The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella.

The injection of laboratory animals with pathogenic microorganisms poses a significant safety risk because of the potential for injury by accidental needlestick. This is especially true for researchers using invertebrate models of disease due to the required precision and accuracy of the injection. The restraint of the greater wax moth larvae (Galleria mellonella) is often achieved by grasping a larva firmly between finger and thumb. Needle resistant gloves or forceps can be used to reduce the risk of a needlestick but can result in animal injury, a loss of throughput, and inconsistencies in experimental data. Restraint devices are commonly used for the manipulation of small mammals, and in this manuscript, we describe the construction of two devices that can be used to entrap and restrain G. mellonella larvae prior to injection with pathogenic microbes. These devices reduce the manual handling of larvae and provide an engineering control to protect against accidental needlestick injury while maintaining a high rate of injection.

Introduction

The larvae of the greater wax moth Galleria mellonella is an important animal model for studying host-pathogen interactions and for the discovery of novel antimicrobial therapeutics. The popularity of this model organism is driven by the low cost of purchase and the reduced ethical concerns for the experimental manipulation of insects. This allows the challenge of a large number of larvae in a single experiment, which can improve the statistical power of an assay. Starting in the 1940s, a diversity of viral, bacterial, fungal, and nematode pathogens, have been studied for their ability to cause disease in G. mellonella larvae [1-15]. Importantly, G. mellonella can be maintained at mammalian body temperature and the outcomes of infection can reproduce that of mammalian animal models [16-18]. This is likely due to similarities in the innate immune response to pathogens mediated by elements of cellular and humoral immunity between insects and mammals [19-21]. G. mellonella has also been used extensively for compound toxicity screening [22]. There are several methods to introduce compounds and pathogenic microorganisms into G. mellonella, including topical application, feeding, baiting, oral gavage, submersion, and direct injection into the hemocoel [9,11,23-26]. The latter method is often favored because of the ability to control the dosage and timing of injections.

Despite the many benefits, there are challenges with using G. mellonella larvae as an animal model, most notably standardizing the health and developmental stage of the larvae. This is especially difficult when larvae are purchased from commercial sources that are primarily focused on providing feed and bait for the pet and angling communities [27,28], although there are commercial pipelines for scientific-grade larvae [29]. Another difficulty is the manipulation and restraint of small larvae during experimental injections. These experiments require both biological containment and the adequate protection of personnel from needlestick injuries and laboratory-acquired infections. The most basic technique of larval injection calls for the restraint of a larva between finger and thumb during the injection process [9,11,14,30]. With the operator’s hands protected by latex gloves, this method offers maximum dexterity. However, the close proximity of a pathogen-filled needle to inadequately protected fingers presents a significant biological safety hazard that exposes personnel to a high risk of accidental needlestick injury. This particular restraint procedure is in conflict with biosafety guidelines for the implementation of policies for improved work practices that minimize needlestick injuries whenever possible [31]. Alternatively, the safe handling of G. mellonella larvae can be achieved with the use of needle resistant gloves or forceps, but with a loss of manual dexterity and the potential to cause animal injury or stress that can alter pathogen susceptibility [32]. Furthermore, needle-resistant gloves are made of porous materials that require covering with disposable laboratory gloves to prevent biological contamination, which further limits manual dexterity. To maximize safety, humane physical restraint devices have been used routinely for the handling and manipulation of laboratory animals [33]. For the injection of G. mellonella, there is also a device named the “Galleria grabber” that has been developed for the restraint of larvae between layers of a sponge [34]. This method enables injection without the need for grasping larvae between finger and thumb and offers the user protection from accidental needlestick injury. However, the use of a porous sponge for multiple injections increases the chance of its contamination by pathogenic microbes, which presents a challenge for effective decontamination.

In this study we present two simple restraint devices that can be fabricated from micropipette tips or acrylic glass (also known as poly(methyl methacrylate) or Plexiglass). These devices are easy to assemble and can be used to restrain large numbers of G. mellonella larvae in preparation for injection. The described protocol reduces the manual handling of larvae, enables a rapid injection speed, and allows the effective decontamination and sterilization of the devices for reuse. Both devices provide increased protection of the operator from accidental needlestick injury and laboratory-acquired infection.

Materials and methods

Culturing and preparation of yeast cells for injection

C. glabrata ATCC2001 was maintained using yeast extract peptone dextrose growth media (YPD). Several days prior to injection, C. glabrata was streaked out to clones from a -80°C frozen stock in YPD with 15% glycerol. A single colony of yeast cells was grown overnight at room temperature to stationary phase in a 2 mL culture of liquid YPD medium. Stationary phase cultures were diluted 1/20 into a 125 mL flask and grown at room temperature until an OD600 of 1.5 was reached. Hemocytometer counts of these cultures were used to determine the number of yeasts used for each injection (8.0 × 105, 3.0 × 106, 4.6 × 106, and 5.0 × 106 C. glabrata cells per injection). Prior to injection, yeast cells were harvested by centrifugation at 8,000 × g for 1 min (25°C) and suspended in filter sterilized PBS (pH 7).

G. mellonella larva handling, care, and disposal

The described experiments followed the ARRIVE guidelines for reporting the use of animals in research [35]. Larvae were ordered from www.premiumcrickets.com using the “weather protect” service to maintain the temperature of the larvae during overnight shipping. Upon arrival, larvae were stored without light in wood shavings at 17°C and were allowed to acclimatize for at least 2 days to control for the adverse physiological consequences of shipping [32]. G. mellonella larvae were used within 1 week due to the known physiological consequences of long-term storage [36]. Healthy G. mellonella larvae were selected by weight (175–225 mg), uniformity in color (little to no melanization), and responsiveness to touch. Prior to injection, larvae were incubated at 37°C for 16 hours to allow for acclimatization to the assay temperature. Dead or unhealthy larva that are observed after the pre-incubation period were removed from the study prior to injection. Larvae exposed to pathogenic microorganisms were disposed of by placing them in secondary containment and incubating at -20°C for 24 hours before sterilization by autoclaving.

Fabrication of restraint devices for G. mellonella

The primary consumable used for the creation of the restrain device was the 250 μL VistaLab micropipette tip (catalog number: 4058–2000). Other brands of pipette tips have been tested for their compatibility with this method (S1 Table). Micropipette tips were cut at predefined points to enable assembly (Fig 1). To construct the restraint device fabricated from acrylic glass, transparent clear acrylic glass was purchased from AliExpress (1 mm × 100 mm × 100 mm) and was cut to the desired shape with a CO2 laser cutter (BOSS Laser LS-1416) using software provided by the manufacturer (available online in SVG format at https://www.thingiverse.com/ design ID 4170068, “LarvaWormCorralV4”).

Fig 1

Entrapment of G. mellonella in a restraint device constructed from a micropipette tip.

(A) Dimensions of a 250 μL VistaLab pipette tip indicating the cut site to create two 5 mm openings. (B) Assembly of the restraint device without a larva. (C) Top: Assembly of the restraint device with a larva captured abdomen-first into the larger half of the micropipette tip. Middle: A larva is entrapped by enclosing the chamber. Bottom: A larva is released by opening the chamber. Measurements are presented in millimeters. (D) Storage of multiple larvae in restraint devices prior to injection.

Injection of G. mellonella larvae

Injections were performed with Hamilton 700 syringes (Model 701 N, Volume: 10 μL, Point Style: 2, Gauge: 26s and Model 1750 LTSN SYR, Volume: 500 μL, Point style: 4 Gauge: 26s) with a repeating dispenser used for multiple injections (Hamilton PB600-1). We also expect that this method is fully compatible with the use of insulin syringes with shorter needles as prolegs are almost always positioned close to the opening in the restraint devices. During injection, fingers were protected by a HexArmor PointGuard® Ultra 4041 glove (Performance Fabrics Incorporated).

Data analysis

The graphical representation of the average survival rates of G. mellonella larvae after injection and the Kaplan-Meier log-rank analysis were performed using R (version 1.1.419) with the packages “ggplot2”, “dplyr”, “survival”, and “survminer”. LT50 was calculated using the package “MASS”. A power analysis was performed to assess the required sample size using G*Power (V3.1) (One-tailed T-Test, α = 0.05, β = 0.2, effect size = 0.8).

Results and discussion

The restraint of Galleria mellonella larvae using reusable restraint devices

A central challenge to the manipulation and injection of Galleria mellonella larvae is the ability to restrain them without injury prior to injection. We have previously observed that manual handling and restraint of larvae by inexperienced laboratory personnel can often cause animal injury and death. To minimize these undesirable consequences, we have designed and tested two types of devices for the restraint of individual G. mellonella larvae. These chambers can be constructed either from a disposable 250 μL or 1,000 μL micropipette tip of the required dimensions (Fig 1A and S1 Table) or assembled from laser cut acrylic glass (Fig 2). Both devices are designed to be fully reusable. To make the micropipette tip device, a tip is cut, and the two halves are used to entrap a larva with minimal handling (S1 Movie). Specifically, a larva is captured within the wider end of the cut pipette tip and entrapped by inserting the second half of the pipette tip to seal the restraint device at one end (Fig 1B). This procedure takes an average of 7 seconds (SD = +/- 3 seconds, n = 30). After entrapment, the escape time of larvae from the device was measured under ambient light conditions. After 30 and 60 minutes, 7% and 20% of larvae were observed to have exited the device, respectively (n = 30). This long occupancy time allows the loading of multiple larvae before injection and allows easier injection due to their predictable positioning within the device (Figs 1 and 2). Moreover, once entrapped, larvae generally wedge themselves into the device and remain motionless, even when turned to reveal their ventral side, which is likely due to their known aversion to light (Figs 1D and 2D) [37]. Larvae that are attempting to exit the device prior to injection can be persuaded to re-enter with a gentle touch to their abdomen. After injection, a larva can be released by gently pulling the two halves of the device apart to allow egress (Fig 1C). To assemble the acrylic glass restraint device, the layers are stacked to construct a chamber, using a binder clip to secure them together (Fig 2). Depending on the size of the larvae, the height of the chamber can be adjusted by removing or adding notched layers. Importantly, a chamber that is too large will enable a larva to turn in the device and not present its ventral side for injection. Larvae are coaxed into entering the chamber head-first by gently pushing their abdomen towards the chamber opening. This procedure takes an average of 12 seconds (SD = +/- 5 seconds, n = 30).

Fig 2

Entrapment of G. mellonella in a restraint device constructed from laser cut acrylic glass.

(A) Dimensions of the acrylic glass restraint device. Measurements are presented in millimeters. (B) Schematic overview of the assembly of acrylic glass without a larva. (C) Assembly of the restraint device without a larva (D) A restraint device with larva captured head-first into the chamber. The ventral side of the larva is visible upon rotation of the chamber.

The injection of restrained Galleria mellonella larvae

To test whether the fabricated restraint devices are suitable for the injection of G. mellonella, larvae were first restrained using the described devices. Each larva was then injected with either phosphate-buffered saline (PBS) or the opportunistic fungal pathogen C. glabrata and the degree of insect mortality was measured. The relative performance of these devices was measured compared to larval restraint by finger and thumb using PBS. Injections were performed with Hamilton 700 syringes. Prior to injection, 70% ethanol was used to wash the syringe three times followed by a single wash with sterile distilled water before loading with PBS or C. glabrata. Fingers holding the restrain device or a larva were protected during injection with a needle resistant glove covered with a disposable laboratory glove to prevent contamination. To inject a larva immobilized within a restraint device, the needle is inserted through the wide end of the restraint device and used to pin down the larva at the last left proleg before puncturing the cuticle (Fig 3 and S2 Movie). The needle is inserted along the long axis of the larva at a shallow angle of 10–20° beneath the cuticle to avoid puncturing the midgut. The needle penetrates to a depth of <5 mm, whereupon the plunger is depressed to eject the contents of the syringe into the hemocoel. The shallow angle and depth of injection can be verified as the needle is visible through the cuticle. The injection time with a single channel Hamilton syringe takes an average of 62 seconds per larva (SD +/- 12 seconds, n = 11) for the micropipette device, which includes loading the syringe, injection, larval release, and decontamination of the needle and syringe. Using a repeat dispenser increases the rate of injection to 8 seconds per larva for both the micropipette tip device (SD +/- 2 seconds, n = 30) and the acrylic glass device (SD +/- 3 seconds, n = 30). This is compared to 30 seconds (SD +/- 16 seconds, n = 30) to restrain and inject larvae between finger and thumb, using a needle resistant glove. After the withdrawal of the needle, the larva is placed in a 9 cm petri dish fitted with two 9 cm diameter disks of paper towel in the dark to promote the egress from the restraint device. Restraint devices can be easily decontaminated with 70% ethanol or 10% bleach after every use. While it is possible to repeatedly sterilize the micropipette devices using an autoclave, this method is not recommended for acrylic glass [37].

Fig 3

A ventral view of a G. mellonella larva restrained within a micropipette device during the injection of C. glabrata into the last proleg.

The injection point in the last proleg of the larvae is punctured by a Hamilton syringe needle.

Once injected, larvae were observed for up to 7 days to assess the physiological consequences of the injection of 4.6 × 106 C. glabrata cells or PBS (Fig 4). Of the 90 larvae injected with PBS using different restraint methods, 96% of larvae survived injection (86/90) after 7 days and with no significant difference in survival of the larvae restrained by any method (Fig 4 and Table 1). There was also no significant difference in the survival curves of the larvae that were injected with C. glabrata (Fig 4 and Table 2), with an average time to 50% lethality (LT50) of 1.8 and 1.4 days for the micropipette tip and acrylic glass devices, respectively (Table 2). In an independent experiment, there was also a dose-dependent lethality with the injection of different numbers of C. glabrata (8 × 105, 3 × 106, and 5 × 106) using the micropipette tip device (Fig 5). The LT50 was calculated as 2.7 and 1.7 days upon injection of 3 × 106, and 5 × 106 C. glabrata cells, respectively, which was significantly different from the PBS control (p < 0.0001) (Fig 5). These data are comparable to previous studies that used alternative injection methods to infect G. mellonella with C. glabrata ATCC 2001 [7].

Fig 4

Survival rates of G. mellonella larvae over seven days post-injection with PBS or C. glabrata cells after using different methods of larval restraint.

Statistical significance was judged by the Kaplan-Meier log rank test and compared three restraint methods after injection with PBS and the restraint devices after injection with C. glabrata (ns = not significant). Results presented for PBS injections were from a single biological replicate (n = 30). Results presented for C. glabrata injections were from two independent biological replicate (n = 15 for each replicate).

Table 1

A comparison of larval mortality using restrain devices compared to restraint by finger and thumb after the injection of PBS.

Device	n	Log rank test (P value)	Device loading time (seconds)	Injection time (seconds)
Finger	30	-	-	30
Micropipette tip	30	0.56	7	8
Acrylic glass	30	0.99	12	8

Table 2

A comparison of larval mortality using two types of restraint device during the injection of C. glabrata.

Device	n	Log rank test (P value)	LT50 (days)
Micropipette tip	30	0.14	1.8
Acrylic glass	30		1.4

Fig 5

Survival rates of G. mellonella larvae over five days post-injection with three different inocula of C. glabrata cells or with PBS.

Statistical significance relative to PBS was judged by the Kaplan-Meier log rank test (** p < 0.0001, ns = not significant). Results presented for these injections were from at least two independent biological replicates.

Conclusion

The method described in this report uses engineering controls to spatially separate an injection needle from the fingers of laboratory personnel. When combined with a needle resistant glove, this almost completely eliminates the risk of needle injury and the accidental infection of laboratory personnel with pathogenic microbes. The devices that we have described to restrain G. mellonella larvae are inexpensive, non-porous, and can be reused multiple times after sterilization or decontamination. We have found that the ability to entrap multiple larvae enables a high rate of injection when using a repeat dispenser with a Hamilton syringe. The described devices are most effective with two people working together, one loading larvae into the restraint devices and the other performing injections. We find that the rate of injection is comparable to the Galleria grabber (20–25 seconds per injection) [34] and is close to the traditional methods of immobilization between finger and thumb protected by a needle resistant glove (30 seconds). The described method for the restraint of G. mellonella larvae offers a rapid and reproducible workflow for the injection of hazardous microorganisms.

Entrapment of a G. mellonella larva in an injection device constructed from a micropipette tip.

(MOV)

Click here for additional data file.

Injection of a restrained G. mellonella larva in the last proleg with a Hamilton syringe.

(MOV)

Click here for additional data file.

Brand compatibility with construction of the micropipette tip restraint device.

(DOCX)

Click here for additional data file.

10 May 2020

PONE-D-20-06694

The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella.

PLOS ONE

Dear Dr. Rowley,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

Three experts in this field reviewed your paper and found that this Galleria mellonella injection method presented by this study could be very useful for those working with this model organism. Having said that, the reviewer 1 and 3 raised several critical concerns that you need to pay attention to. In particular, the comparative data among suggested techniques appear to be critical to me. Other comments should also be addressed point-by-point.

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Reviewer #1: This manuscript provides the community working with Galleria mellonella larvae with 2 alternative ways of larval injection which aim to reduce, or even fully prevent needle injuries for the person injecting larvae, and as a consequence, transmission of pathogens/microbes to the injecting person.

Overall I agree with the authors that both tools, the modified pippette tips and the plexi-glass chamber, are useful Tools to facilitate Galleria injection and reduce the potential danger of needle injuries. An Information /method very valuable to the community.

Regardless of the Tools shown here, I have some comments I d like the authors to address:

Major:

In my opinion, this manuscript is solely a description of a method/in fact a tool to facilitate a method already known to the community - nevertheless the article is structures like a Research paper in Methods/Results and Discussion, this might be due to the outline the journal recommends, but what has been shown here as "results" is basically a Repetition of the method part, so I suggest a re-structuring of the manuscript

The actual Galleria-survivial Assay Shows, that Candida glabrata kills larvae, injected with the help of one tool - but there has not been carried out a comparision between the 2 Tools (pippette tip or plexiglass chamber), nor to a method without any of These Tools. I therefore strongly encourage the authors to Show comparative data on this - with only one inocolum size.

Also, it is important to include: how many larvae per sample (per inocolum) have been infected, do the survival curves represent one Experiment? or an average survival of 3 Experiments - has the Assay been repeated at all? And at what temperature have the larvae been incubated.

As discussion Point the authors might add that many Groups use Insuline syringes to inject larvae - could they also be used? or is their needle to short to reach the larvae trapped in the two different Chambers

Minor:

line 83: "In this study...should start as a new Paragraph

line 87: I dont fully agree with the conclusion "significantly resuces the Manual handling"...as to me urging a larva into one of the Chambers could be similarly stressful to the animal as Holding it between the fingers? and the authors do not Show comparative data that proof this conclusion - the only obvious Advantage of both chamber is - and that is important - that piercing yourself with a needle is almost impossible

line 124 ff: many Groups have described that they inject larvae through one of the prolegs - because this was explained to cause least harm to the larvae (Fallon et al, Methods MolBiol 2012) - this would not be possible to do with larvae in a chamber - please comment on this - did the authors do comparative assays to compare if injecting via the cuticle is more harmful thatn through the opening of the prolegs?

line 146: define LT50

Reviewer #2: The paper tediously describes a novel technique of injection of restraint Galleria larvae which is the model for studies toxicology, immunology, pathology. The approach offered is advantageous as compared to the previous ones. The paper is well organized, the data are convincing and the substantial body of relevant literature is cited. There is a high probability that the article, if published, will be referenced in future research.

Reviewer #3: General comments:

The text submitted by Fredericks and colleagues is a technical paper focussed on reducing the risk of accidental inoculation of potentially harmful pathogens when using Galleria larvae as an in vivo model. Their solution is a set of ‘cheap and cheerful’ handling devices – described herein. The text is mostly, clearly written if a little superficial at times. The take-home message is straightforward but clarity is needed in a couple of areas.

Overall, this paper follows a very similar approach to another manuscript by Dalton et al. (2017) – albeit their designs are more elegant.

Dalton, J. P., Uy, B., Swift, S., & Wiles, S. (2017). A novel restraint device for injection of Galleria mellonella larvae that minimizes the risk of accidental operator needle stick injury. Frontiers in cellular and infection microbiology, 7, 99.

There are many administration techniques used for Galleria, and these can differ according to lab, e.g., chilling on ice prior to inoculation, using a pipette tip taped to the bench where the insect larva is held against (to avoid needlestick injury), brief submersion in ‘baths’ containing entomopathogens, etc.

This reviewer does agree that more standardisation for Galleria work is needed (having worked on these insects since 2008). However, some of the arguments used for suggesting these devices are better than others available are askew.

When referring to Dalton et al. (2017), the authors are critical of the need for multiple de-contamination rounds. The Galleria grabber, which is comprised of a large (15 mm) thick sponge and bulldog clip, is perhaps more environmentally friendly than the new devices explained herein, where there is a sizeable volume of single-use plastic.

Lines 52-55. Submersion is also quite common with biopesticide work. It would be prudent to include a reference here, perhaps a recent paper comparing gavage and direct injection e.g., Coates et al. (2019).

Coates, C. J., Lim, J., Harman, K., Rowley, A. F., Griffiths, D. J., Emery, H., & Layton, W. (2019). The insect, Galleria mellonella, is a compatible model for evaluating the toxicology of okadaic acid. Cell biology and toxicology, 35(3), 219-232.

Lines 70-72: this may be through, but intrahaemocoelic injection can be achieved using automated micro-injection platforms.

Lines 80 - 83: see comments above on the perceived betterment of the proposed designs versus that of Dalton et al (2017).

Line 87 (and elsewhere): It is unclear how ‘injection speed’ was measured

Lines 93-99: Unless this protocol has been published previously the authors must either cite the original text, or, provide specific details, e.g., centrifugation (speed, duration, temperature), PBS, pH? (filter-sterilised?), how many yeast were injected per larva?

Why did you culture C. glabrata at room temperature (~20oC) when it is a microbe usually found in human mucosa? Additionally, you then inoculate/incubate (Line 108) the insects at 37 degrees C. Consistency is needed.

There appears to be only one strain/species used – contrary to the title of the section.

Line 109: replace larva with ‘larvae’, and are with ‘were’

Line 144: please include the sample sizes, and number of technical versus biological replicates. Did the authors follow the ARRIVE guidelines regarding sample size and power calculations?

Line 157: re-usable acrylic sheets will surely need to be de-contaminated, as with the sponge-based device developed by Dalton et al (2017), it can act as a fomite (refer back to critical comments, lines 80-83).

Lines 160 – 163:

Although essential to the argument being made, the authors do not describe their protocol in the methods section, or the sample size used to generate this information.

Lines 163- 165: how are the authors determining ‘faster rate of injection’, qualitatively, arbitrary benchmark?

Line 203: the so-called ‘white solids’ represent insect fat body

Lines 221: provide specific detail to evidence this statement.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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25 Jun 2020

Response to reviewers’ comments

Reviewer #1:

This manuscript provides the community working with Galleria mellonella larvae with 2 alternative ways of larval injection which aim to reduce, or even fully prevent needle injuries for the person injecting larvae, and as a consequence, transmission of pathogens/microbes to the injecting person.

Overall I agree with the authors that both tools, the modified pippette tips and the plexi-glass chamber, are useful Tools to facilitate Galleria injection and reduce the potential danger of needle injuries. An Information /method very valuable to the community.

Regardless of the Tools shown here, I have some comments I d like the authors to address:

Major:

In my opinion, this manuscript is solely a description of a method/in fact a tool to facilitate a method already known to the community - nevertheless the article is structures like a Research paper in Methods/Results and Discussion, this might be due to the outline the journal recommends, but what has been shown here as "results" is basically a Repetition of the method part, so I suggest a re-structuring of the manuscript

Manuscript organization revised: We have now removed a large section from the Materials and Methods section detailing the injection process and incorporated it into the Results/Discussion section. The Materials and Methods section is now only used to describe details relating to the more granular details of our study that are not mentioned in the Results/Discussion section (i.e. strains used, details of chamber construction, and data analysis). We have also added three new subheadings in the Results/Discussion section to compare different methods of injection, to describe the loading of our restraint devices, and the injection process using our devices. This new organization avoids the somewhat redundant description of the injection process that was written in the initial submission.

The actual Galleria-survivial Assay Shows, that Candida glabrata kills larvae, injected with the help of one tool - but there has not been carried out a comparision between the 2 Tools (pippette tip or plexiglass chamber), nor to a method without any of These Tools. I therefore strongly encourage the authors to Show comparative data on this - with only one inocolum size.

DONE: New data included for better comparison between the methods: Figure 4, tables 1 and 2 are now included and comparisons between the methods are made in the text at lines: 216-228. We have added additional data that directly compares the performance of the two injection chambers using the opportunistic pathogen C. glabrata. We did not compare the injection using finger and thumb because of the risk of laboratory acquired infection. We did however, compare the restrain devices to manual restraint between finger and thumb for injections of PBS.

Also, it is important to include: how many larvae per sample (per inocolum) have been infected, do the survival curves represent one Experiment? or an average survival of 3 Experiments - has the Assay been repeated at all? And at what temperature have the larvae been incubated.

Additional details have been provided. We now more clearly detail the numbers of larvae used in each experiment in Table 1 and Table 2.

As discussion Point the authors might add that many Groups use Insuline syringes to inject larvae - could they also be used? or is their needle to short to reach the larvae trapped in the two different Chambers

Added: Line 134 “We also expect that this method is fully compatible with the use of insulin syringes with shorter needles as prolegs are almost always positioned close to the opening in the injection chambers”

Minor:

line 83: "In this study...should start as a new Paragraph

Done.

line 87: I dont fully agree with the conclusion "significantly resuces the Manual handling"...as to me urging a larva into one of the Chambers could be similarly stressful to the animal as Holding it between the fingers? and the authors do not Show comparative data that proof this conclusion - the only obvious Advantage of both chamber is - and that is important - that piercing yourself with a needle is almost impossible

Done. We have removed this statement based on the reviewers concerns. The sentence now reads: (line 91) “Both devices provide increased protection of the operator from accidental needlestick injury and laboratory-acquired infection.”

line 124 ff: many Groups have described that they inject larvae through one of the prolegs - because this was explained to cause least harm to the larvae (Fallon et al, Methods MolBiol 2012) - this would not be possible to do with larvae in a chamber - please comment on this - did the authors do comparative assays to compare if injecting via the cuticle is more harmful thatn through the opening of the prolegs?

Comment: We describe the targeting of the proleg in the paper (lines 199-201) and demonstrate the technique in the supplementary movie S1. In our method the angle of injection is quite shallow because of the restraint of the larva in the injection chamber.

line 146: define LT50

Done: We have now more completely defined LT50. Line 221: “...with an average time to 50% lethality (LT50)...”

Reviewer #2:

The paper tediously describes a novel technique of injection of restraint Galleria larvae which is the model for studies toxicology, immunology, pathology. The approach offered is advantageous as compared to the previous ones. The paper is well organized, the data are convincing and the substantial body of relevant literature is cited. There is a high probability that the article, if published, will be referenced in future research.

Reviewer #3:

General comments:

The text submitted by Fredericks and colleagues is a technical paper focussed on reducing the risk of accidental inoculation of potentially harmful pathogens when using Galleria larvae as an in vivo model. Their solution is a set of ‘cheap and cheerful’ handling devices – described herein. The text is mostly, clearly written if a little superficial at times. The take-home message is straightforward but clarity is needed in a couple of areas.

Overall, this paper follows a very similar approach to another manuscript by Dalton et al. (2017) – albeit their designs are more elegant.

Dalton, J. P., Uy, B., Swift, S., & Wiles, S. (2017). A novel restraint device for injection of Galleria mellonella larvae that minimizes the risk of accidental operator needle stick injury. Frontiers in cellular and infection microbiology, 7, 99.

There are many administration techniques used for Galleria, and these can differ according to lab, e.g., chilling on ice prior to inoculation, using a pipette tip taped to the bench where the insect larva is held against (to avoid needlestick injury), brief submersion in ‘baths’ containing entomopathogens, etc.

This reviewer does agree that more standardisation for Galleria work is needed (having worked on these insects since 2008). However, some of the arguments used for suggesting these devices are better than others available are askew.

When referring to Dalton et al. (2017), the authors are critical of the need for multiple de-contamination rounds. The Galleria grabber, which is comprised of a large (15 mm) thick sponge and bulldog clip, is perhaps more environmentally friendly than the new devices explained herein, where there is a sizeable volume of single-use plastic.

Done: We have now clarified that both of our devices are fully reusable to address the environmental concerns of the reviewer. Lines 253 “The devices that we have described to restrain G. mellonella larvae are inexpensive, non-porous, and can be reused multiple times after sterilization or decontamination.”

Lines 52-55. Submersion is also quite common with biopesticide work. It would be prudent to include a reference here, perhaps a recent paper comparing gavage and direct injection e.g., Coates et al. (2019).

Done: Our We now included additional references that demonstrate the various methods of introducing pathogens and chemicals into G. mellonella (including the reference suggested by the reviewer) (Line 55)

Lines 70-72: this may be through, but intrahaemocoelic injection can be achieved using automated micro-injection platforms.

Comment: The authors are aware of autoinjection platforms for C. elegans and Zebrafish embryos, but not for Galleria. If the reviewer could provide us with a specific description and reference we would be happy to include it in the paper.

Lines 80 - 83: see comments above on the perceived betterment of the proposed designs versus that of Dalton et al (2017).

Done: We have reworded this section to better describe the Galleria grabber. We focus our evaluation of the device on its porosity, which is of primary concern when dealing with pathogenic microbes that require biological containment. Lines 79-84: “For the injection of G. mellonella, there is also a device named the “Galleria grabber” that has been developed for the restraint of larvae between layers of a sponge [34]. This method enables injection without the need for grasping larvae between finger and thumb and offers the user protection from accidental needlestick injury. However, the use of a porous sponge for multiple injections increases the chance of its contamination by pathogenic microbes, which presents a challenge for effective decontamination.”

Line 87 (and elsewhere): It is unclear how ‘injection speed’ was measured

Done: We have rearranged and added to the description of the injection process to better describe the process that was timed for each injection. Lines 204-214 “The injection time with a single channel Hamilton syringe takes an average of 62 seconds per larva (SD +/- 12 seconds, n = 11) for the micropipette device, which includes loading the syringe, injection, larval release, and decontamination of the needle and syringe. Using a repeat dispenser increases the rate of injection to 8 seconds per larva for both the micropipette tip device (SD +/- 2 seconds, n = 30) and the acrylic glass device (SD +/- 3 seconds, n = 30). This is compared to 30 seconds (SD +/- 16 seconds, n = 30) to restrain and inject larvae between finger and thumb, using a needle resistant glove.”

Lines 93-99: Unless this protocol has been published previously the authors must either cite the original text, or, provide specific details, e.g., centrifugation (speed, duration, temperature), PBS, pH? (filter-sterilised?), how many yeast were injected per larva?

Done: Lines102-105 “Hemocytometer counts of these cultures were used to determine the number of yeasts used for each injection (8.0 × 105, 3.0 × 106, 4.6 × 106, and 5.0 × 106 C. glabrata cells per injection). Prior to injection, yeast cells were harvested by centrifugation at 8,000 × g for 1 min (25°C) and suspended in filter sterilized PBS (pH 7).

Why did you culture C. glabrata at room temperature (~20oC) when it is a microbe usually found in human mucosa? Additionally, you then inoculate/incubate (Line 108) the insects at 37 degrees C. Consistency is needed.

Comment: Initial growth at room temperature was chosen to prevent overgrowth of C. glabrata cultures to enable more consistent results and to accommodate the schedules of undergraduate researchers that spearheaded this project. In addition, C. glabrata is also found in the environment in diverse habitats, not just within the mammalian digestive tract. Growth at 37oC initiates rapid expression of virulence genes (such as adhesins) that enable host invasion.

There appears to be only one strain/species used – contrary to the title of the section.

Done: Title changed to “Culturing and preparation of yeast cells for injection.”

Line 109: replace larva with ‘larvae’, and are with ‘were’

Done

Line 144: please include the sample sizes, and number of technical versus biological replicates. Did the authors follow the ARRIVE guidelines regarding sample size and power calculations?

Done: We now include a new table (Table 1) that states the number of larvae used in each experiment. Figure legends for Fig 4 and Fig 5 describe the number of technical vs biological replicate for each experiment.

In line with the ARIVE guidelines, we also added details of the power analysis that we performed. Line 142 “A power analysis was performed to assess the required sample size using G*Power (V3.1) (One-tailed T-Test, � = 0.05, � = 0.2, effect size = 0.8).

Line 157: re-usable acrylic sheets will surely need to be de-contaminated, as with the sponge-based device developed by Dalton et al (2017), it can act as a fomite (refer back to critical comments, lines 80-83).

Done: see previous comment above.

Lines 160 – 163:

Although essential to the argument being made, the authors do not describe their protocol in the methods section, or the sample size used to generate this information.

Comment: based on other reviewers comments that this is a methods paper we have fully described all materials used and the details of larval handling etc. in the methods and described the method fully in the results section.

Lines 163- 165: how are the authors determining ‘faster rate of injection’, qualitatively, arbitrary benchmark?

Done: Rephrased the sentence Line 162 “This long occupancy time allows the loading of multiple larvae before injection and allows easier injection due to their predictable positioning within the device (Fig 1 and 2).”

Line 203: the so-called ‘white solids’ represent insect fat body

Sentence revised – see next comment.

Lines 221: provide specific detail to evidence this statement. “In the rare case that an injected larva exudes excessive amounts of hemolymph or any white solids (from the fat body), then the individual is discarded because of the increased likelihood of injection-induced injury.”

Done: We have removed this sentence because of a lack of data. We initially included it to indicate that we sometimes have off-target injections that we have observed cause injury to larvae as described previously (Fallon et al, Methods Mol Biol (2012)). However, this user error is infrequent so we feel it is not a major concern (less than 1 in 1000 injections).

Submitted filename: Mollifier paper_response to reviewers_final.docx

Click here for additional data file.

15 Jul 2020

The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella.

PONE-D-20-06694R1

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20 Jul 2020

PONE-D-20-06694R1

The design and implementation of restraint devices for the injection of pathogenic microorganisms into Galleria mellonella

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