Key words

1 Introduction

There are many published examples of the use of lepidopteran larvae for baculovirus-mediated protein production. The use of larvae was pioneered with the silkworm, Bombyx mori (e.g., α-interferon [1] and mouse interleukin-3 [2]), and this host insect continues to be extensively used both for research and for commercial protein production, e.g., by the Japanese firm Katakura (e.g., PON1 [3], protein complex [4] and 45 recombinant proteins from six categories [5]). Other lepidopteran hosts have also been investigated, including the tobacco hornworm Manduca sexta [6], the Cecropia moth Hyalophora cecropia [7], the beet armyworm Spodoptera exigua [8], the tobacco budworm Heliothis virescens [9], the saltmarsh caterpillar Estigmene acrea [10], and, probably most importantly, the cabbage looper Trichoplusia ni.

Autographa californica multiple nucleopolyhedrovirus (AcMNPV) is by far the most widely used baculovirus expression vector. While AcMNPV infects a wide range of lepidopteran hosts, not all moths are good hosts for this virus. For example, while being susceptible to a mutant [11], B. mori is not susceptible to wild type AcMNPV, and M. sexta, H. cecropia, and S. exigua are all much less susceptible than H. virescens [12]. T. ni is an excellent host for AcMNPV [13] and has been extensively used to produce a variety of proteins (e.g., insulin receptor kinase domain [14], adenosine deaminase [15], Phospholipase A2 [16], interleukin-2 [17], the cardiac sodium-calcium exchanger [18], Fab antibody [19], and phosphatase 2A subunit [20]).

While commercial-scale protein production in T. ni larvae is available (Chesapeake PERL Inc., Savage, MD, and Entopath, Easton, PA), this chapter is aimed at small to medium scale production on the bench-top. Compared to production in cultured insect cells, larvae can produce a large amount of protein at reduced cost and without the need for investment in expensive equipment [5, 21]. In addition, the scale-up issues typical of bioreactor-based production do not occur when using insect larvae [13]. Potential applications for proteins produced in larvae include use in vaccine preparation [22–24] and crystallography [25].

We limit the discussion in this chapter to production of protein in cabbage looper larvae, using three different methods that differ in the manner of larval inoculation. Traditionally, larva-mediated protein expression has been done by generating a recombinant AcMNPV vector in cultured cells (commonly, S. frugiperda cells), followed by budded virus amplification and the establishment of an infection by injecting the budded virus into late-instar larvae. This first method is effective but tedious: even a skilled investigator can inject only a few hundred larvae. A second method, useful for producing larger amounts of recombinant protein, is based on oral inoculation rather than injection. It is possible to generate orally infective inoculum, so-called pre-occluded virus (POV), by processing a few dozen injected larvae and to use this inoculum to infect thousands of T. ni larvae via the diet . Finally, in a third method the intermediate steps in cultured cells are eliminated completely by generating recombinant AcMNPV DNA (Bacmid) in bacteria and using this Bacmid DNA to transfect T. ni larvae (Liu and van Beek, unpublished results).

2 Materials

2.1 Rearing of Trichoplusia ni Larvae

  1. 1.

    We recommend the purchase of eggs or larvae from any of several commercial suppliers (Benzon Research, Carlisle, PA, www.benzonresearch.com; Bio-Serv, Frenchtown, NJ, www.bio-serv.com; Entopath, Easton, PA, www.entopath.com) (see Notes 1 and 2 ).

  2. 2.

    Corn cob grits (e.g., Bio-Serv). A bulking agent that is needed only if the larvae are reared starting with “loose” T. ni eggs. If not sterilized, then autoclave before use.

  3. 3.

    General Purpose Lepidoptera Diet (#F9772, Bio-Serv, Frenchtown, NJ), a dry mix insect diet (see Note 3 ). We recommend purchase of insect eggs and diet from the same vendor.

  4. 4.

    Transfer forceps (e.g., BioQuip, Rancho Dominguez, CA: #4750): soft tweezers for handling of insect larvae.

  5. 5.

    Eight-oz cups and fitting lids (e.g., Solo Cup Company, Urbana, IL) for rearing larvae in groups of approximately 25 individuals (see Note 4 ).

  6. 6.

    Dissecting microscope with a micrometer scale engraved in one ocular lens (see Note 5 ).

2.2 Inoculation by Injection

  1. 1.

    Spodoptera frugiperda Sf-9 (ATCC: #CRL-1711) or Sf-21 cells can be used for generating recombinant virus, as well as for titration of the extracellular virus stock (see Note 6 ).

  2. 2.

    Insect cell culture media. There are many types of insect media commercially available, and any medium recommended by the manufacturer for maintaining Sf-9 or Sf-21 cells is suitable (see Chapter 8 and Note 7 ).

  3. 3.

    Ten-microliter syringe (Hamilton, Reno, NV), fitted with a 26 s gauge needle.

2.3 Oral Inoculation

  1. 1.

    Mortar and pestle.

  2. 2.

    FD&C Blue #1 (Hilton-Davis, Cincinnati, OH) or blue food coloring dye.

  3. 3.

    Plastic screen (100 mesh) or cheesecloth.

2.4 Transfection of Larvae with Bacmid DNA

  1. 1.

    Cellfectin II reagent (Life Technologies, CA): a transfection reagent.

  2. 2.

    Wizard Plus Minipreps DNA Purification System (Promega, Madison, WI).

2.5 Homogenization and Clarification

  1. 1.

    Anti-melanizing agent: Either β-mercaptoethanol (BME) (Sigma-Aldrich, St. Louis, MO), or a stock solution of 25 mM phenylthiourea in ethanol (PTU) (Sigma-Aldrich) (see Note 8 ).

  2. 2.

    Complete Protease Inhibitor Cocktail tablets (Roche Applied Sciences, Indianapolis, IN).

  3. 3.

    Extraction buffer : 50 mM Tris–HCl, pH 7.5, 100 mM NaCl, 1 mM EDTA.

3 Methods

3.1 The Host Insect

The cabbage looper is a suitable and widely used insect for recombinant protein production. When the insects are reared on a suitable diet at their optimal temperature (~29 °C), either in the dark or under a day/night regimen, they develop from egg to pupa in approximately 13 days. After about 2 days in the egg stage, larvae will hatch and progress through five instars over a period of about 9 days. Late in the fifth instar, larvae change in appearance when they enter the prepupal stage, which lasts approximately 2 days. Metamorphosis takes place in the pupal stage, and adult insects emerges after about 4 days.

3.2 Diet Preparation

Prepare the diet as recommended by the supplier. As an example, the following is a slightly modified version of the manufacturer’s protocol for preparation of General Purpose Lepidoptera Diet.

  1. 1.

    For 1 L diet: add 17 g agar to 400 mL water and mix thoroughly.

  2. 2.

    Heat until boiling, e.g., in a microwave. Stir the mixture occasionally during the process. The suspension should appear white and foaming when boiling.

  3. 3.

    Add 160 mL cold water and mix.

  4. 4.

    While the agar is being heated, mix the bulk diet ingredients (144 g) in 300 mL cold water.

  5. 5.

    Combine the agar and the bulk diet ingredient suspensions, top off with water to 1 L, and mix thoroughly.

  6. 6.

    Before the diet sets at approximately 37 °C, pour ~20 mL diet into each 8-oz cup and let stand for 15 min to allow the diet to solidify and the condensate to evaporate.

  7. 7.

    Close the cups with the proper lid. Freshly prepared diet may be stored under refrigeration for 3 weeks without any detrimental effects. Remember to equilibrate the diet to room temperature for at least 1 h and let condensed water evaporate before placing insects or eggs on the diet.

3.3 Trichoplusia ni Rearing

T. ni can be purchased as surface-sterilized eggs attached to a substrate such as paper towel or muslin (Subheading 3.3.1), as “loose” eggs (Subheading 3.3.2), or as larvae on diet (Subheading 3.4).

3.3.1 Larval Rearing Starting with Eggs on a Substrate

  1. 1.

    Cut the substrate into small strips (50–100 eggs). Egg density can be estimated by counting under a dissecting microscope. To this end mark an area of approximately 1 in2 and count under the lowest magnification.

  2. 2.

    Staple a strip to the inside of the lid of each 8-oz plastic cup filled with 20 mL diet.

  3. 3.

    Incubate at 29 °C.

  4. 4.

    After 3–4 days of incubation , begin to monitor larval development as described under Subheading 3.4.

3.3.2 Larval Rearing Starting with Loose Eggs

  1. 1.

    Weigh out the corn cob grits to be used (1 g grits per 8-oz cup).

  2. 2.

    Add 1 mL water for each 15 g grits and mix until the clumps have disappeared.

  3. 3.

    Mix insect eggs with the moistened corn cob grits. Needed per cup: 1 g corn cob grits and 10–15 mg eggs (1 mg contains ~10 eggs).

  4. 4.

    Incubate the mixture for 16–24 h at 29 °C.

  5. 5.

    For each cup, spread approximately 1 g egg-grits mixture onto the diet. The diet should be at room temperature and show no excessive moisture on its surface.

  6. 6.

    Incubate at 29 °C.

  7. 7.

    After 3–4 days of incubation , begin to monitor larval development as described under Subheading 3.4.

3.4 Determination of Developmental Stage

T. ni larval development consists of five instars , which are developmenta l stages separated by a molt. Since injection of larvae is carried out preferentially during early fifth instar, and oral inoculation at late fourth instar, it is important to be able to determine the developmental stage of the larvae; this is also convenient for planning experimentation. It is possible to manipulate developmental speed by changing incubation temperature.

  1. 1.

    Determine the instar of the larvae at 3 or 4 days after seeding the eggs onto the diet . To this end, take a small sample of larvae and measure their head capsule width under a dissecting microscope with a micrometer scale engraved in the ocular (find the correct magnification via calibration with the aid of a ruler). Determine the larval instar after comparison of measured and tabulated values (Table 1). Larval weight is also an indicator of developmental stage , albeit a less reliable one. When using weight it is important to note that larvae early in a particular stage are lighter than they were late in the preceding stage (Table 1).

    Table 1 Weight and head capsule width of Trichoplusia ni larvae
    Full size table
  2. 2.

    Estimate the number of larvae per cup and reduce to approximately 25 (see Note 9 ).

  3. 3.

    At 29 °C it will take 6–7 days between seeding of the eggs and the molt from fourth to fifth instar (see Note 10 ). Daily monitoring of the larvae will help in choosing the correct stage for inoculation. Whether the larva is early or late in a particular instar can be judged by the width of the head capsule in relation to the width of the body. Larvae that have recently molted possess a relatively wide head capsule and slender body, whereas late in the instar the width of the body exceeds that of the head.

3.5 Recombinant Virus

The construction of recombinant baculovirus vectors is described elsewhere in this book (Chapter 4), and a number of different baculovirus vector cloning kits are available from various commercial sources (e.g., Invitrogen, BD Biosciences/Pharmingen, Novagen, NextGen Sciences, and AB Vector). The kits introduce heterologous coding sequences either by homologous recombination or transposition insertion. It is possible to eliminate bacterial amplification of a transfer vector plasmid and/or bacmid and to introduce heterologous coding sequences by direct ligation into a baculoviruses modified with unique restriction endonuclease cut sites (e.g., see [26]). This direct ligation method has been successfully used to generate customized baculovirus vectors for the whole insect platform (Chesapeake PERL Inc., Savage, MD). The choice of baculovirus cloning vector depends on availability, ease of the procedures (screening), flexibility (number and types of expressible promoters, availability of purification tags and secretion signals, etc.), or even on the presence/absence in the vector of a virally encoded protease or chaperones. However, the choice of a particular system is not influenced by whether the recombinant virus will be used in cultured cells or in larvae; thus, each system will yield recombinant AcMNPV suitable for infection of T. ni larvae and the expression of recombinant protein. See Note 11 for safety aspects of recombinant baculoviruses.

3.6 Inoculation

There are three different methods for inoculating larvae: (a) injection with extracellular virus; (b) oral inoculation; and (c) transfection with viral DNA. Injection with extracellular virus is used most commonly. If a large amount of recombinant protein is desired, or if the yield of the target protein is expected to be relatively low (as may be the case with membrane proteins), then large numbers of larvae may be needed to produce the desired amount of protein. Injection of thousands of larvae can be avoided by using a small number (e.g., 20) of injected larvae to prepare orally infective inoculum for a mass inoculation. The orally infectious virus morphotype in preoccluded virus (POV) inoculum consists of virions that are produced late in the infection cycle and remain in the nucleus as they are destined to be incorporated into polyhedral occlusion bodies [27–29]. However, almost all recombinant baculovirus expression vectors lack the polyhedrin gene and therefore no polyhedral occlusion bodies are formed.

  1. 1.

    Extracellular virus . Inoculum consisting of extracellular virus (also referred to as budded virus) is obtained by collecting the medium from infected cell culture. The working stock of recombinant AcMNPV for injection of larvae consists of extracellular virus with a titer of 106 pfu/mL or higher. Methods for extracellular virus titration are described elsewhere in this volume (Chapters 4, 5, 10, 11 and 22).

  2. 2.

    Preoccluded virus. The working stock of recombinant AcMNPV for oral inoculation consists of POV. This form of the virus cannot be titered in vitro. Its potency can be determined only by larval bioassay, but this is usually not necessary. POV inoculum is most easily prepared from a small batch of larvae that have been injected with extracellular virus.

  3. 3.

    Recombinant viral DNA. The third inoculation method, larval transfection, is accomplished with viral DNA, in the form of Escherichia coli-produced Bacmid (Bac-to-Bac™, Invitrogen, Carlsbad, CA) (for an alternative method see Note 12 ). DNA is purified from the bacterial culture using a miniprep method, and DNA concentration in the inoculum can be estimated, e.g., by spectroscopy.

3.6.1 Larval Injection

Larval injection may seem difficult at first, but after a little practice (and the appropriate syringe with a sharp needle) it appears to be remarkably easy on the insect. Early fifth instar larvae are injected with approximately 1000 pfu of extracellular virus. This dose leads to a synchronous infection in all treated larvae. Typically, a small droplet of hemolymph will appear over the wound site immediately after injection. The hemolymph will melanize over a period of a few hours and wound healing takes place underneath; however, some larvae (usually less than 10 %) do not survive inoculation. Control larvae will pupate after 2–3 days, but in virus-infected larvae pupation (in fact, any molt) is blocked by a virus-encoded ecdysteroid UDP-glucosyltransferase [30]. Infected larvae will die from the virus infection after approximately 3–3.5 days (or, if lower doses are used, after as long as 4–5 days).

  1. 1.

    Prepare control inoculum (insect medium), and viral inoculum consisting of extracellular virus at a titer of 106 pfu/mL (if necessary dilute stock with insect medium).

  2. 2.

    Select 100 early fifth instar larvae (head capsule width 1.9 mm, weight about 110 mg). Leave the selected larvae on their diet , until ~10 min before inoculation, when they may be cooled on ice in groups of 5 larvae (see Note 13 ).

  3. 3.

    Inject four groups of five larvae with 1 μL control inoculum as follows: wearing latex gloves, hold the insect lightly between thumb and forefinger and insert the needle at a low angle (to avoid puncturing the insect’s midgut) into the body cavity at the posterior half of the larva. Release the insect, inject about 1 μL inoculum by advancing the plunger, and then slide the larva carefully off the needle onto fresh diet.

  4. 4.

    Repeat step 3 for the remaining 80 larvae using the recombinant budded virus stock.

  5. 5.

    Incubate the larvae at 29 °C.

  6. 6.

    After approximately 24 h, remove any larvae that died from the injection procedure.

  7. 7.

    Harvest infected larvae when approximately 10 % of the remaining insects are dead, typically between 72 and 84 h after inoculation. If the larvae are not to be processed immediately, then they should be stored at −80 °C.

3.6.2 Oral Inoculation

  1. 1.

    Follow the procedure for injection of larvae as outlined under Subheading 3.6.1, but inject only 20 larvae with viral inoculum.

  2. 2.

    Harvest the infected larvae approximately 3.5 days after inoculation.

  3. 3.

    Freeze the larvae at −20 °C.

  4. 4.

    Just before inoculation, weigh the frozen larvae.

  5. 5.

    For each 200 cups with larvae to be inoculated, place 0.5 g frozen larvae and 0.5 mL water in a mortar, and homogenize to a slurry using a pestle.

  6. 6.

    Prepare approximately 700 mL of a solution of blue dye in water, using either 0.5 mg/mL FD&C #1 or ~1 % food coloring dye.

  7. 7.

    Dilute the slurry with dyed water to 660 mL and pour the inoculum through a 100 mesh screen or four layers of cheesecloth to remove large pieces of insect debris. This mixture is the POV inoculum. The remaining 40 mL of dyed water is the negative control inoculum.

  8. 8.

    Apply 1 mL control inoculum to each of three diet-filled cups. Swirl the cups to cover the entire surface with inoculum and let it soak into the diet. Mark these cups as control treatments.

  9. 9.

    In the same manner, apply 1 mL POV inoculum to each of the remaining cups.

  10. 10.

    Using soft tweezers, place 25 late fourth insta r T. ni larvae onto the treated diet .

  11. 11.

    Inspect the larvae daily and harvest after approximately 4–5 days, when approximately 10 % of them have died from the virus infection. If the larvae are not to be processed immediately, then they should be stored at −80 °C.

3.6.3 Larval Transfection

Infection of insect larvae with recombinant viral DNA is the fastest way to express recombinant protein in larvae. It has two advantages over the other methods described in the preceding paragraphs: (a) it eliminates the need to set up and maintain a system of cultured insect cells, and (b) it cuts almost a week off the time between cloning of the heterologous gene and expression of recombinant protein in T. ni larvae (see Note 14 ).

Since this method is new, we illustrate it using a specific example, the expression under the control of the AcMNPV polyhedrin promoter of DsRed, a red fluorescing protein derived from Discosoma coral [31].

  1. 1.

    Amplify by PCR a fragment containing the open reading frame of DsRed from pDsRed (BD Biosciences/Clontech, Palo Alto, CA), adding Bpl I restriction enzyme sites on both ends.

  2. 2.

    Digest the resulting fragment of approximately 700 base pairs with BplI, then treat with Klenow enzyme, digest with BglII, and ligate into the vector pFastBac1 (Bac-to-Bac, Invitrogen) previously cut with BamHI and StuI. This results in the donor vector pFB1DsRed, with the DsRed gene under the control of the AcMNPV polyhedrin gene promoter.

  3. 3.

    Follow the manufacturer’s recommendations for the Bac-to-Bac system to make recombinant Bacmid DNA carrying the DsRed gene in DH10Bac cells.

  4. 4.

    Isolate Bacmid DNA with the aid of the Wizard Plus Minipreps DNA Purification System.

  5. 5.

    Measure the concentration of the DNA in the resulting miniprep and adjust to 1 mg/mL with sterile water.

  6. 6.

    For control inoculum, mix gently 10 μL Cellfectin and 15 μL Sf-900 II medium (Invitrogen) and let sit at room temperature for at least 15 min before use.

  7. 7.

    For transfection inoculum, mix gently 40 μL Cellfectin, 10 μL miniprep DNA (1 mg/mL), and 50 μL Sf-900 II medium and let sit at room temperature for at least 15 min before use.

  8. 8.

    Select approximately 100 early fifth instar T. ni larvae as under Subheading 3.4.

  9. 9.

    Inject 20 larvae with 1 μL control inoculum each, and 80 larvae with 1 μL transfection inoculum each, as described under Subheading 3.6.1.

  10. 10.

    Incubate the larvae, and remove any dead larvae after 24 h as described under Subheading 3.6.1.

  11. 11.

    Harvest the infected larvae between 96 and 110 h after inoculation. The success rate of transfection can easily be monitored by the change in color of infected larvae, from pale green to bright red, caused by the fluorescence of DsRed under ambient light. In our hands the infection rate was 81 % while mortality caused by the injection procedure was 10 % (see Note 15 ).

  12. 12.

    Store harvested larvae at −80 °C until processing.

3.7 Incubation and Harvest Time

Controlling the incubation conditions of the larvae during infection is very important. Infected larvae are more vulnerable to bacteria and fungi, and it is therefore necessary to allow sufficient air exchange so that no condensation occurs in the containers. However, drying out of the diet should also be avoided.

The time chosen to harvest the infected larvae may be critical for the amount and quality of target protein recovered. Protein synthesis occurs until shortly before the death of the insect, but at the same time protein degradation may also increase towards the end of the infection cycle. The ideal time to harvest is different for each larva, since the course of virus infection and expression of a heterologous protein in a group of larvae are not completely synchronous. For a protein of average stability, we recommend that larvae should be harvested just prior to death: in practice, when ~ 10 % of the population has died.

3.8 Homogenization, Clarification, Extraction and Purification

A comprehensive description of protein separation and purification methods is beyond the scope of this work. Typically, homogenization of small batches in an extraction buffer is accomplished with a tissue grinder or a blender, followed by centrifugation to remove non-soluble material and lipids. These steps are carried out while the sample is kept cold, and may include the use of protease inhibitors to prevent in-process degradation. One aspect of downstream processing that differs from cultured insect cell-based systems is worth noting here. Insects maintain a complex pathway in their hemolymph that, when triggered in the presence of oxygen, leads to melanization. A homogenate of larvae will become dark gray to black in a matter of hours, even when refrigerated. Melanization can be prevented by the addition of either 25 μM PTU (1/1000 volume of 25 mM PTU in alcohol) or 5 mM BME.

After the homogenate has been clarified, the target protein may be purified by affinity chromatography or any other suitable method.

4 Notes

  1. 1.

    Purchasing from a commercial vendor guarantees a certain level of quality and consistency. It is usually possible to purchase eggs or larvae, and eggs may be delivered either attached to a substrate (e.g., muslin or paper towel) or loose in a container. In either case the eggs should have been surface-sterilized by the vendor. Handling of loose eggs is somewhat more complex than handling eggs on substrate, whereas purchase of larvae is obviously the easiest.

  2. 2.

    USDA APHIS permit 526 is required to house and transport cabbage looper specimens (T. ni). For additional information regarding how to apply for your permit visit the following web site: http://www.aphis.usda.gov/plant_health/permits/organism/plantpest_howtoapply.shtml .

  3. 3.

    Several suitable insect diets have been developed for rearing cabbage loopers. These are usually agar-based and contain carbohydrates, plant proteins, vitamins, micronutrients, and antibiotics. For the production of recombinant proteins that are free of mammalian components it may be necessary to ascertain that no animal-derived components are included in the diet.

  4. 4.

    If a type of container is used other than the recommended 8-oz cups, then the number of larvae placed in the cup should be between two and three per in2 diet surface.

  5. 5.

    A balance can also be used to determine the larval development stage. However, measurement of head capsule width under a dissecting microscope is a more reliable method.

  6. 6.

    Using Bacmid DNA (Bac-to-Bac, Invitrogen), transfection of Sf-9 cells has, in our hands, been more efficient than that of Tn-5 cells. Methods for growing insect cell types and for virus titer determination (TCID50 or overlay plaque assay) can be found elsewhere in this book (Chapter 9 and Chapters 4, 5, 10, 11 and 22, respectively).

  7. 7.

    Serum -containing and serum-free media are available for these cell lines. Since insect cells are ostensibly free of mammalian pathogens, it may be important under certain circumstances to exclude any possibility of contamination via serum derived from animal sources.

  8. 8.

    Insect hemolymph undergoes a cascade reaction when exposed to oxygen, resulting in the formation of melanin. This “blackening” of hemolymph and the entire insect homogenate should be prevented. Two commonly used inhibitors are β-mercaptoethanol (BME) and phenylthiourea (PTU). BME is also a potent disruptor of protein disulfide bridges, and this function often precludes its use. PTU is extremely toxic, but it is effective even at very low concentrations.

  9. 9.

    Larvae feed continuously except when molting, during which time they will migrate away from the diet. By removing larvae that are feeding, while retaining those that are molting, this behavioral trait can be used to improve developmental homogeneity of the population.

  10. 10.

    Larval development can be slowed (but not increased) by changing the incubation temperature. Larval development essentially stops at 11 °C, and at room temperature development takes approximately twice as long. Note that larval development decreases rapidly at temperatures above 29 °C.

  11. 11.

    With regard to the safety of recombinant baculoviruses, it is important to note that baculoviruses are not infectious to humans. They are classified under Biosafety Level 1, which represents a basic level of containment that relies on standard microbiological practices, with no special primary or secondary barriers recommended other than a sink for handwashing. The NIH classifies experiments with recombinant baculoviruses in the lowest Risk Group, RG1. Material contaminated with recombinant baculovirus should be autoclaved before disposal.

  12. 12.

    Another method that enables in vitro generation of recombinant DNA is direct ligation (Baculodirect™, Invitrogen). We have not tested whether this DNA is suitable for larval transfection.

  13. 13.

    Injection can be done without cooling the larvae; in fact, if one has acquired some experience, it is faster.

  14. 14.

    A similar method has recently been described for the transfection of B. mori larvae [32].

  15. 15.

    By injecting fourth-instar instead of fifth-instar larvae, we achieved infection in over 90 % of the larvae. However, due to their smaller size, injecting these larvae was more tedious and led to significantly higher control mortality.