This research was supported by the National Key Research and Development Program of China (2021YFC2600100) and the Natural Science Foundation of Zhejiang Province (LY21C040001).

1. Isolation of fungi from M. alternatus (Figure 1)
<ol>
	<li>Collect the beetle sample
	<ol>
		<li>Collect the pines sawyer beetles M. alternatus using commercial traps (see Table of Materials) baited with attractants before the predicted emergence periods of the beetle populations in naturally infected pine forests. 
		NOTE: In this study, beetles were collected from five geographical regions of southern China (Huzhou/Zhejiang, Liangshan/Sichuan, Xiamen/Fujian, Shaoguan/Guangdong, Yulin/Guangxi). The trap setting from top to bottom is as follows: a round top (50 cm in diameter), a cross-shaped panel (35 cm in width and 66 cm in length), a funnel (35.5 cm in top round diameter and 5.5 cm in bottom round diameter), a collection cup (10.5 cm in diameter and 26.5 cm in length). The host volatiles (e.g., ethanol and &#945;-pinene) and the aggregation pheromone (2-undecyloxy-1-ethanol) are used as the bait30.</li>
		<li>Label the specimen before transferring it to the laboratory. Put each beetle in individual sterilized tubes with fresh twigs. Replace twigs with fresh ones every 2 days.</li>
		<li>Rear the alive beetles at 25 &#177; 1 &#176;C in a non-humidified incubator (16-8 L/D cycles) and observe them daily.</li>
		<li>Record samples showing decreased feeding and mobility. Transfer dead M. alternatus that become hardened and stiff to wet chambers to observe the growth of fungal mycelia and conidia. 
		NOTE: Once infected by entomopathogenic&#160;fungi, the beetles displayed a decrease in feeding and mobility at the initial stage of infection31,32. After death, their bodies became hardened and stiff, with mycelia and conidia appearing several days after storage in a wet chamber33.</li>
		<li>Store beetle cadavers with fungal infections at 4 &#176;C for future use. To follow this protocol, process the samples immediately within a few hours to avoid contamination with more saprotrophic fungi.</li>
	</ol>
	</li>
	<li>Preparation of growth medium
	<ol>
		<li>Add 39 g of potato dextrose agar (PDA) powder into 1 L of pure water and autoclave it for 30 min at 121 &#176;C.</li>
		<li>Cool to an operable temperature (~50 &#176;C), add 0.05 g of streptomycin, 0.05 g of penicillin G, and 0.05 g of tetracycline, and shake to distribute the medium.</li>
		<li>Plate the medium into Petri dishes under a clean bench and use after solidification. 
		NOTE: The PDA medium with antibiotics is used for isolation from beetles, which prevents the growth of bacteria without affecting fungi growth during isolation. However, the PDA medium without antibiotics is used for daily cultivation and conservation.</li>
		<li>Put 35 g of potato dextrose broth (PDB) powder into 1 L of ddH2O and autoclave it for 30 min at 121 &#176;C. Cool it for use.</li>
	</ol>
	</li>
	<li>Dissection of the beetle sample with fungal infection symptoms
	<ol>
		<li>Prepare sterilized scissors, scalpels, and insect pins; then, work under a clean bench with an alcohol burner. 
		NOTE: The use of dissection tools can be adjusted according to personal preferences. Insect pins are sharper than standard dissecting needles, and different specifications can meet the need for dissection.</li>
		<li>Dissect the beetle body integuments with tools in sterile and single-use Petri dishes. Take care to avoid possible damage to the midgut and hindgut tissues that may exude internal contents.</li>
		<li>Divide the beetle bodies into the main positions as follows: antennae, head, thorax, abdomen, wings (each pair), and legs.</li>
	</ol>
	</li>
	<li>Fungi purification
	<ol>
		<li>Cut the integuments of each position into small pieces with scissors. Gently press the outer surface onto the surface of PDA plates with antibiotics. 
		NOTE: Make sure that fungal mycelia on the integuments have been inoculated to the plate.</li>
		<li>Seal with parafilm carefully, and culture in an incubator at 25 &#177; 1 &#176;C until the tissues are thoroughly covered by mycelium.</li>
		<li>Transfer single colonies according to phenotypic properties (color, shape) onto a new PDA with antibiotics for culture at 25 &#177; 1 &#176;C.</li>
		<li>Repeat step 1.4.3 twice or three times on PDA with antibiotics until pure colonies are isolated separately. 
		&#8203;NOTE: If multiple fungal strains grow in the original plates, pick out the mycelium precisely to facilitate the sorting of more fungal species.</li>
	</ol>
	</li>
	<li>Storage of the fungal isolates
	<ol>
		<li>Transfer agar blocks (~5 mm in diameter) from colonies to new PDA plates.</li>
		<li>Culture in an incubator at 25 &#177; 1 &#176;C for 1-2 weeks.</li>
		<li>Place in a 4 &#176;C refrigerator for daily storage for further use.</li>
		<li>Inoculate fungal strains into 50 mL of sterile PDB in a 250 mL flask, shaking at 180 rpm/min at 25 &#176;C for 5-7 days.</li>
		<li>Harvest 1 mL of the freshly grown fungal mycelium and suspend it in 1 mL of sterilized 20% glycerol in 2 mL sterile freezer tubes (the final concentration of glycerol is 10%).</li>
		<li>Seal the tubes with parafilm and precool them at -20 &#176;C and -40 &#176;C. Then, maintain the tubes at -80 &#176;C as stocks.</li>
	</ol>
	</li>
</ol>
2. Molecular and morphological identification of fungal isolates
<ol>
	<li>Extraction of fungal genomic DNA
	<ol>
		<li>Culture fungi in PDB as described in step 1.5.4.</li>
		<li>Harvest the mycelium and filter it to separate it from the PDB. Homogenize in liquid nitrogen using a precooled mortar and pestle.</li>
		<li>Extract the genomic DNA using the Fungi Genomic DNA Isolation Kit according to the manufacturer&#39;s instructions (see Table of Materials).</li>
	</ol>
	</li>
	<li>PCR amplification and DNA sequencing
	<ol>
		<li>Prepare the PCR reaction mixture as follows: 25 &#181;L of Taq DNA polymerase, 1 &#181;L of template, 2 &#181;L of forward and reverse primers, and ddH2O to a total volume of 50 &#181;L.</li>
		<li>Amplify the rDNA-ITS region of the DNA sample using primer pairs ITS1 and ITS434 (see Table 1) with the following procedure: initial denaturing at 95 &#176;C for 4 min, followed by 35 cycles of denaturing at 94 &#176;C for 60 s, annealing at 58 &#176;C for 60 s, elongation at 72 &#176;C for 2 min, and a final elongation at 72 &#176;C for 10 min.</li>
		<li>Electrophoresis of amplified products using 1.5% agarose gel at 100 V, 100 mA.</li>
		<li>Sequence the PCR.</li>
		<li>Align the sequence using Clustal X2.0 and MEGA 6.0. 
		NOTE: During sequence alignment, sites with ambiguous alignment are excluded, and gaps are treated as missing data.</li>
		<li>Compare the obtained sequence with those in the ITS sequence database in GenBank using BLAST on the National Center for Biotechnology Information (NCBI) website. Identify the fungal strain species information preliminarily.</li>
	</ol>
	</li>
	<li>Morphological Identification
	<ol>
		<li>Use a camera to capture the mature fungal pure colony morphology on both the front and reverse sides of the PDA plates.</li>
		<li>Pluck conidia from the pure culture fungal colonies with an inoculation needle and transfer them to a glass slide with a drop of sterile water.</li>
		<li>Hold the cover slip and slowly put it down at an angle of ~45&#176;, so that the cover slip can cover the specimen without bubbles.</li>
		<li>Cut a 5 mm2 agar block with a scalpel from colonies at the edge of the fungal colony, and transfer it to a clean glass slide with a drop of sterile water. Carefully tease apart fungi with a fine needle to see specific structures.</li>
		<li>Repeat step 2.3.3.</li>
		<li>Observe the asexual morph of the fungi under an optical microscope (OM), including the shape, transparency, and posture of the hyphae, conidiophores, phialides, and conidia. Record and measure the shape and size of the asexual characteristics to distinguish the different fungal isolates from each other. 
		&#8203;NOTE: To maximize the ability to observe fungal structures, use stains and mounting medium35.</li>
	</ol>
	</li>
</ol>
3. Induction of the fungal infection symptoms on M. alternatus to observe their behavioral phenotypes
<ol>
	<li>Preparation of conidial suspension
	<ol>
		<li>Prepare 0.01% Tween-80 with pure water and autoclave it for 25 min at 121 &#176;C. Use after cooling.</li>
		<li>Add an appropriate amount of sterile small glass beads and 0.01% Tween-80 solution to a 2 mL centrifuge tube.</li>
		<li>Scrape fungal colonies into the tube and shake it sufficiently with a vortex shaker until the colony is broken up. Filter through two layers of gauze to remove the mycelium. 
		NOTE: Shaking is done to ensure the suspension of conidiospore in 0.01% Tween-80 solution.</li>
		<li>Count the number of spores under the OM with a hemocytometer, adjust to 1 &#215; 108 conidial/mL with sterile 0.01% Tween-80, and keep at 4 &#176;C for use.</li>
	</ol>
	</li>
	<li>Preparation of beetles
	<ol>
		<li>Autoclave the pine twigs (about the same size) and dry in the oven (~ 60 &#176;C).</li>
		<li>Keep field-collected M. alternatus adults with pine twigs in an incubator at 25 &#177; 1 &#176;C.</li>
		<li>Starve beetles for 24 h and sterilize the beetles&#39; surface with bleach, ethanol, and distilled water [10:10:80 (v:v)] before use.</li>
	</ol>
	</li>
	<li>Induction of the fungal infection
	<ol>
		<li>Work under a clean bench, immerse the pine twigs into conidial suspension for 10 s, and dry them in the air.</li>
		<li>Dip beetles in conidial suspension for 10 s, then transfer to 50 mL sterile tubes (one beetle and one twig per tube). Replace the twigs with new ones every 2 days.</li>
		<li>For negative control, change the conidial suspension into only sterile 0.01% Tween-80 solution. Make five replicates for each fungal strain and control group.</li>
		<li>Assess the activity of beetles based on the amount of frass produced on pine twigs. When feeding ceases, consider them dead.</li>
		<li>Transfer the dead beetles into new sterile 50 mL tubes and place a piece of sterile moist cotton. Incubate at 25 &#177; 1 &#176;C. 
		&#8203;NOTE: The wet cotton is used to maintain humidity, as the growth of fungi requires appropriate moisture, but not too much.</li>
		<li>Observe and photograph the change of beetle surface at the same time every day.</li>
	</ol>
	</li>
	<li>Re-isolation and confirm fungal species
	<ol>
		<li>Until clear fungal infection phenotypes appear on the surface of beetles, pick mycelium from different tissues individually into new PDA plates.</li>
		<li>Culture at 25 &#177; 1 &#176;C and observe to ensure the morphology is consistent with that of the inoculated fungi.</li>
	</ol>
	</li>
	<li>Treatment of model beetle
	<ol>
		<li>Repeat steps 3.2.1-3.3.6 on the model beetle Tribolium castaneum, using wheat bran as food, and sterilize wheat bran with UV light.</li>
		<li>Touch beetles lightly with sterile tweezers. Assume they are dead if there is no response.</li>
	</ol>
	</li>
</ol>
4. Confirm the infection phenotypes of fungi on M. alternatus and model beetle
<ol>
	<li>Sample preparation for observation
	<ol>
		<li>Dissect the infected M. alternatus cadavers carefully as in step 1.3.</li>
		<li>Pick T. castaneum bodies with obvious symptoms of fungal infection.</li>
		<li>Cut 5 mm disc plugs of four mature fungal colonies growing on PDA.</li>
	</ol>
	</li>
	<li>Sample pretreatment
	<ol>
		<li>Put plugs, infected beetle tissues, and bodies into prechilled 2.5% glutaraldehyde at 4 &#176;C for 2 days.</li>
		<li>Wash samples thrice in 0.1% phosphate buffer (pH 7.2-7.4) for 5 min and dehydrate in 30%, 50%, 70%, 80%, 90%, 95%, 100% ethanol for 10 min.</li>
		<li>Dry the samples in a vacuum freeze dryer. 
		&#8203;NOTE: The entire pretreatment process should be careful, including cutting, soaking, dehydration, washing, and drying, to prevent sample damage.</li>
	</ol>
	</li>
	<li>Scanning electron microscopy (SEM) observation
	<ol>
		<li>Coat the pretreated samples with platinum using a sputter coater, and observe under a scanning electron microscope36.</li>
		<li>Record the morphological characteristics of hyphae and conidia growing on PDA, M. alternatus tissues, and model beetle bodies.</li>
		<li>Compare if the results are consistent with those under OM in step 2.3.</li>
	</ol>
	</li>
</ol>
5. Evaluation of entomopathogenic activity
<ol>
	<li>Preparation of conidial suspension
	<ol>
		<li>The preparation of conidial suspension is the same as in steps 3.1.1-3.1.4.</li>
	</ol>
	</li>
	<li>Pretreatment of model beetle
	<ol>
		<li>Culture, feed, sterilize, and starve T. castaneum as in step 3.5.1.</li>
		<li>Sterilize wheat bran by UV, and place equal weight to sterile Petri dishes.</li>
	</ol>
	</li>
	<li>Entomopathogenic activity test
	<ol>
		<li>Treat wheat bran with the conidial suspension and dry them in the air under a clean bench at room temperature.</li>
		<li>Immerse T. castaneum beetles in the conidial suspension for 10 s and transfer them to a Petri dish (n = 20 in each Petri dish). Replace new wheat bran with the same conidial treatment every 4 days.</li>
		<li>For negative control, apply only sterile 0.01% Tween-80 solution. 
		NOTE: Set aside at least three replicates for each treatment group and the control.</li>
		<li>Count the dead beetles daily. 
		NOTE: Touch beetles lightly with sterile tweezers. Assume they are dead if there is a lack of response.</li>
	</ol>
	</li>
	<li>Multi-gene phylogenetic analysis
	<ol>
		<li>Extract entomopathogenic fungal genomic DNA as described in step 2.1. 
		NOTE: Multi-gene identification is conducted for the parasitic EPFs that show significant infection phenotypes and strong entomopathogenic activities against the model beetle.</li>
		<li>Amplify the following genes, including a region spanning the nuclear ribosomal SSU34 gene, a segment of the large subunit rRNA gene (LSU37,38), part of the elongation factor 1-alpha (tef-1&#945;39) gene, the second largest subunit sequences of RNA polymerase &#1030;&#1030; (rpb240), and part of the &#946;-tubulin41 gene, using the primer pairs NS1/NS4, LR7/LROR, EF-983F/EF-2218R, RPB2-5&#39;F/RPB2-5&#39;R, and TUB1/TUB22 (see Table 1), respectively.
		<ol>
			<li>Prepare the PCR reaction mixture as in step 2.2.1.</li>
			<li>Carry out amplification as follows: initial denaturing at 95 &#176;C for 4 min, followed by 35 cycles (tef-1&#945;, rpb2, SSU), 38 cycles (LSU) or 39 cycles (&#946;-tubulin) of denaturing at 94 &#176;C for 60 s, annealing at 47 &#176;C for 60 s (LSU), 55 &#176;C for 60 s (tef-1&#945;), 54 &#176;C for 40 s (&#946;-tubulin), and 50 &#176;C for 30 s (rpb2, SSU), elongation at 72 &#176;C for 1 min, and a final elongation at 72 &#176;C for 10 min.</li>
		</ol>
		</li>
		<li>Sequence and align following the same steps as shown in steps 2.2.4-2.2.5.</li>
		<li>Perform the phylogenetic analysis by MEGA 6.0 based on the Maximum Likelihood (ML) method42.</li>
	</ol>
	</li>
</ol>

Exploring Entomopathogenic Fungi Resources from Forest Wood Borers

The authors declare no conflicts of interest.

Different geographical populations of FWB may develop varied interactions with the natural entomopathogenic fungi, due to long-term environmental adaptation of EPF species to local climate factors and the specific genotypic population of the host insect44,45. Expansion of the sampling sites to multiple insect occurrence regions helps increase the possibility of acquiring diverse strains or species of EPF from their natural hosts, as described by previous studies ...

The devastation caused by insect pests has led to great ecological and economic losses in both forest and agricultural ecosystems. Most agricultural pests expose themselves to natural enemies or artificial control agents while damaging host plants. Instead, forest wood borers (FWB) nearly complete their whole developmental cycles inside host tree trunks1, which raises large challenges to explore efficient biocontrol organisms from FWB in the wild field. What is even worse is that FWBs carry a great number of phytopathogens2 or have an intimate relationship with these pathogens as their potential vectors3,4, dramatically amplifying the negative effects of FWB on forest health. Excessive use of chemical insecticides can alleviate FWB severity, but the emergence of insecticidal resistance5,6 limits their environmental application. In certain cases, insect parasitoids, predatory arthropods as well as entomopathogenic microbes were released as biocontrol agents to the distribution areas of FWB7 and were proven to be efficient and economically acceptable alternatives to chemical control8,9,10.
Entomopathogenic fungi (EPF) are regarded to have the advantage in controlling FWB over most other microbial groups. Their spores can be carried by insect hosts and stably fixed on body surfaces via penetration into the cuticle or integument8,11. EPF also present excellent adaptability to environmental stresses and some species colonize well in the tissue of trees as endophytes12,13, facilitating their growth, survival, and transmission. However, compared to that in agricultural industries, the species diversity of EPF used in natural forest ecosystems is remarkably restricted14,15,16. Beauveria bassiana (strain PPRI 5339) was evidenced as the most promising strain to promote an IPM program to Eucalyptus weevils in South Africa17 and the combination of two promising isolates of B. bassiana provided an opportunity for the practical microbial control of red palm weevil, Rhynchophorus ferrugineus, at different life stages in palm tree fields18. In addition to Beauveria and the well-known Metarhizium, other EPF genera of the order Hypocreales, especially species of Lecanicillium (many of which are now classified into the genus Akanthomyces19,20), showed strong pathogenicity and high potential in management of forest pests, such as the Cypress aphid in Chile21.
The pine sawyer beetle Monochamus alternatus is a notorious pine forest pest in China and neighboring countries, which burrows into branches and trunks of pine trees to impede the transportation of nutrients and water22,23,24. Moreover, M. alternatus also promotes the invasion of the plant-parasitic pine wood nematode (Bursaphelenchus xylophilus, PWN) as its main vector beetle. Another congeneric species of the beetle, M. galloprovincialis, has spread PWN in several countries in Europe in recent years25. Previous research reported several genera of natural EPFs from Monochamus spp., such as Beauveria, Metarhizium, and Lecanicillium (Verticillium, an even former name of Lecanicillium), in Spain, Japan, and the Anhui/Zhejiang Provinces of China26,27,28,29. Nevertheless, these collections of EPFs seem to be commonly restricted in a certain location, compared to the wide occurrence of Monochamus beetles in natural fields. As the M. alternatus beetle has a wide geographical distribution in China, it could be regarded as a representative wood borer to explore more potential EPFs across different populations.
In the present protocol, we introduce a specific procedure exploring EPFs from several geographical populations of M. alternatus in southern China. This protocol uses a model Coleopteran beetle as a substitute to perform entomopathogenicity assays, under the condition that the tested fungal species has a consistent behavioral phenotype on both beetle species. This protocol can also provide insights into EPF exploration for other forest wood borers, in which the diversity of their entomopathogenic fungal species is underestimated or less investigated.

<table><tbody><tr><td>1.5 mL, 2 mL centrifuge tubes</td><td>Biosharp</td><td>BS-15-M</td><td></td></tr><tr><td>10 &amp;micro;L pipet tips</td><td>Sangon Biotech</td><td>F601216</td><td></td></tr><tr><td>10 &amp;micro;L, 20 &amp;micro;L, 100 &amp;micro;L, 200 &amp;micro;L, 1,000 &amp;micro;L pipettes</td><td>Rainin</td><td></td><td></td></tr><tr><td>1,000 &amp;micro;L pipet tips</td><td>Sangon Biotech</td><td>F630102</td><td></td></tr><tr><td>2 mL cryogenic vials</td><td>Corning</td><td>430659</td><td></td></tr><tr><td>20x PBS buffer</td><td>Sangon Biotech</td><td>B548117-0500</td><td></td></tr><tr><td>200 &amp;micro;L pipet tips</td><td>Sangon Biotech</td><td>F601227</td><td></td></tr><tr><td>2,000 bp maker</td><td>TaKaRar</td><td>SD0531</td><td></td></tr><tr><td>50 mL tubes</td><td>Nest</td><td>602052</td><td></td></tr><tr><td>50% glutaraldehyde solution</td><td>Sangon Biotech</td><td>G916054</td><td></td></tr><tr><td>50x TAE buffer</td><td>Sangon Biotech</td><td>B548101</td><td></td></tr><tr><td>6x loading buffer</td><td>TaKaRar</td><td>SD0503</td><td></td></tr><tr><td>Agarose</td><td>Sangon Biotech</td><td>A610013</td><td></td></tr><tr><td>Anhydrous ethanol</td><td>Jkchemical</td><td>LB10V37</td><td></td></tr><tr><td>Biochemistry Cultivation Cabinet</td><td>Shanghaiyiheng</td><td>LRH-250F</td><td></td></tr><tr><td>Chloroform</td><td>Juhua</td><td>61553</td><td></td></tr><tr><td>Commercial beetle traps</td><td>FEIMENGDI</td><td>BF-8</td><td>www.yinyouji.com</td></tr><tr><td>Gel imager</td><td>Bio-Rad</td><td>GelDoc XR+</td><td></td></tr><tr><td>Glycerol</td><td>Sangon Biotech</td><td>A600232</td><td></td></tr><tr><td>High speed refrigerated centrifuge</td><td>Sigma</td><td>D-37520</td><td></td></tr><tr><td>High-Pressure Steam Sterilization Pot</td><td>Mettler Toledo</td><td>JA5003</td><td></td></tr><tr><td>Isopropyl alcohol</td><td>General-reagent</td><td>G75885B</td><td></td></tr><tr><td>Nucleic acid dye</td><td>Sangon Biotech</td><td>A616696</td><td></td></tr><tr><td>Optical Microscope, OM</td><td>Leica</td><td>DM2000</td><td></td></tr><tr><td>Parafilm</td><td>Parafilm</td><td>PM996</td><td></td></tr><tr><td>PCR meter</td><td>Heal Force</td><td>Trident960</td><td></td></tr><tr><td>Penicillin G</td><td>Marklin</td><td>GB15743</td><td></td></tr><tr><td>Potato dextrose agar, PDA</td><td>Oxoid</td><td>CM0139</td><td></td></tr><tr><td>Potato dextrose broth, PDB</td><td>Solarbio</td><td>P9240</td><td></td></tr><tr><td>Primers</td><td>Sangon Biotech</td><td>/</td><td></td></tr><tr><td>Primers Taq</td><td>TaKaRar</td><td>RR902A</td><td></td></tr><tr><td>Rapid Fungi Genomic DNA Isolation Kit</td><td>Sangon Biotech</td><td>B518229</td><td></td></tr><tr><td>Scanning Electron Microscope, SEM</td><td>Hitachi</td><td>S-3400N</td><td></td></tr><tr><td>Streptomycin</td><td>Marklin</td><td>S6153</td><td></td></tr><tr><td>Tetracycline</td><td>Marklin</td><td>T829835</td><td></td></tr><tr><td>Tween-80</td><td>Marklin</td><td>T6336</td><td></td></tr><tr><td>Vacuum freeze dryer</td><td>Yamato</td><td>DC801</td><td></td></tr><tr><td>Vortex Shaker</td><td>HLD</td><td>WH-861</td><td></td></tr><tr><td>&amp;beta;-Mercaptoethanol</td><td>Marklin</td><td>M6230</td><td></td></tr></tbody></table>

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.

Isolation and identification of fungal isolates from M. alternatus 
With the aid of attractant traps, a large number (approximately 500 beetles in total) of M. alternatus were collected from five geographical regions. Beetle cadavers with typical symptoms of infection by entomopathogenic fungi were picked; then, body integuments of every beetle were dissected into several positions as described in protocol step 1.3. As a result, more than 600 fungal isolates were ...

Read this Scientific Article Protocol about Isolation, Behavioral Identification, and Pathogenicity Assessment of Entomopathogenic Fungi from a Forest Wood Borer at JoVE.com

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.

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 ...

isolation-behavioral-identification-pathogenicity-assessment

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Biology

656 Views.  Zhejiang A&F University. We onderzoeken entomopathogene schimmelbronnen van wilde Monochamus alternatus-populaties. Door deze soort als voorbeeld te nemen, zouden we de diversiteit aan entomopathogene schimmels van andere boshoutboorders kunnen verrijken voor toekomstige biologische bestrijdingstoepassingen. We hebben onlangs drie schimmelsoorten verkregen die zijn gecertificeerd als parasitaire entomopathogene schimmels van Monochamus alternatus, waarvan er twee niet zo bekend zijn,...

Video: Auteur in de schijnwerpers: Evaluatie van entomopathogene schimmels in wilde Monochamus alternatus-populaties voor biologische bestrijdingstoepassingen in

Isolation and Analysis of Hematopoietic Stem Cells from the Placenta

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. All HSCs emerge during embryonic development, after which their pool size is maintained by self-renewing cell divisions. Identifying the anatomical origin of HSCs and the critical developmental events regulating the process of HSC development has been complicated as many anatomical sites participate during fetal hematopoiesis. Recently, we identified the placenta as a major hematopoietic organ where HSCs are generated and expanded in unique microenvironmental niches (Gekas, et al 2005, Rhodes, et al 2008). Consequently, the placenta is an important source of HSCs during their emergence and initial expansion.
In this article, we show dissection techniques for the isolation of murine placenta from E10.5 and E12.5 embryos, corresponding to the developmental stages of initiation of HSCs and the peak in the size of the HSC pool in the placenta, respectively. In addition, we present an optimized protocol for enzymatic and mechanical dissociation of placental tissue into single-cell suspension for use in flow cytometry or functional assays. We have found that use of collagenase for single-cell suspension of placenta gives sufficient yields of HSCs. An important factor affecting HSC yield from the placenta is the degree of mechanical dissociation prior to, and duration of, enzymatic treatment.
We also provide a protocol for the preparation of fixed-frozen placental tissue sections for the visualization of developing HSCs by immunohistochemistry in their precise cellular niches. As hematopoietic specific antigens are not preserved during preparation of paraffin embedded sections, we routinely use fixed frozen sections for localizing placental HSCs and progenitors.

We have identified the placenta as a major hematopoietic organ during development. We found that hematopoietic stem cells (HSCs) are both generated and expanded in the placenta in unique microenvironmental niches. Here, we describe experimental techniques required for isolation and visualization of HSCs in the mouse placenta.

Hematopoietic stem cells (HSCs) have the ability to self-renew and generate all cell types of the blood lineages throughout the lifetime of an individual. ...

Psychophysiological Stress Assessment Using Biofeedback 

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous system, previously thought to be outside of conscious control. By letting people observe signals from their own bodies, biofeedback enables them to develop greater awareness of their physiological and psychological reactions, such as stress, and to learn to modify these reactions. Biofeedback practitioners can facilitate this process by assessing people s reactions to mildly stressful events and formulating a biofeedback-based treatment plan. During stress assessment the practitioner first records a baseline for physiological readings, and then presents the client with several mild stressors, such as a cognitive, physical and emotional stressor. Variety of stressors is presented in order to determine a person's stimulus-response specificity, or differences in each person's reaction to qualitatively different stimuli. This video will demonstrate the process of psychophysiological stress assessment using biofeedback and present general guidelines for treatment planning.

Psychophysiological Stress Assessment Using Biofeedback

In this video we will describe one method of assessing person's psychophysiological reaction to stress using biofeedback. We will also present general guidelines for treatment planning.

In the last half century, research in biofeedback has shown the extent to which the human mind can influence the functioning of the autonomic nervous ...

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ease of growth and small size. This small size, though, has disallowed a number of technical approaches found in other model systems. For example, blood transfusions in mammalian systems and grafting techniques in plants enable asking questions of circulatory system composition and signaling. The circulatory system of the worm, the pseudocoelom, has until recently been impossible to assay directly. To answer questions of intercellular signaling and circulatory system composition C. elegans researchers have traditionally turned to genetic analysis, cell/tissue specific rescue, and mosaic analysis. These techniques provide a means to infer what is happening between cells, but are not universally applicable in identification and characterization of extracellular molecules. Here we present a newly developed technique to directly assay the pseudocoelomic fluid of C. elegans. The technique begins with either genetic or physical manipulation to increase the volume of extracellular fluid. Afterward the animals are subjected to a vampiric reverse microinjection technique using a microinjection rig that allows fine balance pressure control. After isolation of extracellular fluid, the collected fluid can be assayed by transfer into other animals or by molecular means. To demonstrate the effectiveness of this technique we present a detailed approach to assay a specific example of extracellular signaling molecules, long dsRNA during a systemic RNAi response. Although characterization of systemic RNAi is a proof of principle example, we see this technique as being adaptable to answer a variety of questions of circulatory system composition and signaling.

Vampiric Isolation of Extracellular Fluid from Caenorhabditis elegans

The model organism C. elegans uses pseudocoelomic fluid as a passive circulatory system. Direct assay of this fluid has not been previously possible. Here we present a novel technique to directly assay the extracellular space, and use systemic silencing signals during an RNAi response as a proof of principle example.

The genetically tractable model organism C. elegans has provided insights into a myriad of biological questions, enabled by its short generation time, ...

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, fatigability, and recovery after fatigue can be obtained to assess specific aspects of the excitation-contraction coupling (ECC) process such as excitability, contractile machinery and Ca2+ handling ability. This method removes the nerve and blood supply and focuses on the isolated skeletal muscle itself. We routinely use this method to identify genetic components that alter the contractile property of skeletal muscle though modulating Ca2+ signaling pathways. Here, we describe a newly identified skeletal muscle phenotype, i.e., mechanic alternans, as an example of the various and rich information that can be obtained using the in vitro muscle contractility assay. Combination of this assay with single cell assays, genetic approaches and biochemistry assays can provide important insights into the mechanisms of ECC in skeletal muscle.

Ex Vivo Assessment of Contractility, Fatigability and Alternans in Isolated Skeletal Muscles

We describe a method to directly measure muscle force, muscle power, contractile kinetics and fatigability of isolated skeletal muscles in an in vitro system using field stimulation. Valuable information on Ca2+ handling properties and contractile machinery of the muscle can be obtained using different stimulating protocols.

 
Described here is a method to measure contractility of isolated skeletal muscles. Parameters such as muscle force, muscle power, contractile kinetics, ...

Isolation and Functional Analysis of Mitochondria from Cultured Cells and Mouse Tissue

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of cell heath due to their role in both cell metabolism and energy production as well as control of apoptosis. Therefore, direct evaluation of isolated mitochondria and mitochondrial perturbation offers the ability to determine if organelle-specific (dys)function is occurring. The methods described in this protocol include isolation of intact, functional mitochondria from HEK cultured cells and mouse liver and spinal cord, but can be easily adapted for use with other cultured cells or animal tissues. Mitochondrial function assessed by TMRE and the use of common mitochondrial uncouplers and inhibitors in conjunction with a fluorescent plate reader allow this protocol not only to be versatile and accessible to most research laboratories, but also offers high throughput.

Comparison of mitochondrial membrane potential between samples yields valuable information about cellular status. Detailed steps for isolating mitochondria and assessing response to inhibitors and uncouplers using fluorescence are described. The method and utility of this protocol are illustrated by use of a cell culture and animal model of cellular stress.

Comparison between two or more distinct groups, such as healthy vs. disease, is necessary to determine cellular status. Mitochondria are at the nexus of ...

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. To improve the mating efficiency of B. Xylophilus and to increase the chances of mating observation, virgin adults were cultured and used for the investigation. Eggs were obtained by keeping the nematodes in water and allowing the females to lay eggs for 10 min. The second-stage juveniles (J2) were synchronized by incubating the eggs for 24 h at 25 &#176;C in the dark, and the early J4 were obtained by culturing the J2 with grey mold, Botrytis cinerea, for another 52 h. At this time point, most J4 nematodes could be clearly distinguished as being male or female using their genital morphology. The male and female J4 were collected and cultured separately in two different Petri dishes for 24 h to get virgin adult nematodes. A virgin male and a virgin female were paired in a drop of water in the well of a concave slide. The mating behavior was filmed with a video recorder under a stereomicroscope. The whole period of the mating process was 82.8 &#177;3.91 min (mean &#177;SE) and could be divided into 4 different phases: searching, contacting, copulating, and lingering. The mean minutes of duration were 21.8 &#177; 2.0, 28.0 &#177; 1.9, 25.8 &#177; 0.7 and 7.2 &#177; 0.5, respectively. Eleven sub-behaviors were described: cruising, approaching, encountering, touching, hooping, locating, attaching, ejaculating, separating, quiescence, and roaming. Interestingly, obvious intra-sexual competition was observed when one female was grouped with 3 males or one male with 3 females. This protocol is useful and valuable, not only in investigating the mating behavior of B. xylophilus, but also in acting as a reference for ethological studies of other nematodes.

Observation and Quantification of Mating Behavior in the Pinewood Nematode, Bursaphelenchus xylophilus

A protocol for investigating the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, is presented. Behavioral features of B. xylophilus are described in the mating process.

A method for observing and quantifying the mating behavior of the pinewood nematode, Bursaphelenchus xylophilus, was established under a stereomicroscope. ...

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 ...

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.

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.

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

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 ...

Isolation and Screening from Soil Biodiversity for Fungi Involved in the Degradation of Recalcitrant Materials

Environmental pollution is an increasing problem, and identifying fungi involved in the bioremediation process is an essential task. Soil hosts an incredible diversity of microbial life and can be a good source of these bioremediative fungi. This work aims to search for soil fungi with bioremediation potential by using different screening tests. Mineral culture media supplemented with recalcitrant substances as the sole carbon source were used as growth tests. First, soil dilutions were plated on Petri dishes with mineral medium amended with humic acids or lignocellulose. The growing fungal colonies were isolated and tested on different substrates, such as complex mixtures of hydrocarbons (petrolatum and used motor oil) and powders of different plastic polymers (PET, PP, PS, PUR, PVC). Qualitative enzymatic tests were associated with the growth tests to investigate the production of esterases, laccases, peroxidases, and proteases. These enzymes are involved in the main degradation processes of recalcitrant material, and their constitutive secretion by the examined fungal strains could have the potential to be exploited for bioremediation. More than 100 strains were isolated and tested, and several isolates with good bioremediation potential were found. In conclusion, the described screening tests are an easy and low-cost method to identify fungal strains with bioremediation potential from the soil. In addition, it is possible to tailor the screening tests for different pollutants, according to requirements, by adding other recalcitrant substances to minimal culture media.

Here, we present a protocol for screening soil biodiversity to look for fungal strains involved in the degradation of recalcitrant materials. First, fungal strains able to grow on humic acids or lignocellulose are isolated. Their activity is then tested both in enzymatic assays and on pollutants such as hydrocarbons and plastics.

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

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