both M. pinghaense and M. robertsii (adapted from Jaronski and Jackson18).</li>
		<li>Prepare the fermentation bags as detailed above, in steps 2.1.1-2.1.3, and for each bag, add 1 kg of rice and 300 mL of sterile distilled water.</li>
		<li>Gently mix the contents of the fermentation bags and place in outer autoclave bags in an upright position, and autoclave at 121 &#176;C for 55 min. Following the autoclaving, allow the substrates to cool down for &#177; 45 min under sterile conditions.</li>
	</ol>
	</li>
	<li>Inoculation and fermentation
	<ol>
		<li>Remove the closure of each of the liquid culture flasks of both M. pinghaense and M. robertsii under a laminar flow and flame the rim of each flask for 10 s.</li>
		<li>Pour the 100-mL liquid cultures into the fermentation bags by removing the cotton wool plugs from their necks (Figure 2). Place the cotton plugs back on and cover the top of the bag&#39;s neck with surgical paper secured with a rubber band. 
		NOTE: Determine blastospore concentration for each flask using a hemocytometer, and use blastospore concentrations of 1 &#215; 107 - 5 &#215; 108 blastospores/mL to innoculate the substrates28.</li>
		<li>Twist the top part of the bag and mix the contents of the bag by shaking and light manipulation of the substrate by massage, and incubate the bags at &#177; 25 &#176;C, by flattening the substrate in the bags to prevent the formation of thick beds18.</li>
		<li>Break the substrate in the fermentation bags by massaging contents of the bags, 2-3 days following the inoculation and the incubation, when visible mycelial growth and the binding of the substrate by the fungus had occurred (adapted from the technique of Jaronski and Jackson18). 
		NOTE: Allow fermentation to continue for 4 weeks.</li>
	</ol>
	</li>
</ol>
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63246/63246fig01.jpg" /> 
Figure 1: Liquid culture medium in 250-mL flasks. (A) Before autoclaving. (B) After autoclaving and inoculation with EPF spores. (C) Turbid medium with fungal blastospores. <a href="https://www.jove.com/files/ftp_upload/63246/63246fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/63246/63246fig02.jpg" /> 
Figure 2: Prepared blastospore liquid culture medium. (A) Metarhizium robertsii and (B) Metarhizium pinghaense prior to the inoculation of rice as a solid substrate. <a href="https://www.jove.com/files/ftp_upload/63246/63246fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
4. Drying of fungal cultures
<ol>
	<li>Dry the fungal cultures for 10-12 days following fermentation, prior to their use in trials, by transferring the sporulated cultures into 26-30 kg (30 x 43 x 15250 cm3) brown paper bags.</li>
	<li>To improve the tensile strength of the paper bags, horizontally cut off one-third of the top part of each bag, and line the bottom of the bag (Figure 3A, B).</li>
</ol>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/63246/63246fig03.jpg" /> 
Figure 3: Preparation of paper bags, drying procedure of cultures, and packaging. (A,B) The preparation of brown paper bags. The drying procedure of Metarhizium species cultures grown on (C,E) parboiled rice and (D,F) flaked barley. (G) Paper bags closed with staples to create a triangular tent structure. <a href="https://www.jove.com/files/ftp_upload/63246/63246fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="3">
	<li>Gently crumble the substrate in each fermentation bag, cut off the corner of each bag and transfer the whole culture to the paper bags through the space left by the cut-off corner (Figure 3C-F). To avoid the excessive escaping of fungal spores into the air, perform this process slowly. 
	NOTE: Conduct this process under sterile conditions, using laminar flow, to avoid contamination.</li>
	<li>Label each paper bag and fold over the top end of each bag twice and close with staples to create a triangular tent structure, and place the bags on wire drying racks to allow proper, even drying, at a controlled temperature of &#177; 25 &#176;C and low humidity of 30-40%.</li>
	<li>Turn the bags daily to allow even drying of the cultures and to avoid any vegetative regrowth that might take place, which would lower the yield of harvestable fungal spores.</li>
	<li>Weigh each drying bag after every 2 days during the drying process, and continue the drying process for each bag until little to no change is observed in the mass of the bags between the successive days.</li>
</ol>
5. Harvest of fungal conidia
<ol>
	<li>Harvest fungal conidia mechanically from the cultures using three nested sieves, a test sieve (ETS) mesh no. 35 (with 500-&#181;m aperture), nested on a test sieve (with 212-&#181;m aperture), nested on ETS mesh no. 100 (with 150-&#181;m aperture), mounted on a collection pan.</li>
	<li>Load the dry culture sample on the ETS mesh no. 35 sieve slowly and place a lid on the sieve to prevent the release of fungal conidia into the air.</li>
	<li>Add 10-12 glass marbles to the sieves to assist the passage of the fungal conidia through the mesh screens and avoid the retention of the conidia in the sieve, which can result in reduced spore recovery.</li>
	<li>Tape and seal the sieve joints using electrical tape to prevent the escape of conidial dust.</li>
	<li>Place the sieves on a vibratory shaker, fitted with a sticky pad, to secure the collection pan and sieves, for 20-25 min, at a motion of 560-640 vibrations per min (Figure 4). 
	NOTE: Technique adapted from Jaronski and Jackson18.</li>
</ol>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/63246/63246fig04.jpg" /> 
Figure 4: Harvesting of fungal spores from dried Metarhizium robertsii cultures on rice and flaked barley. (A)&#160;10-12 glass marbles added to the sieves to assist the passage of the fungal conidia through the mesh screens. M. robertsii conidia harvested from cultures on (B) rice, and (C) flaked barely. (D) Sieves on a vibratory shaker. <a href="https://www.jove.com/files/ftp_upload/63246/63246fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<ol start="6">
	<li>Remove the test sieves from the collection pan, collect conidia and store the collected conidia in airtight and water-impermeable zipper-lock bags for long-term storage (3-6 months).</li>
</ol>
6. Quantification of fungal conidia produced
<ol>
	<li>Measure and record the mass of each grain substrate prior to the harvesting of the fungal conidia from each substrate.</li>
	<li>Measure the overall weight of the collected conidia by subtracting the mass of the collected spores from the mass of the solid substrate. 
	NOTE: The total conidia yield does involve not only the harvested conidia but also the fungal conidia left on the solid substrate.</li>
	<li>Weigh the substrate and remove 10 g from the weighed substrate. Suspend the 10 g of the substrate in 0.05% Tween 20 and dilute in 10 mL of sterile distilled water.</li>
	<li>Vortex-mix the suspension for 2 min, and use a hemocytometer to do the spore count to determine the number of conidia washed from the substrate.</li>
	<li>Conduct further dilutions by transferring 1000 &#181;L of the 10 mL washed conidial suspension to 9 mL of sterile distilled water to make up 10 mL of dilution suspensions.</li>
	<li>Vortex-mix the conidial suspensions for 2 min, and determine the conidial concentrations. 
	NOTE: Follow the procedure and formula described by Inglis, Enkerli, and Goettel30 to determine the conidial concentration of the suspensions.</li>
	<li>Collect and suspend a total of 0.1 g of the collected conidial powder from each culture in 10 mL of sterile distilled water supplemented with 0.05% Tween 20, and vortex-mix the conidial suspension for 2 min, and determine the conidial concentration using a hemocytometer.</li>
	<li>Conduct further dilutions by transferring 1000 &#181;L of the 10 mL conidial suspension to 9 mL of sterile distilled water to make up the 10 mL suspension dilutions.</li>
	<li>Vortex-mix the conidial suspensions for 2 min, calculate the conidial concentrations and determine the number of conidia per gram.</li>
	<li>Multiply the number of conidia per gram of harvested powder by the initial mass of the harvested conidial powder. Multiply the number of conidia washed from the substrate by the total weight of the spent substrate, being the substrate from which the conidia were harvested.</li>
	<li>Add the two given values together and divide by the initial dry weight of the substrate to calculate the number of conidia per kg or g of the substrate18. 
	NOTE: The calculations were done mainly for the rice substrate. The germination or conidial viability test was conducted for both the M. pinghaense and the M. robertsii isolates to determine the viability of the produced conidia.</li>
</ol>
7. Data analysis
<ol>
	<li>Use an appropriate computer software program to conduct the statistical analysis of the obtained results. 
	NOTE: Statistical analysis of the data was done using STATISTICA version 13.5.0.17.</li>
</ol>

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E., M&#252;ller, E. J., Brown, H. D., D'Uamba, P., Jone, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+first+trial+of+a+Metarhizium+anisopliae+var.+acridum+mycoinsecticide+for+the+control+of+the+red+locust+in+a+recognised+outbreak+area.">The first trial of a Metarhizium anisopliae var. acridum mycoinsecticide for the control of the red locust in a recognised outbreak area.</a> International Journal of Tropical Insect Science. 19 (4), 323-331 (1999).</li><li>Hatting, J. L., Moore, S. D., Malan, A. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+control+of+phytophagous+invertebrate+pests+in+South+Africa.+Current+status+and+future+prospects.">Microbial control of phytophagous invertebrate pests in South Africa. Current status and future prospects.</a> Journal of Invertebrate Pathology. 165, 54-66 (2019).</li><li>Mathulwe, L. L., Malan, A. P., Stokwe, N. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+screening+of+entomopathogenic+fungi+and+nematodes+for+pathogenicity+against+the+obscure+mealybug,+Pseudococcus+viburni+(Hemiptera:+Pseudococcidae).">Laboratory screening of entomopathogenic fungi and nematodes for pathogenicity against the obscure mealybug, Pseudococcus viburni (Hemiptera: Pseudococcidae).</a> Biocontrol Science and Technology. , (2021).</li><li>Inglis, G. D., Enkerli, J., Goettel, M. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Laboratory+techniques+used+for+entomopathogenic+fungi:+Hypocreales.">Laboratory techniques used for entomopathogenic fungi: Hypocreales.</a> Manual of Techniques in Invertebrate Pathology. , 189-253 (2012).</li><li>Mehta, J., 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=Impact+of+carbon+&amp;+nitrogen+sources+on+the+Trichoderma+viride+(Biofungicide)+and+Beauveria+bassiana+(entomopathogenic+fungi).">Impact of carbon &amp; nitrogen sources on the Trichoderma viride (Biofungicide) and Beauveria bassiana (entomopathogenic fungi).</a> European Journal of Experimental Biology. 2 (6), 2061-2067 (2012).</li><li>Burges, H. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formulation+of+mycoinsecticides.">Formulation of mycoinsecticides.</a> Formulation of Microbial Biopesticides. , 131-185 (1998).</li></ol>

The authors have nothing to disclose.

The successful integration of microbial agents for the biological control of important agricultural insect pests in an agroecosystem depends on both success and ease of mass production of the entomopathogens as the first step under laboratory conditions. The mass production of EPF is important for the large-scale application and availability of EPF products for IPM programs using biological control9,10,11,<sup class="...

Entomopathogenic fungi (EPF) have gained importance as crop protection agents in the biological control of important agricultural insect pests1,2. The entomopathogens, which occur naturally in soil, cause epizootics in the populations of various pest species3. The species of EPF are host-specific and pose relatively few risks in terms of attacking nontarget species, and they are nontoxic to the environment4. EPF have a unique mechanism for invading their host, as well as for propagating and persisting in their immediate environment1. They attack the host mainly through asexual spores that attach to and penetrate the host cuticle to invade and proliferate in the host hemocoel. The host eventually dies due to depletion of the hemolymph nutrients or as a result of the toxemia caused by the toxic metabolites released by the fungus. Following death, under ideal environmental conditions, the fungus emerges on the outer surface (overt mycosis) of the host cadaver5,6.
Growing concerns regarding the negative effects of chemical residues on human health, environmental pollution, and the development of pest resistance have led to the global drive to reduce inputs of chemical-based insecticides and to find alternative, novel, and sustainable strategies for crop protection and pest control6,7,8. This has provided opportunities to develop microbial-based insecticides for use in Integrated Pest Management (IPM) programs, which are more ecologically favorable strategies than conventional chemical control3,8.
To develop a successful microbial control agent for an agricultural pest, a suitable organism must first be isolated, characterized, identified and its pathogenicity for the&#160;target pest confirmed. However, an easy, cost-effective method for large-scale production of the microbial agent is required to produce a viable product for use in biological control programmes9,10,11,12,13. Mass production of substantial quantities of good-quality entomopathogens depends on the microbial strain, the environment, the target pest, the formulation, the market, the application strategy, and the desired end product14,15,16. EPF can be mass-produced using liquid substrate fermentation to produce blastospores or the solid substrate fermentation process to produce aerial conidia6,17,18. However, the mass production and formulation process of entomopathogens directly influences the virulence, the cost, the shelf life, and the field efficacy of the final product. For successful use in IPM, the production process of the entomopathogens must be easy to run, require minimal labor, produce a high-yield concentration of virulent, viable, and persistent propagules, and be low in cost4,13,14,16.
Understanding the nutritional requirements of entomopathogens is important for mass cultivation with all culturing methods4,12. The nutritional components of the production medium have a significant impact on the attributes of the resulting propagules, including biocontrol efficacy, yield, desiccation tolerance, and persistence8,19,20,21. The optimization of production procedures is designed to address such factors22. For EPF, the main requirements for good growth, sporulation, and mass production of fungal conidia are adequate moisture, optimum growth temperature, pH, gas exchange of CO2 and O2, and nutrition, including good phosphorous, carbohydrate, carbon, and nitrogen sources18.
Jaronski and Jackson18 describe the solid substrate fermentation method as the most efficient and the closest approximation method to the natural process for EPF production relative to the liquid substrate fermentation method because, under natural conditions, the fungal conidium is borne on solid erect structures, like the surface of insect cadavers. Agricultural products and by-products containing starch are mostly used for the mass production of hypocrealean fungi, as the fungi readily decompose starch through secretion of highly concentrated hydrolytic enzymes from their hyphal tips, to penetrate the solid substance, and to access the nutrients present in the substance11,17,18,23. The grain products also provide the requirements for healthy biomass production because, when they are hydrated and sterilized, the substrates can absorb further nutrients from any liquid medium16,18,24.
Previously, several studies attempted to mass culture EPF species like Beauveria bassiana (Bals.) Vuil., Cordyceps fumosorosea (Wize) Kelper B. Shrestha &#38; Spatafora, Verticillium lecanii (Zimm.) Viegas and some of the Metarhizium anisopliae (Metschn.) Sorokin species complex isolates on various substrates16,23,24. Such mass-produced and commercially developed isolates include Green Muscle&#174; (strain IMI 330189), developed from M. anisopliae var Metarhizium acridum (Driver &#38; Milner) J.F. Bisch, Rehner &#38; Humber, Metarhizium 69 (Meta 69 strain ICIPE69), and Real Metarhizium 69 (L9281), developed from M. anisopliae, and Broadband&#174; (strain PPRI 5339) and Eco-Bb&#174;, developed from B. bassiana25,26. However, limited attempts have been made to mass culture Metarhizium robertsii J.F. Bisch., S.A. Rehner &#38; Humber and Metarhizium pinghaense Chen &#38; Guo. These two isolates were selected in a previous study as the most effective for the control of the mealybug, Pseudococcus viburni Signoret (Hemiptera: Pseudococcidae)27. Therefore, the current study aimed to formulate and mass-produce a sufficient number of resilient infective propagules of the local isolates of M. robertsii and M. pinghaense for commercial application against insect pests. The solid substrate fermentation method was used to mass-produce the fungal conidia for both EPF isolates. Two EPF inoculation methods, using conidial suspensions and the liquid fungal culture of blastospores, were used to inoculate the solid substrates.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.05% Tween 20</td>
			<td>Lasec</td>
			<td></td>
			<td>Added to conidial suspensions to allow fungal spores to mix with water</td>
		</tr>
		<tr>
			<td>20 mL McCartney bottles</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to make conidial suspensions</td>
		</tr>
		<tr>
			<td>Aluminium foil</td>
			<td></td>
			<td></td>
			<td>Used as a cover of the cotton wool plugs on 250-mL flask</td>
		</tr>
		<tr>
			<td>Autoclave</td>
			<td></td>
			<td></td>
			<td>Used to sterilize materials and ingredients used for the conidia production process</td>
		</tr>
		<tr>
			<td>Autoclave bags</td>
			<td>Lasec</td>
			<td></td>
			<td>Fermentation bags or solid substrate containers</td>
		</tr>
		<tr>
			<td>Autoclave tape</td>
			<td>Lasec</td>
			<td></td>
			<td>To secure PVC pipes on the fermentation bags</td>
		</tr>
		<tr>
			<td>Brown Kraft paper bags</td>
			<td></td>
			<td></td>
			<td>Used to dry conidia cultures on agricultural grains</td>
		</tr>
		<tr>
			<td>Bunsen burnner</td>
			<td>Labnet (Labnet International, Inc.)</td>
			<td></td>
			<td>Used to flame equipment (surgical blades,inoculating loops and rims of flasks)</td>
		</tr>
		<tr>
			<td>Clear edge test sieve</td>
			<td></td>
			<td></td>
			<td>Used to separate fungal conidia from agricultural grain substrates</td>
		</tr>
		<tr>
			<td>Corn steep liquor</td>
			<td>SIGMA</td>
			<td>66071-94-1</td>
			<td>Ingredient of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Cotton Wool</td>
			<td>Lasec</td>
			<td></td>
			<td>Used as plug of the neck for fermentation bags</td>
		</tr>
		<tr>
			<td>Duran laboratory bottles</td>
			<td>Neolab</td>
			<td></td>
			<td>Used to autoclave SDA medium and distilled water</td>
		</tr>
		<tr>
			<td>Electrical tape</td>
			<td></td>
			<td></td>
			<td>Used to tape and seal the sieve joints to prevent the escape of conidial dust</td>
		</tr>
		<tr>
			<td>ENDECOTTS test sieve</td>
			<td></td>
			<td></td>
			<td>Used to separate fungal conidia from agricultural grain substrates</td>
		</tr>
		<tr>
			<td>Erlenmeyer Flasks, Narrow neck,250-mL flask</td>
			<td>Lasec</td>
			<td></td>
			<td>Carrier of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Ethanol (99%)</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to sterilize surgical blades and inoculating loops</td>
		</tr>
		<tr>
			<td>Flaked barley</td>
			<td>Health Connection Wholefoods</td>
			<td></td>
			<td>Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii</td>
		</tr>
		<tr>
			<td>Flaked oats</td>
			<td>Tiger brands</td>
			<td></td>
			<td>Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii</td>
		</tr>
		<tr>
			<td>Glucose</td>
			<td>Merck</td>
			<td></td>
			<td>Ingredient of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Growth Chamber/ incubators</td>
			<td></td>
			<td></td>
			<td>For growing fungal conidia culture</td>
		</tr>
		<tr>
			<td>Haemocytometer</td>
			<td></td>
			<td></td>
			<td>Used to determine conidial concentrations</td>
		</tr>
		<tr>
			<td>Inoculating loops</td>
			<td>Lasec</td>
			<td></td>
			<td>For harvesting spores to innoculate liquid medium for blastospores growth</td>
		</tr>
		<tr>
			<td>Kitchen rolling pin</td>
			<td></td>
			<td></td>
			<td>Used to manipulate the solid grain substrate bed</td>
		</tr>
		<tr>
			<td>Laminar flow Cabinet</td>
			<td>ESCO Laminar Flow Cabinet</td>
			<td></td>
			<td>Provide as sterile environment during substrate inoculation</td>
		</tr>
		<tr>
			<td>Metarhizium pinghaense conidia</td>
			<td>Stellenbosch University</td>
			<td>5HEID</td>
			<td>Cultures used to mass culture conidia of Metarhizium pinghaense</td>
		</tr>
		<tr>
			<td>Metarhizium robertsii conidia</td>
			<td>Stellenbosch University</td>
			<td>6EIKEN</td>
			<td>Cultures used to mass culture conidia of Metarhizium robertsii</td>
		</tr>
		<tr>
			<td>Microscope</td>
			<td>ZEIZZ (Scope. A1)</td>
			<td></td>
			<td>Used to determine conidial concentrations and conidial viability</td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>IncoShake- LABOTEC</td>
			<td></td>
			<td>Used for the blastospore production process</td>
		</tr>
		<tr>
			<td>Parboiled rice</td>
			<td>Spekko</td>
			<td></td>
			<td>Agricultural grain used as a solid substrate growth medium for conidia of both M. pinghaense and M. robertsii</td>
		</tr>
		<tr>
			<td>Penicillin-Streptomycin</td>
			<td>SIGMA</td>
			<td></td>
			<td>Added to the SDA medium to prevent bacterial contamination</td>
		</tr>
		<tr>
			<td>Petri-dishes</td>
			<td>Lasec</td>
			<td></td>
			<td>Containers for the SDA medium</td>
		</tr>
		<tr>
			<td>Pipettes and pipette tips</td>
			<td>Labnet (BioPette PLUS)</td>
			<td></td>
			<td>Used to measure liquids ingredients</td>
		</tr>
		<tr>
			<td>Polyvinylchloride Marley waste pipe</td>
			<td></td>
			<td></td>
			<td>Used to create a neck for the fermentation bag</td>
		</tr>
		<tr>
			<td>Potassium phosphate dibasic (K2HPO4)</td>
			<td>SIGMA-ALDRICH</td>
			<td></td>
			<td>Ingredient of the blastospore liquid medium</td>
		</tr>
		<tr>
			<td>Rubber band</td>
			<td></td>
			<td></td>
			<td>Used to secure the secure the surgical paper over the fermentation bag PVC pipe necks</td>
		</tr>
		<tr>
			<td>Sabaroud dextrose agar (SDA)</td>
			<td>NEOGEN Culture Media</td>
			<td></td>
			<td>Medium used to culture spores of both Metarhizium pinghaense and Metarhizium robertsii</td>
		</tr>
		<tr>
			<td>Sterile distilled water</td>
			<td></td>
			<td></td>
			<td>To hydrate agricultural grains, to make conidial suspensions</td>
		</tr>
		<tr>
			<td>Sticky pad</td>
			<td></td>
			<td></td>
			<td>Used to secure the seives on the vibratory shaker</td>
		</tr>
		<tr>
			<td>Surgical blade</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to scrape off spores from fungal cultures</td>
		</tr>
		<tr>
			<td>Surgical paper</td>
			<td>Lasec</td>
			<td></td>
			<td>Used to cover the PVC necks and cotton wool plugs of the fermentation bag</td>
		</tr>
		<tr>
			<td>Vibratory shaker</td>
			<td></td>
			<td></td>
			<td>Used to shake conidia off the agricultural grain substrates</td>
		</tr>
		<tr>
			<td>Vortex mixer</td>
			<td>Labnet (Labnet International, Inc.)</td>
			<td></td>
			<td>Used to mix conidial suspensions in Mc Cartney bottles</td>
		</tr>
		<tr>
			<td>Yeast extract</td>
			<td>Biolab</td>
			<td></td>
			<td>Added to the SDA medium to improve spore germination and growth</td>
		</tr>
		<tr>
			<td>Zipper-lock bags</td>
			<td>GLAD</td>
			<td></td>
			<td>Used to to store harvested fungal conidia</td>
		</tr>
	</tbody>
</table>

mass production of entomopathogenic fungi, metarhizium robertsii and metarhizium pinghaense, for commercial application against insect pests

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. The increase in pest resistance to chemical insecticides, the growing concerns regarding the negative effects of insecticides on human health, and the environmental pollution from pesticides have led to a global drive to find novel sustainable strategies for crop protection and pest control. Previously, attempts to mass culture such entomopathogenic fungi (EPF) species as Beauveria bassiana have been conducted. However, only limited attempts have been conducted to mass culture Metarhizium robertsii and M. pinghaense for use against insect pests. This study aimed to mass-produce a sufficient number of resilient infective propagules of South African isolates of M. robertsii and M. pinghaense for commercial application. Three agricultural grain products, flaked oats, flaked barley, and rice, were used as the EPF solid fermentation substrates. Two inoculation methods, conidial suspensions and the liquid fungal culture of blastospores were used to inoculate the solid substrates. Inoculation using conidial suspensions was observed to be relatively less effective, as increased levels of contamination were observed on the solid substrates relative to when using the blastospore inoculation method. Flaked oats were found not to be a suitable growth substrate for both M. robertsii and M. pinghaense, as no dry conidia were harvested from the substrate. Flaked barley was found to favor the production of M. robertsii conidia over that of M. pinghaense, and an average of 1.83 g &plusmn; 1.47 g of dry M. robertsii conidia and zero grams of M. pinghaense conidia was harvested from the substrate. Rice grains were found to favor the conidial mass production of both M. pinghaense and M. robertsii isolates, with an average of 8.2 g &plusmn; 4.38 g and 6 g &plusmn; 2 g harvested from the substrate, respectively.

A decline in the content mass of the cultures on rice for both the M. pinghaense and the M. robertsii was observed over time during the drying stage of the fungal cultures, with no, or little, change being observed in the mass once the cultures were dry (Figure 5). The harvested dry fungal conidia powder of both the M. pinghaense and the M. robertsii is shown in Figure 6.
<p class="jove_content" fo:keep-together.within-pag...

Watch this Scientific Journal Video about Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests at JoVE.com

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi have gained importance as the biological control agents of agricultural insect pests. In this study, the mass production of a sufficient number of resilient infective propagules of South African isolates of both Metarhizium robertsii and M. pinghaense for commercial application against insect pests was successfully conducted using agricultural grain products.

Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

Entomopathogenic fungi of the Metarhizium anisopliae species complex have gained importance as the biological control agents of agricultural insect pests. ...

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6.6K Views.  Private Bag X1. Entomopathogenic fungi have gained importance as the biological control agents of agricultural insect pests. In this study, the mass production of a sufficient number of resilient infective propagules of South African isolates of both Metarhizium robertsii and M. pinghaense for commercial application against insect pests was successfully conducted using agricultural grain products.

Video: Mass Production of Entomopathogenic Fungi, Metarhizium robertsii and Metarhizium pinghaense, for Commercial Application Against Insect Pests

A Method For Production of Recombinant mCD1d Protein in Insect Cells.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated heavy chains that are noncovalently associated with beta2-microglobulin. They bind lipids rather than peptides. Although their structure confirms the similarity of CD1 proteins to MHC class I and class II antigen presenting molecules, the mCD1d groove is relatively narrow, deep, and highly hydrophobic and it has two binding pockets instead of the several shallow pockets described for the classical MHC-encoded antigen-presenting molecules. Based upon their amino acid sequences, such a hydrobphobic groove provides an ideal environment for the binding of lipid antigens. The Natural Killer T (NKT) cells use their TCR to recognize glycolipids bound to or presented by CD1d. T cells reactive to lipids presented by CD1 have been involved in the protection against autoimmune and infectious diseases and in tumor rejection. Thus, the ability to identify, purify , and track the response of CD1-reactive NKT cell is of great importance . The generation of tetramers of alpha Galactosyl ceramide (a-Galcer) with CD1d has significant insight into the biology of NKT cells. Tetramers constructed from other CD1 molecules have also been generated and these new reagents have greatly expanded the knowledge of the functions of lipid-reactive T cells, with potential use in monitoring the response to lipid-based vaccines and in the diagnosis of autoimmune diseases and other treatments.

A Method to prepare Insect cells and infect them with baculovirus for the the purpose of production of recombinant mCD1d proteinand generating mCD1d tetramers.

CD1 proteins constitute a third class of antigen-presenting molecules. They are cell surface glycoproteins, expressed as approximately 50-kDa glycosylated ...

Efficient Production and Purification of Recombinant Murine Kindlin-3 from Insect Cells for Biophysical Studies

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and the control of gene transcription in the cell nucleus. The kindlins are ~75 kDa multidomain proteins and bind to an NPxY motif and upstream T/S cluster of the integrin &#946;-subunit cytoplasmic tail. The hematopoietically-important kindlin isoform, kindlin-3, is critical for platelet aggregation during thrombus formation, leukocyte rolling in response to infection and inflammation and osteoclast podocyte formation in bone resorption. Kindlin-3&#39;s role in these processes has resulted in extensive cellular and physiological studies. However, there is a need for an efficient method of acquiring high quality milligram quantities of the protein for further studies. We have developed a protocol, here described, for the efficient expression and purification of recombinant murine kindlin-3 by use of a baculovirus-driven expression system in Sf9 cells yielding sufficient amounts of high purity full-length protein to allow its biophysical characterization. The same approach could be taken in the study of the other mammalian kindlin isoforms.

Kindlins are fundamental to cell adhesion through integrins but studies of them have been hampered by the difficulty encountered in expressing them recombinantly in bacterial hosts. We describe here methods for their efficient production in baculovirus-infected insect cells.

Kindlins are essential coactivators, with talin, of the cell surface receptors&#160;integrins and also participate in integrin outside-in signalling, and ...

Application of Membrane and Cell Wall Selective Fluorescent Dyes for Live-Cell Imaging of Filamentous Fungi

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two decades ago and since then continues to contribute greatly to our understanding of the filamentous fungal lifestyle. This paper provides a practical guide for the utilization of the two membrane dyes FM 1-43 and FM 4-64 and the four cell wall stains Calcofluor White M2R, Solophenyl Flavine 7GFE 500, Pontamine Fast Scarlet 48 and Congo Red. The focus is on their low-dose application to ascertain artefact-free staining, their co-imaging properties, and their quantitative evaluation. The presented methods are applicable to all filamentous fungal samples that can be prepared in the described ways. The fundamental staining approaches can serve as starting points for adaptations to species that might require different cultivation conditions. First, biophysical and biochemical properties are reviewed as their understanding is essential for using these dyes as truly vital fluorescent stains. Secondly, step-by-step protocols are presented that detail the preparation of various fungal sample types for fluorescent live-cell imaging. Finally, example experiments illustrate different approaches to: (1) identify defects in the spatio-temporal organization of endocytosis in genetic mutants, (2) comparatively characterize shared and distinct co-localization of GFP-labeled target proteins in the endocytic pathway, (3) identify morphogenetic cell wall defects in a genetic mutant, and (4) monitor cell wall biogenesis in real time.

Vital fluorescent dyes are essential tools for live-cell imaging analyses in modern fungal cell biology. This paper details the application of established and lesser-known fluorescent dyes for tracking plasma membrane dynamics, endo-/exocytosis and cell wall morphogenesis in filamentous fungi.

The application of membrane and cell wall selective fluorescent dyes for live-cell imaging analyses of organelle dynamics in fungal cells started two ...

Immunofluorescent Labeling of Plant Virus and Insect Vector Proteins in Hemipteran Guts

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are highly efficient at virus transmission plays a key role in virus epidemics in fields. Studying virus-insect vector interactions can advance our understanding of virus transmission and epidemics with the aim of designing novel strategies to control plant viruses and their vector insects. Immunofluorescence labeling has been widely used to analyze interactions between pathogens and hosts and is used here in the white-backed planthopper (WBPH, Sogatella furcifera), which efficiently transmits the southern rice black streaked dwarf virus (SRBSDV, genus Fijivirus, family Reoviridae), to locate the virions and insect proteins in the midgut epithelial cells. Using laser scanning confocal microscopy, we studied the morphological characteristics of midgut epithelial cells, cellular localization of insect proteins, and the colocalization of virions and an insect protein. This protocol can be used to study virus activities in insects, functions of insect proteins, and interactions between virus and vector insect.

This protocol for immunofluorescent labeling of both plant virus proteins and vector insect proteins in excised insect guts can be used to study interactions among virus and vector insects, insect protein functions and molecular mechanisms underlying virus transmission.

Most plant viruses in nature are transmitted from one plant to another by hemipteran insects. A high population density of the vector insects that are ...

Isolation and Selection of Entomopathogenic Fungi from Soil Samples and Evaluation of Fungal Virulence against Insect Pests

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important to isolate and select indigenous EPF. Therefore, the soil bait method combined with the insect bait (mealworm, Tenebrio molitor) system was used in this study with some modifications. The isolated EPF were then subjected to the virulence test against the agricultural pest Spodoptera litura. Furthermore, the potential EPF strains were subjected to morphological and molecular identifications. In addition, the conidia production and thermotolerance assay were performed for the promising EPF strains and compared; these data were further substituted into the formula of effective conidia number (ECN) for laboratory ranking. The soil bait-mealworm system and the ECN formula can be improved by replacing insect species and integrating more stress factors for the evaluation of commercialization and field application. This protocol provides a quick and efficient approach for EPF selection and will improve the research on biological control agents.

Here we present a protocol based on the&#160;mealworm (Tenebrio molitor)-bait system that was used for isolating and selecting entomopathogenic fungi (EPF) from soil samples. An effective conidia number (ECN) formula is used to select high stress tolerant EPF based on physiological characteristics for pest microbial control in the field.

Entomopathogenic fungi (EPF) are one of the microbial control agents for integrated pest management. To control local or invasive pests, it is important ...

Comparison of Methods for Isolating Entomopathogenic Fungi from Soil Samples

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic fungi (EPF) from soil samples. The soil is a rich habitat for microorganisms, including EPF particularly belonging to the genera Metarhizium and Beauveria, which can regulate arthropod pests. Biological products based on fungi are available in the market mainly for agricultural arthropod pest control. Nevertheless, despite the high endemic biodiversity, only a few strains are used in commercial bioproducts worldwide. In the present study, 524 soil samples were cultured on potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium). The growth of fungal colonies was observed for 3 weeks. All Metarhizium and Beauveria EPF were morphologically identified at the genus level. Additionally, some isolates were molecularly identified at the species level. Twenty-four out of these 524 soil samples were also surveyed for EPF occurrence using the insect bait method (Galleria mellonella and Tenebrio molitor). A total of 51 EPF strains were isolated (41 Metarhizium spp. and 10 Beauveria spp.) from the 524 soil samples. All fungal strains were isolated either from croplands or grasslands. Of the 24 samples selected for comparison, 91.7% were positive for EPF using Galleria bait, 62.5% using Tenebrio bait, and 41.7% using CTC. Our results suggested that using insect baits to isolate the EPF from the soil is more efficient than using the CTC medium. The comparison of isolation methods in addition to the identification and conservation of EPF has a positive impact on the knowledge about biodiversity. The improvement of EPF collection supports scientific development and technological innovation.

Entomopathogenic fungal colonies are isolated from tropical soil samples using Tenebrio bait, Galleria bait, as well as selective artificial medium, i.e., potato dextrose agar enriched with yeast extract supplemented with chloramphenicol, thiabendazole, and cycloheximide (CTC medium).

The goal of the present study is to compare the effectiveness of using insect baits versus artificial selective medium for isolating entomopathogenic ...

Optimized Bioassay for Entomopathogenic Fungi Against Mustard Aphid

Assessment of Aphidicidal Effect of Entomopathogenic Fungi against Parthenogenetic Insect, Mustard Aphid, Lipaphis erysimi (Kalt.)

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, entomopathogenic fungi (EPF) are potential microbial control agents for controlling this pest. Therefore, virulence screening of EPF isolates under Petri dish conditions is necessary before field application. However, the mustard aphid is a parthenogenetic insect, making it difficult to record data during Petri dish experiments. A modified system for detached-leaf bioassays was developed to address this issue, using a micro-sprayer to inoculate conidia onto aphids and prevent drowning by facilitating air-drying after spore suspension. The system maintained high relative humidity throughout the observation period, and the leaf disc remained fresh for over ten days, allowing parthenogenetic reproduction of the aphids. To prevent offspring buildup, a process of daily removal using a painting brush was implemented. This protocol demonstrates a stable system for evaluating the virulence of EPF isolates against mustard aphids or other aphids, enabling the selection of potential isolates for aphid control.

Author Spotlight: Eco-Friendly Pest Management Using Entomopathogenic Fungi Against Mustard Aphids 

This protocol presents an optimized detached-leaf bioassay system for evaluating the effectiveness of entomopathogenic fungi (EPF) against the mustard aphid (Lipaphis erysimi (Kalt.)), a parthenogenetic insect. The method outlines the data collection process during Petri dish experiments, enabling researchers to consistently measure the virulence of EPF against mustard aphids and other parthenogenetic insects.

The mustard aphid (L. erysimi) is a pest that infests various cruciferous crops and transmits plant viruses. To achieve eco-friendly pest management, ...

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence period is considered an acceptable alternative to chemical control. However, EPF resources have been significantly less explored for FWBs, in contrast to agricultural insect pests. This paper presents a protocol for exploring EPF resources from FWBs using wild Monochamus alternatus populations as an example. In this protocol, the assignment of traps baited with M. alternatus attractants to different populations guaranteed the collection of adequate samples with natural infection symptoms, during the emergence periods of the beetle. Following finely dissecting integuments and placing them onto a selective medium, fungal species were isolated from each part of beetle bodies and identified based on both molecular and morphological traits.
Several fungal species were certified as parasitic EPFs via re-infection of healthy M. alternatus with spore suspensions. Their behavioral phenotypes on M. alternatus were observed using scanning electron microscopy and further compared with those on the Coleopteran model insect Tribolium castaneum. For EPFs that present consistent parasitism phenotypes on both beetle species, evaluation of their activities on T. castaneum provided valuable information on lethality for future study on M. alternatus. This protocol helped the discovery of EPF newly reported on M. alternatus populations in China, which could be applied as an efficient approach to explore more EPF resources from other FWBs.

Author Spotlight: Evaluation of Entomopathogenic Fungi in Wild Monochamus alternatus Populations for Biocontrol Applications in Forest Wood Borers

Here we present a protocol for obtaining entomopathogenic fungi from a forest wood borer and a substitutive way to evaluate their entomopathogenic activities using a Coleopteran model insect. This method is efficient and convenient for exploring entomopathogenic fungal resources from wood-boring insect pests in natural forests.

Forest wood borers (FWB) cause severe tree damage and economic losses worldwide. The release of entomopathogenic fungi (EPF) during the FWB emergence ...

Advancing RNAi Techniques in Trichogramma dendrolimi for Gene Function Analysis

RNA Interference in the Egg Parasitoid, Trichogramma dendrolimi Matsumura

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and forests. The immature stages of Trichogramma offspring develop within the host egg, exhibiting remarkable diminutiveness (approximately 0.5 mm in adult length). RNA-interference (RNAi) methodology has emerged as a crucial tool for elucidating gene functions in numerous organisms. However, manipulating RNAi in certain small parasitoid species, such as Trichogramma, has generally posed significant challenges. In this study, we present an efficient RNAi method in Trichogramma denrolimi. The outlined procedure encompasses the acquisition and isolation of individual T. dendrolimi specimens from host eggs, the design and synthesis of double-stranded RNA (dsRNA), the in vitro transplantation and cultivation of T. dendrolimi pupae, the micro-injection of dsRNA, and the subsequent assessment of target gene knockdown through RT-qPCR analysis. This study furnishes a comprehensive, visually detailed procedure for conducting RNAi experiments in T. dendrolimi, thereby enabling researchers to investigate the gene regulation in this species. Furthermore, this methodology is adaptable for RNAi studies or micro-injections in other Trichogramma species with minor adjustments, rendering it a valuable reference for conducting RNAi experiments in other endoparasitic species.

Author Spotlight: Investigating Wolbachia-Induced Thelytokous Parthenogenesis and Genetic Toolkit Development Through RNA Interference

The manipulation of RNA interference (RNAi) presents a formidable challenge in many parasitoid species with diminutive size, such as Trichogramma wasps. This study delineated an efficient RNAi method in Trichogramma denrolimi. The present methodology provides a robust model for investigating gene regulation in Trichogramma wasps.

The egg parasitoids, Trichogramma spp, are recognized as efficient biological control agents against various lepidopteran pests in agriculture and ...

Protocol for Wet Beveling of Microinjection Needles Under Constant Air Pressure

Wet Beveling of Microinjection Needles Utilizing Constant Air Pressure for Feedback on Needle Opening

Microinjection needles are a critical tool in the delivery of genome modification reagents, CRISPR components (guide RNAs, Cas9 protein, and donor template), and transposon system components (plasmids and transposase mRNA) into developing insect embryos. Sharp microinjection needles are particularly important during the delivery of these modifying agents since they help minimize damage to the embryo being injected, thereby increasing the survival of these embryos as compared to injection with non-beveled needles. Further, the beveling of needles produces needles that are more consistent from needle to needle as compared to needles opened by randomly breaking the needle tip by brushing the tip against an object (side of a coverslip, the surface of the embryo to be injected, etc.). The process of wet beveling of microinjection needles with constant pressure air delivered to the needle allows the person beveling the needle to know when the needle is open (presence of bubbles) and also gives a relative indication of how large a needle opening has been created. The relative opening size in the needle can be determined by adjusting the air pressure delivered to the needle until an equilibrium is reached and bubbles stop flowing from the tip of the needle. The lower the pressure at which the equilibrium is reached, the larger the needle size; and conversely, the higher the pressure, the smaller the needle size.

Author Spotlight: Enhancing Microinjection Needle Quality by Wet Beveling

This protocol describes the assembly of a pneumatic system for the delivery of pressurized air to a needle during the process of needle beveling. The protocol further describes the beveling process for creating sharp microinjection needles and how to gauge the relative opening size of the needle.