Establishment of a Chilli Thrips (Scirtothrips dorsalis Hood) Rearing System for Virulence Screening of Entomopathogenic Fungi

Summary

A detached-leaf bioassay protocol was established for mass-rearing chilli thrips using a modified detached-leaf method in agar-containing glass containers, ensuring high survival and synchronized development. This system enables efficient screening of entomopathogenic fungi virulence against 2^nd instar larvae and provides a reliable thrips supply for laboratory, greenhouse, and field evaluations.

Abstract

The chilli thrips (Scirtothrips dorsalis Hood) is an economically significant pest of ornamental and food crops in Taiwan. Entomopathogenic fungi (EPF) are working as an eco-friendly biological control agent for managing thrips. It is crucial to screen the virulence of EPF isolates against chilli thrips under laboratory conditions before field application. However, limited data on the rearing method of chilli thrips pose challenges for large-scale laboratory screening. To address this issue, we developed a modified laboratory-rearing method for the mass production of S. dorsalis.

The method utilizes a detached-leaf rearing system using an agar-containing glass container that is suitable for assessing EPF against chilli thrips. This optimized detached-leaf bioassay system is suitable for assessing the efficacy of EPF against chilli thrips. The system has the capacity to produce large numbers of thrips at the same developmental stage for virulence screening. According to the test, the result showed that Cordyceps cateniannulata (Cc-NCHU-213) isolate showed the highest virulence (53.6%) against 2^nd instar larvae of chilli thrips at 7 days post inoculation (d.p.i.). Moreover, the agar-embedded leaf discs remained fresh for over 7 days, allowing thrips to survive (90% survival rate of control treatment) throughout the observation period, indicating the stability of the rearing system. This protocol demonstrates a stable rearing system for mass production and for evaluating the virulence of EPF isolates against chilli thrips and that may have utility for other thrips, enabling screening for potential EPF isolates for controlling thrips in the field.

Introduction

The chilli thrips (Scirtothrips dorsalis Hood; Thysanoptera: Thripidae) is a polyphagous pest infesting over 100 plant species across 40 different families of plants, including vegetables and ornamental and fruit crops¹. It is native to tropical and subtropical Asia-Southeast Asia, India, Japan, and Taiwan². To our knowledge, the chilli thrips is the pest of agricultural and ornamental crops, including mango, tea, blueberry, and lotus in Taiwan. During the past two decades, chilli thrips has become a significant economic pest in several countries because of increased globalization and open agricultural trade. Additionally, the high reproductive potential and adaptability to newly invaded areas contributed to the spread of this pest^1,3.

The chilli thrips feeds primarily on young foliage, shoots, flower petals, and immature fruits, leading to necrosis and death of tissues³. Furthermore, they serve as a vector for various plant diseases, including groundnut bud necrosis virus (GBNV), groundnut chlorotic fan-spot virus (GCFSV), groundnut yellow spot virus (GYSV)⁴, and tobacco streak virus (TSV)⁵. As a result, feeding by chilli thrips can significantly reduce the yield and value of impacted crops. Successful management of chilli thrips relies on an integrated pest management plan that utilizes cultural, biological, and chemical control strategies¹. In Taiwan, organic farmers commonly utilize non-chemical materials, such as narrow-range oil and neem oil, or biological control methods, including the use of predatory mites and minute pirate bugs (by personal observation). However, based on feedback from farmers, application of these oils during the blossom and fruiting season may interfere with pollination and damage pollen, leading to a reduction in the yield and economic value. Therefore, developing effective alternative control measures for chilli thrips is crucial for organic farmers.

Entomopathogenic fungi (EPF) are a group of insect-killing fungi that have been shown to be an effective and sustainable pest management technique^6,7,8. Several studies have demonstrated the effectiveness of EPF against chilli thrips. Commercial EPFs, Beauveria bassiana GHA (BotaniGard_ES), and Metarhizium brunneum F52 (Met-52 EC), have exhibited significant control of chilli thrips on rose plants^9,10. Chilli thrips populations on pepper plants can also be reduced by Cordyceps javanica (= Isaria fumosorosea)¹¹. Other EPFs, such as Lecanicillium lecanii and Purpureocillium lilacinum, have been tested against chilli thrips in different crops, including chilli plant and tea, and were effective in controlling this pest^12,13.

Some previous studies investigating the efficacy of EPFs for thrips control in greenhouses or the field were conducted without first screening for the most effective EPF species/strains, limiting their efficacy. Careful screening of EPFs may greatly enhance pest management. Many studies have reared thrips for bioassays on intact plants within cages^11,14. While an intact plant-rearing system offers a more natural environment for thrips, they pose challenges in managing different developmental stages, as thrips exhibit distinct behaviors at each stage. This variability may complicate the selection of targeted stages for specific experimental purposes. Furthermore, the soil conditions within the intact plant rearing system may influence the pupation and eclosion of chilli thrips, thereby reducing the number of individuals available for subsequent bioassays¹⁵, limiting the resources for further bioassays. Additionally, whole plant rearing systems require more space and continual care for plants, as stressed plants may alter the behavior of thrips and can be more time-consuming and resource-intensive. There are few reports of thrips rearing systems based on non-living plant material (e.g., detached plant tissue or artificial diets) for laboratory bioassay experiments, but the details of these methods were often unclear or omitted^12,16.

This study aimed to develop a simple, cost-effective, and sustainable mass rearing system of chilli thrips under laboratory conditions. In this protocol, a leaf disc rearing method⁷ was modified to offer a more efficient and controlled approach for mass rearing thrips, allowing for precise selection of developmental stages for bioassays via a tight control over environmental conditions. In addition, a stable and reproducible bioassay system was developed to evaluate the virulence of entomopathogenic fungi (EPF) isolates by 50% lethal time (LT₅₀) and 50% median lethal concentration (LC₅₀), which refer to the estimated time under a specific dosage, and the concentration of fungal suspension required to cause 50% mortality of chilli thrips, respectively. Through application of this method, the field-collected thrips can be correctly identified, and the stable laboratory colonies of thrips can be maintained to test the efficacy of different fungal isolates using easier and more reproducible methodology.

Protocol

NOTE: The complete experimental flowchart is shown in Figure 1.

1. Chilli thrips field collection and colony maintenance

1. Collection of chilli thrips
   1. Visually examine both sides of leaves and within flowers for infestation of chilli thrips on blueberry plants in an affected blueberry farm.
   2. Record the details of each sample (e.g., part of the plant and severity of the infestation), and confirm the production practices with farmers, such as conventional farming or organic farming, including application of mineral oil and natural enemies.
   3. Cut the leaves and flowers infested with chilli thrips and transfer them into the plastic zipper bag (Table of Materials), then seal the zipper bag with some air inside to keep the thrips alive. Bring the sample to the laboratory within 3 h.
      NOTE: Put the sample into a cooler when the time of field collection is more than 3 h.
   4. Prepare a glass rearing container (35 mm in height, 45 mm in inner diameter), place three layers of circular paper towel (diam. 40 mm) at the bottom of the glass rearing container, and then add 8-10 drops (400-500 µL) of filtered water (Figure 1 and Table of Materials).
   5. Collect a young, red mango leaf from the mango fields (Figure 1). Wash the leaf and check under an optical microscope (Table of Materials) for insect eggs or other insect contamination before preparing the glass rearing container. Cut the mango leaf with a leather cutter (Table of Materials) into a circular disc (40 mm) and place it on the wet paper towel.
      NOTE: Mango leaves are more stable resources for feeding thrips than young blueberry leaves, which only grow seasonally.
   6. Observe the chilli thrips under an optical microscope at 10x magnification, and transfer 10 adult thrips (five males and five females) using a fine painting brush (Table of Materials) into the glass rearing container.
   7. Seal each glass rearing container with two layers of parafilm (Table of Materials) and make about 40 small holes using a #00 insect pin for ventilation (Table of Materials).
   8. Keep the glass rearing container in an incubator at 25 ± 1 °C, ~70% relative humidity with a photoperiod of light:dark (L:D) = 12 h: 12 h (Table of Materials).
   9. Remove the adults using a paint brush after 48 h.
      NOTE: Based on our observation, the female thrips will lay eggs ~2 days after being transferred to the glass rearing container.
   10. Observe the leaf tissues with eggs under a stereomicroscope to further confirm the oviposition (Figure 1 and Table of Materials). Place a fresh mango leaf disc on the leaf with eggs and add 8-10 drops of filtered water on the paper towel to maintain humidity.
2. Maintenance of the chilli thrips population
   NOTE: The eggs hatch into 1^st instar larvae after ~5-7 days.
   1. Directly transfer the leaf containing 1^st instar larvae into a new glass rearing container.
      NOTE: Using a paint brush, gently transfer 1^st instar larvae into a new glass rearing container, avoiding contamination from the rotten and unexpected fungal growth on the mango leaves. Based on our observation, an adult female lays an average of 6 eggs per day.
   2. Observe the chilli thrips daily. Add 8-10 drops of filtered water every 4 days at the paper towel. Add a fresh mango leaf disc inside the container every 4 days, before the old leaf dries out.
      NOTE: 1^st instar larvae emerge into adults in ~7-10 days, depending on the environmental factors such as temperature and humidity.
   3. Observe the chilli thrips population daily until the thrips develop into adults, and repeat the steps from step 1.1.4 for long-term population maintenance.
3. Mass rearing of chilli thrips
   1. Collect female adults from the maintained population and prepare a glass rearing container based on the above steps.
   2. Transfer 10 female adults into the container and follow the steps from steps 1.1.4 to 1.1.10 to obtain a leaf with eggs.
      NOTE: An adult female lays an average of 6 eggs per day.
   3. Gently transfer ~120 1^st instar larvae into a new glass rearing container using a paint brush.
   4. Observe the chilli thrips daily. Add 8-10 drops of filtered water on the paper towel every 4 days. Add a fresh mango leaf disc into the container every 4 days before the old leaf dries out.
   5. Collect 1^st instar larvae at 5-7 days after the eggs are laid; collect 2^nd instar larvae after 7-10 days; collect adults after ~14 days for the bioassay (Figure 2).
      NOTE: Observe the morphology to distinguish different stages of chilli thrips for further experiments (Figure 2).

2. Molecular identification of chilli thrips

NOTE: Thrips collected from the field have to be identified to the species level by molecular identification using a fragment of the mitochondrial cytochrome oxidase I (COI) gene, a commonly used marker for chilli thrips^17,18.

1. Extraction of chilli thrips genomic DNA
   1. Collect one adult chilli thrips in a 1.5 mL centrifuge tube (Table of Materials).
   2. Homogenize the chilli thrips using a pellet pestle, then isolate genomic DNA using the commercial kit according to the manufacturer's protocol (Table of Materials).
2. PCR amplification and DNA sequencing
   1. Amplify the expected chilli thrips DNA fragment by using PCR Master Mix (2x) with a COI primer pair, forward primer LCO 1490 and reverse primer HCO 2198, which amplifies the partial mitochondrial COI gene (Table of Materials).
      1. Prepare a 20 µL PCR mixture containing 2 µL of thrip genomic DNA template, 1 µL each of forward and reverse primers, 10 µL of 2x PCR Master Mix, and 6 µL of ddH₂O (Table of Materials).
      2. Perform the PCR with a Thermal Cycler with the following cycling conditions: initial denaturation at 94 °C for 2 min, 35 cycles of 94 °C for 30 s, 52 °C for 1 min, 72 °C for 1 min, followed by a final extension of 72 °C for 10 min (Table of Materials).
   2. Check the PCR amplicon by electrophoresis in a 1% agarose gel to verify the result of amplification (Figure 3).
   3. Excise the target band (The expected amplicon size was 658 bp) and purify the COI PCR amplicon of the chilli thrips using a Gel/PCR purification kit according to the manufacturer's protocol. Send the purified product for sequencing through a commercial sequencing service. (Table of Materials).
3. Sequence analysis
   1. Assess the sequence quality using the referenced software (Table of Materials).
   2. Opening the FASTA or TXT file and manually trim the low-quality bases at both the 5′ and 3′ ends by deleting the undesired sequences.
   3. Perform a BLAST search on the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi) using default parameters to confirm species identity.

3. Preparation of entomopathogenic fungi

NOTE: The EPF isolates used in this study are listed in Table 1.

1. Recovery of fungal isolates from fungal resource library
   1. Thaw the preserved fungal conidia stock (conidia suspended in 30% glycerin) from a -80 °C freezer.
   2. Transfer approximately 10 µL of the conidia suspension and evenly spread on a 5.5 cm 1/4 Sabouraud dextrose agar (SDA) plate using a cell spreader in a laminar flow hood. The medium is prepared by mixing 0.75 g of Sabouraud dextrose broth and 1.5 g of agar in 100 mL of double-distilled water (ddH₂O) (Table of Materials).
   3. Seal the Petri dish with parafilm (Table of Materials) and transfer the plate into darkness incubator at 25 °C for 10-14 days.
   4. Subculture the actively growing fungal mycelium by streaking onto a fresh 1/4 Sabouraud dextrose agar (SDA) plate. Incubate fungi into darkness at 25 °C for 10-14 days to allow conidial development before harvest.
      NOTE: Use fungal cultures that are approximately 10 days old for subsequent bioassays. Cultures older than 14 days are not recommended for virulence testing due to potential reductions in conidial viability.
2. Preparation of conidia suspension
   1. Add 2-3 mL of 0.03% Tween 80 to the surface of a 10-14-day-old fungal culture on 1/4 SDA. Gently scrape the conidia using a sterile loop (Table of Materials).
   2. Transfer the fungal suspension into a clean 15 mL centrifuge tube.
   3. Homogenize the suspension by vortexing at the maximum speed and filter it through filter paper to remove hyphal debris and obtain a pure conidia suspension.
   4. Use a hemocytometer (Table of Materials) to check the number of conidia under a light microscope and dilute the suspension to 10⁸ conidia/mL.
   5. Transfer the conidial suspension into a micro-sprayer sterilized with UV light for subsequent bioassay application.
      NOTE: Ensure the conidial suspension is vortexed thoroughly before each transfer to avoid settling. Before use, sterilize the micro-sprayer by exposing it to ultraviolet radiation for 30 minutes inside a laminar flow hood.

4. Virulence screening against chilli thrips

1. Preparation of 2^nd instar larvae
   1. Collect the 2^nd instar larvae by following steps 1.3.1-1.3.5 for the following experiment.
      NOTE: Distinguish the 2^nd instar larvae by their yellow body color and wide abdomen compared to first instar larvae (Figure 2).
2. Preparation of inoculation container
   NOTE: Select the leaf species based on the target crop of chilli thrips. In this study, chilli thrips were collected from the field of infested blueberry 'Biloxi' (V. corymbosum L. × V. darrowii Camp) leaves. Therefore, the bioassay of EPF against chilli thrips will be carried out on blueberry leaves.
   1. Collect young blueberry leaves from a blueberry farm and wash the blueberry leaves to remove surface dust and any other arthropods if present.
   2. Air dry the leaves or use paper towels to wipe out water; then, check the surface of the blueberry leaves for any other arthropods or eggs under a stereomicroscope. Remove any arthropods if present.
   3. Cut a 2.8 cm diameter leaf disc using a leather cutter (Table of Materials), disinfect the leaf using 1% bleach (Sodium hypochlorite solution), and rinse 2x with sterilized water to remove any bleach residue on leaves.
      NOTE: Prepare the leaf disc before agar solidification to ensure the leaf disc can be embedded into the water agar properly.
   4. Autoclave a 2.5% water agar solution (2.5 g agar per 100 mL of ddH₂O) for sterilization.
   5. Pour 3 mL of 2.5% water agar into small glass containers (35 mm in height, 30 mm in inner diameter) inside a laminar flow hood before the agar solidifies. Allow the agar to cool to approximately 40 °C until it reaches a semi-solid state.
   6. Gently place the leaf disc (adaxial side up) onto the agar, embedding it slightly with minimum exposure above the agar surface. Once the water agar fully solidifies, the glass containers are ready for the bioassay.
3. EPF inoculation and observation
   1. Prior to application, vortex the conidial suspension thoroughly. Using a micro-sprayer, evenly apply 0.1 mL of the suspension (prepared in step 3.2.4) onto the surface of each leaf disc from a distance of approximately 10 cm within the glass container.
      NOTE: Test the spraying areas before the experiment. Ensure the spraying evenly covers the entire leaf disc with a thin layer. If droplets accumulate into standing water on the leaf, reduce the inoculation volume to avoid overwetting.
   2. Air-dry the conidia suspension on the leaf for 30 min under sterile conditions (e.g., in a laminar flow hood).
   3. Transfer 10 of 2^nd instar larvae into each container using a fine paint brush and seal each glass rearing container with two layers of parafilm.
   4. Make ~30 small holes by using a #00 insect pin (Table of Materials) for ventilation.
   5. Inoculate the container at 25 ± °C incubator with ~70% relative humidity and a photoperiod of L:D = 12 h: 12 h.
   6. Observe and record the mortality of chilli thrips under a stereomicroscope at 10x magnification daily for 7 days.
   7. Keep the dead larvae inside the glass container for mycosis observation to confirm the EPF infection.

5. Bioassay of selected EPF isolates

NOTE: Either the most virulent EPF isolate or several isolates with highly virulence can be selected for a dose-dependent bioassay against chilli thrips. Three different conidial concentrations, ranging from 10⁶ to 10⁸ conidia/mL, were used to assess the relationship between dose and pathogenicity.

1. Preparation of conidia suspension
   1. Recover the selected EPF isolate following step 3.1.
   2. Prepare three concentrations of conidia suspensions (10⁶, 10⁷, and 10⁸ conidia/mL) as described in step 3.2.
2. Preparation of 2^nd instar larvae
   1. Collect the second-instar larvae by following step 4.1 to obtain newly emerged adults.
3. Preparation of inoculation container
   1. Follow step 4.2. to prepare the inoculation container containing blueberry leaf discs embedded in water agar.
4. EPF inoculation and observation
   1. Conduct the EPF inoculation with three concentrations of conidia suspension, as described in step 4.3.

6. Statistical analysis

1. Calculation of mortality
   1. Transform the mortality data of each fungal strain by Arcsin transformation prior to conducting one-way analysis of variance (ANOVA) to assess differences among fungal treatments (significance level set as p < 0.05).
   2. Perform post hoc comparisons of treatment means using Tukey's Honest Significant Difference (HSD) test.
2. Calculation of median lethal time (LT₅₀) and median lethal concentration (LC₅₀)
   1. Calculate median lethal time (LT₅₀) and median lethal concentration (LC₅₀) values using Probit regression analysis.
   2. Create and populate variables named "total," "response," and "duration" (for LT₅₀) or "concentration" (for LC₅₀) with the observed data in the software interface.
      NOTE: If cumulative mortality does not exceed 50% within the observation period, LT₅₀ and/or LC₅₀ values cannot be estimated reliably.
   3. Perform Probit analysis by navigating to Analyze | Regression | Probit. Set the "total" as the total number of insects tested, "response" as the number of dead insects, and "duration" or "concentration" as the covariate. Set the significance level for the heterogeneity factor to 0.05 by pressing Options and setting the Significance level for use of heterogeneity factor to 0.05.

Results

The experimental flowchart presented in this study demonstrated that the stable rearing system of the chilli thrips could be established, starting from the field collection to the screening of virulence of EPF isolates (Figure 1). Though morphological observation can distinguish chilli thrips from other flower thrips, the use of the partial COI sequence as a molecular marker, including PCR amplicon size and COI sequencing, can further confirm the identificati...

Discussion

Chilli thrips is a globally significant pest affecting numerous economically important crops such as corn, peanut, pepper, soybean, strawberry, sweet potato, and tomato^1,11,19. Current control strategies rely on integrated approaches involving cultural practices, biological control agents, and chemical treatments, including the use of natural enemies, nematodes, and EPFs^11,14<...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#00 insect pin	Entosphinx	DMM0220	Made in UK
1.5 mL centrifuge tube	Corning incorporated	MCT-175-C	Made in USA
10 μL Inoculating Loop	NEST Scientific	718201	Made in USA
100 bp DNA Ladder III	Geneaid	DL007	Made in Taiwan.
2x SuperRed PCR Master Mix	Biotools	TE-SR01	Made in Taiwan
6 cm Petri dish	Alpha Plus Scientific	16021	Made in Taiwan
agar	Bioman Scientific	AGR001.1	Microbiology grade
agarose	Bioman Scientific	PB1200	Made in Taiwan
BioGreen Safe DNA Gel Buffer	Bioman Scientific	SDB001T	Made in Taiwan
Easy DNA High-Speed Extraction Tissue Kit	Fisher Biotec	ET-100	https://www.fisherbiotec.com.au/wp-content/uploads/2021/11/FB-EDNA-DNA-Kits-flyer-11Nov21.pdf (Made in Australia)
Forward primer: LCO 1490	Genomics	PO-PR220919G6	Sequence: 5’-GGT CAA CAA ATC ATA AAG ATA TTG G-3’
GenepHlow Gel/PCR Kit	Geneaid	DFH300	http://www.protech-bio.com/UserFiles/file/Gene-Spin%20Genomic%20DNA%20Kit.pdf
glass rearing container	Sunshine Lab Equipment company		Size: 35 mm in height, 45 mm in inner diameter. Can be repalce by any simular size of plastic or glass cup.
hemocytometer	Paul Marienfeld	640030	Made in Germany
incubator	Hipoint	F740	Made in Taiwan
leather cutter	Real Handmade Workshop		https://shopee.tw/jj891004? categoryId=100636& entryPoint=ShopByPDP& itemId=16023929406
MiniAmp Thermal Cycler	Thermo Fisher Scientific	A37834	Made in USA
optical microscope	Nikon	Ci-L	Made in Japan
painting brush	Tian Cheng brush company	4716608400352	Can be replace by fine-tipped watercolor brush, equivalent to a size 0 or 1 round brush.
paper towel	may flower	BLS007-8	Made in Taiwan
Parafilm M	Bemis	PM-996	Made in USA
pellet pestle	Bioman Scientific	GT100R	Made in Taiwan
Reverse primer: HCO 2198	Genomics	PO-PR220919G6	Sequence: 5’-TAA ACT TCA GGG TGA CCA AAA AAT CA-3’
Sabouraud Dextrose Broth	HiMedia	MH033-500G	Made in India
stereomicroscope	Soptop	SZN71	Made in Taiwan
TAE buffer 50X	Bioman Scientific	TAE501000	Made in Taiwan
Tween 80	PanReac AppliChem	142050.1661	Made in Taiwan
zipper bag	wenchen plastic company	1809000061	size: 12 cm in wide and 17 centimeters in long. (Made in Taiwan)