# Protocol for monitoring Yersinia pestis colonization of the proventriculus in the flea Xenopsylla cheopis using microscopy

Amélie Dewitte, Elisabeth Werkmeister, Florent Sebbane, Sébastien

Bontemps-Gallo

► To cite this version:

Amélie Dewitte, Elisabeth Werkmeister, Florent Sebbane, Sébastien Bontemps-Gallo. Protocol for monitoring Yersinia pestis colonization of the proventriculus in the flea Xenopsylla cheopis using microscopy. STAR Protocols, 2025, 6 (2), pp.103853. 10.1016/j.xpro.2025.103853. hal-05098012

HAL Id: hal-05098012

https://hal.science/hal-05098012v1

Submitted on 4 Jun 2025

HAL is a multi-disciplinary open access
archive for the deposit and dissemination of sci-
entific research documents, whether they are pub-
lished or not. The documents may come from
teaching and research institutions in France or
abroad, or from public or private research centers.

L'archive ouverte pluridisciplinaire HAL, est
destinée au dépôt et à la diffusion de documents
scientifiques de niveau recherche, publiés ou non,
émanant des établissements d'enseignement et de
recherche français ou étrangers, des laboratoires
publics ou privés.

<figure><img src="image_1.png" /></figure>

Distributed under a Creative Commons Attribution 4.0 International License
Protocol

Protocol for monitoring *Yersinia pestis*
colonization of the proventriculus in the flea
*Xenopsylla cheopis* using microscopy

<figure><img src="image_2.png" /></figure>

Colonization of the flea proventriculus is essential for increasing the likelihood of *Yersinia pestis* transmission. Here, we present a protocol for visualizing *Y. pestis* colonization dynamics within the proventriculus of the flea *Xenopsylla cheopis*. We describe steps for quantifying the number of infected proventriculi, the percentage of the surface colonized by bacteria, and an estimation of the bacterial load. These metrics provide a tool for assessing the role of bacterial genes in establishing a transmissible infection.

Publisher's note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Amélie Dewitte,
Elisabeth
Werkmeister,
Florent Sebbane,
Sébastien
Bontemps-Gallo

amelie.dewitte@inserm.fr
(A.D.)
sebastien.
bontemps-gallo@cnrs.fr
(S.B.-G.)

Highlights

Instructions for
quantifying *Y. pestis*
colonization dynamics
in the flea proventriculus

Steps for combining
dissection, fluorescence
microscopy, and
ImageJ-based analysis

Guidelines for
measuring infection
frequency, colonized
surface area, and
bacterial load

Enables standardized
and reproducible
analysis of flea-borne
transmission

Dewitte et al., STAR Protocols
6, 103853
June 20, 2025 © 2025 The

Author(s). Published by
Elsevier Inc.

https://doi.org/10.1016/j.xpro.2025.103853
Protocol

# Protocol for monitoring *Yersinia pestis* colonization of the proventriculus in the flea *Xenopsylla cheopis* using microscopy

Amélie Dewitte,<fnref n="1" />,<fnref n="3" />,* Elisabeth Werkmeister,<fnref n="1" />,<fnref n="2" /> Florent Sebbane,<fnref n="1" /> and Sébastien Bontemps-Gallo<fnref n="1" />,<fnref n="4" />,*

SUMMARY

Colonization of the flea proventriculus is essential for increasing the likelihood of *Yersinia pestis* transmission. Here, we present a protocol for visualizing *Y. pestis* colonization dynamics within the proventriculus of the flea *Xenopsylla cheopis*. We describe steps for quantifying the number of infected proventriculi, the percentage of the surface colonized by bacteria, and an estimation of the bacterial load. These metrics provide a tool for assessing the role of bacterial genes in establishing a transmissible infection.

For complete details on the use and execution of this protocol, please refer to
Dewitte et al.<fnref n="1" />

BEFORE YOU BEGIN

*Yersinia pestis*, the causative agent of plague, is among the most lethal bacterial pathogens. Primarily transmitted by fleas, it circulates among rodent populations. Once ingested by the flea, *Y. pestis* colonizes its proventriculus – a valve between the esophagus and the midgut – to form an undetachable bacterial mass that blocks blood ingestion. This blockage starves the flea, driving it to bite repeatedly in a frenzied attempt to feed, thereby facilitating *Y. pestis* regurgitation into the bite site. Understanding the mechanisms underlying flea-borne transmission of *Y. pestis* is crucial for developing control strategies and potentially eradicating the bacterium.

In 1996, Hinnebusch et al.<fnref n="2" /> were the first to use *Y. pestis* expressing green fluorescent protein (GFP) with microscopy to study the role of genes, notably the *hmsHFRS* locus, in proventricular blockage. Although this empirical approach has since been widely adopted in the scientific community, its reliance on qualitative assessments introduces potential experimenter bias.

Here, we describe a protocol that provides a quantitative and qualitative approach to investigating and analyzing *Y. pestis* colonization dynamics within the flea proventriculus. Our method allows for the measurement of both the colonized surface area of the foregut and the percentage of infected proventriculi. Additionally, fluorescence intensity allows estimation of bacterial load. Indeed, a standard CFU plating assay allows quantification of the total bacterial population in the flea, but not specifically in the proventriculus. Dissecting the digestive tract to isolate only the proventriculus would be highly challenging and raises concerns about potential loss of part of the bacterial population during the procedure. By applying the protocol at different time points post-infection, we can characterize bacterial colonization dynamics within the flea proventriculus.

<figure><img src="image_3.png" /></figure>

<footnote n="1">¹University Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, U1019-UMR9017-CIIL-Centre d'Infection et d'Immunité de Lille, Lille, France</footnote>

<footnote n="2">²University Lille, CNRS, INSERM, CHU Lille, Institut Pasteur de Lille, US41-UMS2014 - PLBS, Lille, France</footnote>

<footnote n="3">³Technical contact</footnote>

<footnote n="4">⁴Lead contact</footnote>

<footnote>*Correspondence: amelie.dewitte@inserm.fr (A.D.), sebastien.bontemps-gallo@cnrs.fr (S.B.-G.)
https://doi.org/10.1016/j.xpro.2025.103853</footnote>
**Institutional permissions**

Regulations for the possession and use of Y. pestis vary by country and strain. For instance, this bacterium is classified as a Tier 1 Select Agent by the U.S. Centers for Disease Control (https://www.selectagents.gov/compliance/guidance/occupational-health/appendix-1.htm) and is also regulated by the French administration (https://ansm.sante.fr/vos-demarches/chercheur/demander-une-autorisation-pour-les-micro-organismes-et-toxines). Therefore, researchers must comply with all local and national guidelines for possessing Y. pestis and conduct work in appropriate containment laboratories.

The protocol described here does not involve the use of live animals. However, the maintenance of infected fleas may require the use of live animals, which is subject to approval from the relevant institutional authorities and validation by an ethics committee.

**Flea infection**

© Timing: 3 days

**Note:** Instructions for flea rearing and infection are available on the BEI Resources website: https://www.beiresources.org/Catalog/VectorResources.aspx. A brief version of the flea infection procedure is provided for convenience.

1. Inoculate 8 mL of Brain Heart Infusion (BHI) broth with Y. pestis harboring the pAcGFP1 plasmid from a frozen stock in a 15 mL tube.

2. Incubate the culture at 28°C overnight (24 h) without shaking.

3. Add 8 mL of the overnight pre-culture to 92 mL of BHI in a 250 mL sterile plastic bottle.

4. Incubate the culture at 37°C overnight (16-20 h) without shaking.

5. Measure the optical density at 600 nm (OD₆₀₀) to determine the bacterial concentration (bacteria per mL).

6. Transfer the culture in 50 mL tubes and centrifuge at 3000 × g for 7 min at room temperature (20°C).

7. Discard the supernatant and suspend the bacterial pellet in 1x PBS to obtain a final concentration of 2.75 × 10⁹ bacteria/mL.

8. Mix 1 mL of the bacterial suspension with 4.5 mL of mouse blood.

9. Load 5.5 mL of the blood into the artificial feeding device.

10. Attach the capsule containing anesthetized fleas that have been starved for at least 5 days prior to the experiment (Figure 1).

▲ **CRITICAL:** step: Fleas are anesthetized by placing the flea-container on ice for 5 min. prolonged exposure beyond 5 min may increase flea mortality.

11. Connect the artificial feeding system to a water circuit to maintain the blood temperature at approximately 37°C.

12. Allow the fleas to feed for 1 h and 15 min.

13. Collect the fleas and select those that have taken a full blood meal.

**Note:** Fleas fully engorged with blood (see Figure 2A) are selected under a stereomicroscope.

14. Place the selected fleas into a suitable container and incubate them in a climatic chamber at 21°C and 75% humidity.

**Note:** The incubation period depends on the experimental design chosen by the investigators. In our initial macro experiment, fleas were incubated for 27 days.
<figure><img src="image_4.png" /><figcaption>Figure 1. Schematic of the artificial feeding device for fleas</figcaption></figure>

KEY RESOURCES TABLE

<table><thead><tr><th>REAGENT or RESOURCE</th><th>SOURCE</th><th>IDENTIFIER</th></tr></thead><tbody><tr><td>Bacterial and virus strains</td><td></td><td></td></tr><tr><td>Yersinia pestis strain KIM6+</td><td>N/A</td><td>N/A</td></tr><tr><td>Experimental models: Organisms/strains</td><td></td><td></td></tr><tr><td>Xenopsylla cheopis (adult)</td><td>N/A</td><td>N/A</td></tr><tr><td>Recombinant DNA</td><td></td><td></td></tr><tr><td>Plasmid pAcGFP1</td><td>Clontech</td><td>632497</td></tr><tr><td>Software and algorithms</td><td></td><td></td></tr><tr><td>ImageJ software</td><td>Schneider et al.<sup>3</sup></td><td>https://imagej.net/ij/</td></tr><tr><td>GraphPad Prism v.10</td><td>GraphPad Software, Inc.</td><td>N/A</td></tr><tr><td>Other</td><td></td><td></td></tr><tr><td>Microscope slides</td><td>Thermo Scientific</td><td>J1800AMNZ</td></tr><tr><td>Cover glasses</td><td>VWR</td><td>ECN 631-1577</td></tr><tr><td>Tweezers (Mini Dumont#M5S, stainless steel, 8.2 cm, 0.09 × 0.05 mm tips)</td><td>World Precision Instruments</td><td>501764</td></tr><tr><td>BD BACTO brain heart infusion</td><td>BD</td><td>237500</td></tr><tr><td>Phosphate-buffered saline (PBS) solution 10x</td><td>Euromedex</td><td>ET330</td></tr><tr><td>250 mL easy grip polystyrene storage bottles with 45 mm caps</td><td>Coming</td><td>430281</td></tr><tr><td>Lithium heparinized mouse blood (OF1, male)</td><td>Charles River Laboratories</td><td>on request</td></tr><tr><td>Glass membrane feeder</td><td>N/A</td><td>N/A</td></tr><tr><td>Flea capsules</td><td>N/A</td><td>N/A</td></tr><tr><td>Nikon ECLIPSE Ci-S microscope</td><td>Nikon</td><td>N/A</td></tr><tr><td>Nikon Sight DS-Fi1c camera</td><td>Nikon</td><td>M554E</td></tr><tr><td>Nikon Intensilight C-HGFI precentered fiber illuminator</td><td>Nikon</td><td>M349E</td></tr><tr><td>Nikon Camera Control Unit DS-L3</td><td>Nikon</td><td>M555E</td></tr><tr><td>Nikon Plan Fluor 20x objective</td><td>Nikon</td><td>N/A</td></tr><tr><td>B-2A fluorescent cube</td><td>Nikon</td><td>MBE44504</td></tr><tr><td>Leica stereomicroscope MZ6</td><td>Leica</td><td>N/A</td></tr></tbody></table>
<figure><img src="image_5.png" /><figcaption>**Figure 2. Morphological and autofluorescent features of fed and unfed fleas**</figcaption></figure>

(A) Image of a not fully fed flea (left) and a fully fed flea (right). The digestive tract of the fed flea is bright red and fills the entire abdominal area.

(B) Image showing the autofluorescence of the Malpighian tubules (MT). Images (4x) were acquired using a bright-field microscope (left image) and a fluorescence microscope (B2-A filter, right image). Scale bars: 100 µm.

## MATERIALS AND EQUIPMENT

* Nikon Eclipse Ci-S microscope, equipped with a Nikon Sight DS-Fi1c camera, a Nikon Intensilight C-HGFI precentered fiber illuminator. All images were acquired using a Nikon Plan Fluor 20x (NA 0.5) objective and a B-2A fluorescent cube (Ex 450-490, DM 505, BA 510, C-FL-C).

* Leica stereomicroscope MZ6.

* Glass membrane feeder and flea capsule are made on request as described in the U.S. patent US4850305A (Georgi & Wade, 1987 - now public domain since 2007). These items can be custom-made by Lillie Glass (for the glass feeder) and by companies like Mobile Mechanics (David Cross) for the flea capsules.

## STEP-BY-STEP METHOD DETAILS

The timing of this protocol depends on the number of conditions and fleas analyzed. Our standard experimental design examines 20 fleas per condition, with timing based on analyzing 20 fleas for a single experimental condition.

### Dissection of *X. cheopis* digestive tract

##### Timing: ~30 min

To evaluate *Y. pestis* colonization of the flea proventriculus, the flea must be dissected to extract the digestive tract, as its exoskeleton exhibits strong autofluorescence. This protocol outlines the procedure from digestive tract extraction to slide preparation for microscopy analysis.

1. Immobilize the fleas (contained in a glass container) on ice for at least 5 min before handling.

**Note:** To fully euthanize the fleas, place them in a freezer at -20°C or -80°C for 5-10 min.
<figure><img src="image_6.png" /><figcaption>**Figure 3.** Annotated schematic of the flea with a yellow dashed line indicating where to place the forceps
Tergites (T) are on the dorsal side, and sternites (S) are on the ventral side.</figcaption></figure>

2. Add 10 µL of Milli-Q water onto a microscope slide.

3. Place one anesthetized flea in the drop of water.

4. Dissect the flea under a stereomicroscope (Figure 3).

a. Using two forceps, firmly grasp the flea.

b. With one forceps, hold the flea transversely behind the head.

c. With the other forceps, grasp along tergites and sternites 6 and 7.

d. Pull the forceps apart while maintaining pressure to separate the chitinous exoskeleton until the digestive tract is released into the water.

e. Release pressure on one of the forceps to extract the digestive tract.

5. Discard the exoskeleton.

6. Remove the Malpighian tubules (which autofluorescence) (Figure 2B) - Critical step.

7. Apply a coverslip.

**Note:** Placing the coverslip should expel midgut contents, while the bacterial mass within the proventriculus should remain intact.

Δ CRITICAL: step: Ensure that the sample does not dry out during imaging and that no water halo effect surround it. If necessary, after placing the coverslip, add water by capillarity using a micropipette and remove excess water by capillarity with a small piece of paper.

8. Place the sample on the microscope stage.

**Imaging**

◎ Timing: ~20 min

9. Acquire images of the proventriculus using widefield light microscopy.

**Note:** We used a Nikon Eclipse Ci-S microscope equipped with a Nikon Sight DS-Fi1c camera. All images were acquired using a Nikon Plan Fluor 20× dry objective. The B2-A fluorescent filter cube was used, with an exposure time of 300 ms and a camera gain of 140. The long-pass B2-A filter cube transmits all wavelengths above 500 nm, allowing the proventriculus autofluorescence to appear in orange while the GFP⁺ Y. pestis remains visible in green.
<figure><img src="image_7.png" /><figcaption>Figure 4. Results of proventriculus colonization analysis at 1-, 2-, 6-, 13-, and 27-days post-infection from Dewitte et al.</figcaption></figure>

(A) Fluorescence images of the proventriculus (in orange) infected with the wild-type strain (in green).

Panel (B) shows the proportion of proventriculi colonized (black) or uncolonized (white) by Y. pestis.

Panel (C) presents the progression of the colonized surface area in fleas infected with the wild-type strain, while panel (D) displays the mean fluorescence intensity (arbitrary units, AU) measured in the proventriculus of fleas colonized by the wild-type strain. Data are adapted from Dewitte et al.¹ Each dot represents an individual flea (n = 20 except at 1-Day post-infection n=16), with data from uninfected proventriculi also shown (labeled as “U”). The dotted line indicates the threshold above which fluorescence intensity (AU) signifies the presence of bacteria, with thresholds set at 1% for surface area and 1.32 × 10⁷ AU for fluorescence intensity. The red line represents the median of all values exceeding the threshold. Statistical analyses were performed using GraphPad Prism with a 2-way ANOVA test (* p < 0.0001). Data from unfed fleas are significantly different from the other conditions (p < 0.0001).

## EXPECTED OUTCOMES

The approach described in this protocol provides three key metrics for assessing Y. pestis colonization of the proventriculus (Figure 4): 1) proventricular surface coverage (i.e. the percentage of the proventricular surface colonized by Y. pestis); 2) infection frequency (i.e. the percentage of infected versus non-infected proventriculi at each time point; and 3) bacterial density (i.e. fluorescence intensity measurements estimate bacterial load and provide insights into bacterial growth dynamics within the foregut). Our observations indicate that Y. pestis colonizes the proventriculus as early as the first day post-infection, with all proventriculi showing bacterial presence up to day 6. By day 2, colonization spreads rapidly across the proventricular surface, while the bacterial population continues to grow, reaching high density by day 6. On days 13 and 27, one flea was found to be uninfected.

## QUANTIFICATION AND STATISTICAL ANALYSIS

### ImageJ macro protocol for proventriculus analysis

Timing: ~15 min

1. Launch ImageJ: Open the ImageJ software on your computer.

2. Run the macro: In the menu bar, navigate to Plugins > Macros > Run...

3. A window will prompt you to select the Macro.

4. Select Image Folder: A window will prompt you to select the folder containing all the images.

5. Image opening: ImageJ will automatically open the first image from the selected folder. Troubleshooting 1.

6. "Action Required" windows: A window titled "Action Required" will appear (Figure 5).
Steps 5 to 7: Selection of the Area

<figure><img src="image_8.png" /><figcaption>Figure 5. Screenshot from the analysis using the macro in ImageJ</figcaption></figure>

7. Select the proventriculus area: Using the Freehand selections tool, manually outline the proventriculus on the image.

8. Validate the selection: Click "Ok" in the "Action Required" window to confirm.

9. Automatic Image Analysis:

a. Removal of unselected Areas.

b. Measurement of the region of interest (ROI).

c. Color threshold processing: a color threshold is applied to select bacteria (green) while excluding proventriculus autofluorescence (orange). Troubleshooting 2.

d. Calculation of the Area occupied by Bacteria.

i. A binary mask of the bacterial area is created.

ii. The area of white pixels (= bacteria) is measured.

iii. The percentage of the ROI covered by bacteria is calculated.

e. Calculation of bacterial load.

i. Color channel splitting: The image is split into red, green, and blue channels.

ii. Channel filtering: The red and blue channels are removed, retaining only the green channel.

iii. Conversion to 32-bit Mode: The green channel is multiplied by the binary mask corresponding to the area occupied by the bacteria, resulting in a 32-bit image of the bacteria's fluorescence intensity, with non-occupied areas (outside the mask) having a value of 0.

iv. Fluorescence intensity measurement: The bacterial area fluorescence intensity is measured.
v. Calculation of average fluorescence intensity.

10. Next Image opens automatically. The next image will open for analysis.

11. Repeat the process. Repeat Steps 6 to 9 for all Images.

12. Output Folder Creation. Once the analysis is complete, a folder named "Mask" containing all processed images will be generated in the same directory selected in Step 3.

13. Results File Generation. A text file named "Result.txt" will be generated in the same directory.
This file contains the following data for each image:

a. Label → Name of the analyzed image.

b. ROI Area → Area of the selected region of interest (ROI).

c. Bacteria Area → Area within the ROI containing bacteria.

d. % Area occupied by Bacteria → (Bacteria Area ÷ ROI Area) x 100.

e. Avg Intensity of Bacteria → Average fluorescence intensity of bacteria in the selected ROI.

## LIMITATIONS

### Selection of the proventriculus region

The size of the proventriculus varies, requiring manual selection of the region for analysis in the first step of the macro. The proventriculus extends from its interface with the esophagus to its distal end. However, defining this distal boundary is challenging. To ensure consistency and minimize subjective interpretation, we limit the selection to the region containing the spines, which are clearly identifiable and reduce experimental bias. This selection also includes any bacterial mass overflow extending into the esophagus.

### Qualitative analysis of bacterial load

Our protocol does not allow for the identification of individual bacteria. Instead, it provides a qualitative assessment of bacterial load. Additionally, when a proventriculus is colonized by only a very small number of bacteria, it may be classified as uncolonized, as the macro would return a value similar to that of a non-infected proventriculus.

### Tracking colonization in a single flea over time

Because fleas must be dissected for analysis, it is impossible to track colonization dynamics within a single flea. Instead, by repeatedly applying this approach to fleas from an infected population at different time points, we can reconstruct an overall view of colonization dynamics.

## TROUBLESHOOTING

### Problem 1

Our Nikon microscope system generates RGB images in TIF format, the only format supported by the macro.

### Potential solution

If the image is in a format other than TIF but still in RGB mode, you can modify the supported image extension on line 18 of the macro.

14 //Creation of a Results folder

15 File.makeDirectory("Mask");

16

17 numero=1;

18 extension=".TIF";
<figure><img src="image_9.png" /><figcaption>Figure 6. Color threshold panel</figcaption></figure>

**Problem 2**

The separation between bacterial fluorescence (green) and proventriculus autofluorescence (orange) is achieved through color threshold settings. These values were initially defined using a reference set of 20 images but may not be optimal for all image sets.

**Potential solution**

Generate a series of images using your own samples and microscope settings to determine the appropriate Hue values for your dataset: 1) open one or more images in ImageJ, 2) navigate to Image > Adjust > Color Threshold to adjust Hue values (Figure 6), and 3) update the macro by modifying the color threshold settings at lines 81-82.

65 selectWindow("Masque");
66 //Using the Color Threshold function
67 // Color Thresholder 2.0.0-rc-61/1.52n
68 // Autogenerated macro, single images only!
69 min=newArray(3);
70 max=newArray(3);
71 filter=newArray(3);
72 a=dessTitle();
73 run("HSB Stack");
74 run("Convert Stack to Images");
75 selectWindow("Hue");
76 rename("0");
77 selectWindow("Saturation");
78 rename("1");
79 selectWindow("Brightness");
80 rename("2");
81 min[0]=47;
82 max[0]=255;
83 filter[0]="pass";
84 min[1]=0;
85 max[1]=255;
86 filter[1]="pass";
87 min[2]=104;
88 max[2]=255;
89 filter[2]="pass";
90 for (i=0;i<3;i++) {
91 selectWindow(""+i);
92 setThreshold(min[i], max[i]);
93 run("Convert to Mask");
94 if (filter[i] == "stop") run("Invert");
95 }
96 imageCalculator("AND create", "0", "1");
97 imageCalculator("AND create", "Result of 0", "2");
98 for (i=0;i<3;i++) {
99 selectWindow(""+i);
100 close();
101}
102 selectWindow("Result of 0");
103 close();
104 selectWindow("Result of Result of 0");
105 rename(a);
106 // Colour Thresholding----------

RESOURCE AVAILABILITY

Lead contact

Further information and requests for resources should be directed to and will be fulfilled by the lead contact, Sébastien Bontemps-Gallo (sebastien.bontemps-gallo@cnrs.fr).
**Technical contact**

Questions about the technical specifics of performing the protocol should be directed to and will be answered by the technical contact, Amélie Dewitte (amelie.dewitte@inserm.fr).

**Materials availability**

This study did not produce any new reagents.

**Data and code availability**

The ImageJ macro is available in the GitHub repository at: https://github.com/sbontemps-gallo/Fleacolonization
https://doi.org/10.5281/zenodo.15323329.

**ACKNOWLEDGMENTS**

The study was funded by the Centre National de la Recherche Scientifique (CNRS), the Institut National de la Santé et de la Recherche Médicale (Inserm), the Institut Pasteur de Lille, the Université de Lille, the Agence National de la Recherche (ANR-15-CE39-0017 and ANR-21-CE15-0047), and the French government's Investissements d'Avenir program (managed by the Agence National de la Recherche, grant ANR-16-IDEX-0004 ULNE). It was also partially supported by the ERC Synergy project – Synergy-Plague (ERC-2023-SyG, GA no. 101118880). Figures were created using BioRender. The flea drawings in the graphical abstract and Figure 1 are from the NIAID Visual & Medical Arts collection (NIAID NIH BIOART, F#159).

**AUTHOR CONTRIBUTIONS**

Conceptualization, A.D., F.S., and S.B.-G.; investigation and data analysis, A.D., S.B.-G., and F.S.; ImageJ macro development, E.W.; writing – original draft, S.B.-G.; writing – review and editing, A.D., E.W., and F.S.; funding acquisition, S. B.-G., and F.S.

**DECLARATION OF INTERESTS**

The authors declare no competing interests.

**DECLARATION OF AI AND AI-ASSISTED TECHNOLOGIES IN THE WRITING PROCESS**

During the preparation of this work, the authors used ChatGPT 4.0 to improve language and readability. After using this service, the authors reviewed and edited the content as needed and take full responsibility for the publication's content.

**REFERENCES**

1. Dewitte, A., Werkmeister, E., Pierre, F., Sebbane, F., and Bontemps-Gallo, S. (2023). A Widefield Light Microscopy-Based Approach Provides Further Insights into the Colonization of the Flea Proventriculus by *Yersinia pestis*. Appl. Environ. Microbiol. 89, e0209122. https://doi.org/10.1128/aem.02091-22.

2. Hinnebusch, B.J., Perry, R.D., and Schwan, T.G. (1996). Role of the *Yersinia pestis* hemin storage (hms) locus in the transmission of plague by fleas. Science 273, 367–370. https://doi.org/10.1126/science.273.5273.367.

3. Schneider, C.A., Rasband, W.S., and Eliceiri, K. W. (2012). NIH Image to ImageJ: 25 years of image analysis. Nat. Methods 9, 671–675. https://doi.org/10.1038/nmeth.2089.