High-throughput screening of caterpillars as a platform to study host–microbe interactions and enteric immunity

Mammalian models of human disease are expensive and subject to ethical restrictions. Here, we present an independent platform for high-throughput screening, using larvae of the tobacco hornworm Manduca sexta, combining diagnostic imaging modalities for a comprehensive characterization of aberrant phenotypes. For validation, we use bacterial/chemical-induced gut inflammation to generate a colitis-like phenotype and identify significant alterations in morphology, tissue properties, and intermediary metabolism, which aggravate with disease progression and can be rescued by antimicrobial treatment. In independent experiments, activation of the highly conserved NADPH oxidase DUOX, a key mediator of gut inflammation, leads to similar, dose-dependent alterations, which can be attenuated by pharmacological interventions. Furthermore, the developed platform could differentiate pathogens from mutualistic gastrointestinal bacteria broadening the scope of applications also to microbiomics and host-pathogen interactions. Overall, larvae-based screening can complement mammals in preclinical studies to explore innate immunity and host-pathogen interactions, thus representing a substantial contribution to improve mammalian welfare.

We also isolated the midgut (x = 0.81 g), the head (x = 0.296 g), as well as fat body tissue and hemolymph (both weighed individually because uniform sampling was not possible), and compared the PUVmaxn values 39 min or 3 h after 18 F-FDG injection in animals 12 h after Bt infection. The removed tissue was washed in 0.9% NaCl.

Statistics
For statistical analysis we generally used PRISM v8, with only the general linear model calculated using Statistica v12. 5.192.7. To evaluate different parameters for CT and MRI diagnostics, all axial slices from a given larvae were measured, and the mean value per animal was calculated. PUVmaxn values were log or square root transformed to achieve normal distribution. Depending on the data distribution (evaluated using the Shapiro-Wilk test), we used one-way analysis of variance (ANOVA) and Tukey's multiple comparisons test or the Kruskal-Wallis test and Dunn's multiple comparisons test. When two treatments were compared, we applied a t-test or Mann-Whitney U-test (two-sided). A Pearson productmoment correlation between parameters was calculated to identify correlations between the CT, MRI, and PET findings. Images were inspected and screened for artifacts directly after image acquisition. Larvae were excluded if a large amount of CA spilled over after injection into the dorsal vessel due to animal movement during image acquisition (MRI control = 2, DSS 5% = 2; CT DSS 5% = 1, E. coli = 1). Additionally, three outliers were excluded using the ROUT (Q (1) 59 = 1%) method (MRI control = 1; PET DSS 5% = 2). To test whether small potential differences in larval size affect the epithelial thickness, we used general linear models with MR/CT gut wall thickness or PET PUVmaxn as the dependent variable, treatment as a category factor, and animal length as a continuous predictor. ROC curve analysis was carried out using PRISM, including sensitivity and specificity for the listed parameters. The corresponding threshold values were used to compare Bt-infected, DSS or E. coli-fed, and control animals using multiple Chi-square tests. The manual thickness measurements were validated via semi-automatic FWHM thickness measurements using OriginPro 2020b and Analyze 14.0. Bland Altman plots have been created using PRISM.

Small animal MRI (μMRI)
The insect larvae were fixed with tape on a standard animal bed and anesthetized with 1-2% isoflurane. Like rodents, the larvae tolerated isoflurane anesthesia very well, and even higher concentrations of up to 4% isoflurane seemed not to harm the animals. For image acquisition, we used a vertical 9.4 Tesla Bruker Wide Bore NMR spectrometer equipped with the actively shielded gradient system Micro 2.5 (1.5 T/m) and a 25-mm 1 H quadrature coil (Bruker, Billerica, MA). The dorsal vessel (heart) of the larvae was cannulated, and following the acquisition of baseline images the CA was injected directly into the heart with the same volume processing, a heart rate of ~25 bpm was preset, which facilitated accurate data reconstruction.

Optoacoustic tomography
For optoacoustic tomography, larvae were handled according to the Arthropod Containment Level 1 guidelines, and work was reviewed and approved by the Memorial Sloan Kettering Cancer Center Institutional Biosafety Committee. The feasibility of optoacoustic tomography in M. sexta was demonstrated by the oral application of black India ink (American Mastertech Scientific, Lodi, CA, #STIINPT PINT) and subsequent imaging of the gut. Black India ink was added to the water at a final concentration of 1% before adding to the food mixture. The larvae were incubated for 12 h with the dyed food and then anesthetized with isoflurane (5 min, 5% v/v in 100% O2) before imaging. Optoacoustic tomography was carried out at 800 nm using a small animal optoacoustic tomography device (MSOT inVision-256 TF, iThera Medical, Munich, Germany). Once anaesthetized, the (limp) larvae were placed into the holder in a supine position with a bed of ultrasound gel (Aquasonic Clear, Parker Laboratories, Fairfield, NJ, #03-08). Ultrasound gel was then added to cover the rest of the larvae. The holder was sealed, and care was taken to ensure no bubbles were present and the ultrasound gel surrounded the larvae. Transverse optoacoustic slices were acquired at 800 nm with a step size of 0.3 mm, a FOV of 25 mm, and a resolution of 75 µm. Slices were acquired covering the entire larvae and 10 laser pulses were averaged for each slice. Two to three larvae at the 3rd pupal stage could readily fit in the holder at a time. Once imaging was complete, larvae were removed from the holder and wiped to remove excess ultrasound gel. No adverse effects of the imaging, immobilization, or anesthesia were observed. The data were reconstructed offline using back projection methods in ViewMSOT (iThera Medical, Munich, Germany). Representative cross sections were chosen to determine optoacoustic intensity. Pixel values across lines covering the diameter of the larvae were recorded (Fiji, ImageJ v2.1.0). Data were then normalized to each corresponding trace, interpolated and smoothened to generate the graphs (Matlab, 2020a).

Survival
Survival kinetics were subject to Kaplan-Meier survival analysis with a log-rank (Mantel-Cox) test to detect differential survival. Each survival experiment was done in triplicate. The Kaplan-Meier plots show the sum of these experiments. The n refers to the number of animals used for the respective analysis.

Three-dimensional reconstruction
For 3D reconstruction, sagittal T1 weighted (n = 5), axial CT sequences (n = 2), axial μCT (ex vivo) sequences, and axial T1 weighted DICOM data were processed and segmented with 3D Slicer v4.8.1 r26813 or Horus v3.3.5. Back projection based optoacoustic reconstructions were exported as 3D tiff stacks from ViewMSOT. The exported stack was then imported to Fiji and visualized using the 3D Viewer plugin, with spacing set to that of the transverse slice steps (0.3 mm).

Confirmation of MR and CT resolution
In the first set of experiments, we quantified the CT and MR resolution using different glass capillaries (Hilgenberg, Malsfeld, Germany). We used capillaries with 4, 4.5, 7, 8 and 9 mm inner diameters, filled with 0.1 mmol Gd-BOPTA in 0.9% NaCl and capillaries with outer diameters of 4.1, 4.7, 10, and 11 mm filled with 50% iodixanol in 0.9% NaCl (Fig. S4). We also used a capillary system with two capillaries stacked one into another. The internal space between these capillaries was filled with 0.1 mmol Gd-BOPTA in 0.9% NaCl. The diameters of the glass capillaries were measured to appraise the empirical resolution of the CT and MR images. (Because the widths of these capillaries were not normally distributed, multiple Mann-Whitney U-tests were performed to estimate the resolution). The mean of the standard deviations of each glass capillary was calculated and then doubled to obtain the empirical CT and MRI resolution for cylindrical or tube-shaped objects.
In addition, we measured an empty glass capillary with a known wall thickness of 1 mm (Hilgenberg, Malsfeld, Germany) in CT with the same imaging settings used for caterpillar imaging. The wall thickness measurements were calculated as FWHM measurements (Fig. S5).
Furthermore, to assess the capability of currently available CT scanners for long cylindrical structures, we used a 3D printed micro PET hot rod phantom (Derenzo phantom, according to PTW specitications, Fig. S6). 3D printing was done using a Anycubic Photon Mono X 3D printer (Anycubic, Shenzhen, China) with ABS-like creamy white 3D printer resin (Phrozen Technology, Taiwan). The Derenzo phantom was created with six differently sized segments of cylindrical holes, ranging from 0.6 0.8, 1.0, 1.2, 1.5 to 2 mm (Fig. S6). After the printing procedure, the object was washed twice with isopropyl alcohol and then hardened under UV light according to the manufacturer's instructions.

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The phantom was initially imaged using our μCT Skyscan 1173 system (Bruker, Billerica, MA). Next, it was imaged with the Photon-counting CT: NAEOTOM Alpha (Siemens Healthineers, Erlangen, Germany), the Dual-source CT SOMATOM Force (Siemens Healthineers, Erlangen, Germany), the signal source CT SOMATOM X.ceed (Siemens Healthineers, Erlangen, Germany) and SOMATOM go.Top (Siemens Healthineers, Erlangen, Germany) (Fig.S6). The bore thickness was measured semiautomatically using the FWHM measurements. The double mean SD of the measurements was used as a proxy for spatial resolution of long cylindrical objects in the mentioned CT scanners.

Verification of Gibbs artifacts
To determine whether the hypointense and hyperintense rings and lines in the larval alimentary tract were Gibbs artifacts, we used water-filled phantoms and took T1 and T2 weighted images with matrices of different sizes. We used a capillary filled with water and two stacked capillaries with the interspace filled with water as phantoms (Fig. S12).

Optical density measurements of the hemolymph and gut lesions
L5d2 larvae were fed with Bt (n = 12) or the control diet (n = 12) and were cooled on ice 36 h later. The surface of the insects was sterilized with 70% ethanol, and half of the dorsal horn was removed. The hemolymph was collected individually in sterile cups, and 10 μl was incubated in standard nutrient broth I (Carl Roth, Karlruhe, Germany, #AE92.1) overnight before measuring the optical density at 560 nm (OD560). L5d6 larvae 24 h after Bt infection (Bt n = 3 and control n = 6) were dissected and the midguts were examined.

Cryosectioning and immunohistochemistry
The medial midgut of L5d2 larvae fed with Bt (n = 7) or the control diet (n = 7) was fixed for 1 h in 3.7% (w/v) paraformaldehyde (PFA) in MS-saline. The tissue was embedded for 30 min in OCT compound (Sakura Finetek, Torrance, CA, #4583) and then frozen using a frigocut cryotome (Cryo Leica CM1950, Leica Biosystems, Wetzlar, Germany). Sections were collected at −26 °C using microscope slides coated with 2% silane in acetone. The freshly-loaded microscope slides were dried for 10 min at room temperature for better tissue adherence. For immunofluorescence staining, the cryosections were incubated for 1 h in 3% (w/v) BSA and 5% (w/v) goat serum in Tris-buffered saline (TBS). The samples were then incubated with the primary monoclonal antibody (plasmatocytes-specific marker Ms#13) and the appropriate secondary antibody (Dylight 549, Abcam, Cambridge, United Kingdom) diluted 1:2500, as previously described. 8

Paraffin-embedded sections and histochemical staining
Larvae were anesthetized on ice and 1 ml of 10% buffered formalin was injected into the midgut. The dorsal horn was removed, and the animal was placed in a Falcon tube containing 10% buffered formalin for 40 min. Abdominal segments 2-4 were then removed and incubated in 10% formalin overnight. Each sample was then placed in a plastic cassette and dehydrated in an ascending ethanol series at 37 °C, followed by 100% isopropanol and three changes of

Scanning electron microscopy
Midguts from L5d2 larvae fed with Bt (n = 3) or the control diet (n = 5) were dissected in PBS and fixed in 2% PFA plus 0.5% glutaraldehyde in PBS for 3 h at room temperature. The fixative was diluted 1:10 and the samples were stored at 4 °C. Next, the midgut samples were rinsed in PBS and ultrapure water and then dehydrated in an ascending ethanol series on ice (30%, 50%, 70%, 80%, 90%, 96%, 99.8%, and 100%) for 10 min at each concentration. The samples were critical point dried (Balzers CPD 030, Balzers, Liechtenstein), sputter-coated with gold (Balzers SCD 004, Balzers, Liechtenstein), and then mounted on aluminum stubs using Ag paste (Electrodag 1415, Plano, Wetzlar, Germany, #16062). The samples were analyzed using a Zeiss EM9DSM982 scanning electron microscope (Zeiss, Oberkochen, Germany).  Table S1. Control reactions were carried out using primers specific for EF1α as a housekeeping gene with an annealing temperature of 75 °C for 30 s. See the source data file for a presentation of complete, uncropped gels.

Quantitative real-time RT-PCR analysis
Total RNA was prepared from different gut regions (foregut, anterior-, median-, posteriormidgut and ileum) of 6 control or 6 animals exposed to uracil (8 h) Table S1). The specificity of the PCR was confirmed by melting-curve analysis and mean normalized expression was determined according to 9

Polyclonal anti-DUOX antibody
The polyclonal rabbit anti-DUOX antibody was custom-created by Davids Biotechnologie (Fig. S26). One rabbit was injected with the DUOX peptide LLRDKHCRYGKAPGGHDAIR (amino acids 342-361, within the extracellular PHD). The affinity-purified antibody was used at a dilution of 1:100 (v/v) for Western blot and immunohistochemistry. Sodium dodecylsulfate polyacrylamide electrophoresis (SDS-PAGE) was carried out as previously described. 10,11 Briefly, we prepared polyacrylamide gels with a 5% stacking gel and and 0.1% Tween-20 for 10 min. The protein bands were visualized by adding bromochloroindoyl phosphate (BCIP) and nitroblue tetrazolium (NBT) as previously described. 12 The ALP reaction was stopped after 10 min and the DUOX signal was assessed as present or absent. Then, the membranes were hydrated in 100% methanol, washed with TBS+T, and rinsed with ultrapure water. Next, the membranes were stained with Ponceau S staining solution (Cell Signaling, Danvers, MA, #59803S) and a protein with a rel. molecular weight of 30 kDa was used as a loading control. Finally, the number of DUOX-positive gut samples of control and uracil-exposed animals were compared ( Fig. 7B+C and S22). The Coomassiestained gels were dehydrated on a vacuum gel dryer. See the source data file for a presentation of complete, uncropped gels.

Detection of HOCl with R19S
HOCl was imaged as previously described. 13 Briefly, larvae fasted 3 h before the treatment were fed on the control diet or with a 0.1 M uracil suspension with or without DPI (65 μM) and NAC (72 μM) for 1 h. The larvae were then exposed to the same diets supplemented with 42 μM R19S (FutureChem, Seoul, South Korea, #FC-8001-0010) for 2 h. After this treatment, the gut was dissected, washed in PBS, and fixed in 4% PFA in PBS for 10 min, before washing in PBS and mounting on glass slides. Images were collected with a Leica SP8 confocal laser scanning microscope (CLSM) with an excitation wavelength of 514 nm and an emission wavelength of 530-603 nm.

CFU count after uracil treatment
L5d6 larvae were raised on a normal diet (n = 15) or on a diet containing 0.1 M uracil (n = 17) for 20.5 h. They were then transferred to a sterile Petri dish in a laminar flow cabinet for fecal collection. One fresh feces pellet per animal was diluted 1:10,000 in standard nutrient broth I and transferred to agar plates based on the same medium (pH 7 and pH 9). The agar plates were incubated at 37 °C for 96 h at pH 7 or for 8 days at pH 9. CFUs were counted with OpenCFU-2.5 26 .

Identification of cultivable bacteria from fecal pellets
All 64 (pH 7 and pH 9) agar plates were scrutinized under a stereomicroscope, and cell morphology was evaluated under a phase-contrast microscope. Colonies were classified and isolated based on colony morphology, cell morphology, and differential growth on selective media (see below). Five different colony archetypes were identified and purified. DNA extraction and 16S ribosomal DNA (rDNA) amplification was performed as previously described 27 using the G7 primer mix or as previously described 28 with the listed primers. PCR products were sequenced (Microsynth SeqLab, Göttingen, Germany), and used as a BLAST search query (https://blast.ncbi.nlm.nih.gov/Blast.cgi). A match was accepted at ≥ 98% sequence identity (see Table S11). Rarefaction curves calculated with EstimateS v9 29 revealed a sufficient sampling of the low-diversity bacterial community in M. sexta fecal pellets (Fig.   9H).

Characterization of host-mutualist and host-pathogen interactions
To further characterize the fecal bacteria of M. sexta, several selective and differential media were inoculated with each isolate (see Table S5). To determine the pathogenicity of these bacteria, L5d5 larvae were fed on a diet spiked with 50 μl of each bacterial suspension (2 × 10 8 cells/ml in standard nutrient broth I, Carl Roth, Karlruhe, Germany, #AE92.1). Each diet group (different bacterial species and control) comprised 24 individual larvae. The diet was renewed daily, and survival was recorded. Antimicrobial susceptibility testing (AST) was performed to determine if the isolated bacterial species produce bactericidal toxins (e.g., bacteriocins). We with 100 μl of each overnight culture and poured the agar into sterile Petri dishes. We punched 4 mm holes into the cooled agar. Overnight cultures of the isolated bacteria were centrifuged twice at 5000 g for 20 min, and 4 μl of the supernatant was added to the holes. The agar plates were incubated for 48 h at 37 °C before we measured the zone of inhibition. The susceptibility of the bacteria against different antibiotics, hemolymph, and different concentrations of HOCl was evaluated using the same method. Linear regression (test for equal elevations) was used to determine differential sensitivity of Enterococcus casseliflavus/gallinarum #1 and Enterococcus sp. #4 against HOCl. HOCl was prepared as described previously 13 (NaClO, Carl Roth, Karlruhe, Germany, #9062.1). Both enterococci inhibited the pathogenic Microbacterium sp., and this was confirmed by spectrophotometry, by measuring the optical density at 415 nm (OD415). The supernatant of both enterococci was diluted (1:1, 1:10, 1:100, and 1:1000) with the overnight cultures in 96-well plates and incubated at room temperature on a platform shaker.
The OD415 was measured after 7 h. To assess whether the inhibitory effect of the enterococci was biologically relevant, we compared the survival of animals fed on (1) a diet inoculated with the pathogenic Microbacterium (2 × 10 8 cell/ml, n = 40) to animals fed with diet inoculated with this strain and supernatants from the two enterococci (1:1 with 2 × 10 8 cell/ml, each n = 24) or control animals (n = 41). To evaluate the potential pleiotropic effect of bacterial 72 maintenance, freshly hatched larvae were fed with penicillin (Sigma-Aldrich, St. Louis, MO, #PENNA, n = 47, control n = 38), which was bactericidal to our isolates at 500 μg/ml in the AST. Accordingly, 50 μl penicillin was spread on the regular diet, and the animals were weighed after 16 days of treatment. The clearance of gut bacteria by penicillin treatment was confirmed by measuring the OD415 of fecal pellets diluted 1:10,000 after 12 h, as discussed above.             References listed in Table S11:                      Table S8.  Table  S8.  Table S8.  Table S8.