Proteomic Insight Into the Ontogeny of Blood-Meal Digestion in the Tick Ixodes ricinus

Abstract

Ticks are important ectoparasites and vectors of a variety of pathogens in both animals and humans, and their increasing global distribution poses a growing health risk. Unlike other blood-feeding vectors, ticks feed for an extended period at each life stage and rely exclusively on blood for development and reproduction. Blood digestion in ticks is mediated by a complex multienzyme network within the endolysosomal system of the midgut (MG) epithelial cells. Previous studies have focused largely on protein digestion during the slow feeding phase. However, the processing of the blood meal after the mating-induced rapid engorgement ("big sip") remains unclear, although the rapid turnover of proteins from host blood proteins into yolk proteins in fully fed females is a crucial step for tick reproduction. In this study, we performed a label-free quantitative proteomic analysis of MG tissue extracts and MG contents of the hard tick Ixodes ricinus to characterize proteases and protease inhibitors expressed during selected timepoints of female feeding and off-host digestion. In addition, we analyzed the distribution of digestive enzymes by activity profiling in MG extracts and contents with specific diagnostic substrates. Our results show that the multienzyme network, mainly based on aspartic acid and cysteine cathepsins and complemented by specific types of serine proteases and metalloproteases, is involved in the intracellular and probably also in the luminal digestion of blood meal proteins in fully engorged female ticks. We also detected different types of protease inhibitors and proposed their regulatory role in controlling both endogenous (tick-derived) and host protease activities in the MG tissue and luminal contents storing ingested blood. These results provide comprehensive insights into the physiology of the tick MG and offer new opportunities for the development of future control strategies against ticks and tick-borne diseases.

Graphical Abstract

Highlights

• •Dynamic proteome profiling of adult tick guts during feeding and off-host stages.
• •Comparative analysis of key proteases involved in the blood digestion.
• •Various protease inhibitors regulate enzymes of both tick and host origin.
• •Major digestive cathepsins are secreted into the lumen upon detachment from the host.
• •Intracellular and extracellular digestion is mediated by various isoenzymes.

In Brief

Longitudinal proteome and activity profiling of the proteolytic system in the midgut (MG) of Ixodes ricinus females during feeding and off-host digestion phases shed light on the fascinating ability of ticks to digest extreme amounts of blood required for enormous egg production. The results suggest that part of the multienzyme digestive apparatus moves from the MG tissue into the lumen prior to egg laying. The new insights into the physiology of the tick MG could contribute to effective tick control.

Ticks and tick-borne diseases are a growing burden on human and animal health worldwide (1). In Europe, the most dangerous tick species is Ixodes ricinus, which serves as the vector for the pathogens causing Lyme borreliosis and tick-borne encephalitis. In contrast to other blood-feeding disease vectors (e.g., mosquitoes), all life stages of the tick feed exclusively on the host's blood, which is the ultimate source of energy and nutrients for the tick's development and reproduction. Hard ticks (Ixodidae) feed on their vertebrate hosts only once per life stage, and blood consumption is the prerequisite for the metamorphosis of the juvenile stages (larva to nymph; nymph to adult male or female). Feeding of the adult female I. ricinus on the host takes about 7 to 8 days and can be divided into two phases: (i) The slow feeding phase (about 6 days after attachment), during which the female ingests about one-third of the total blood meal and the tick's body size increases steadily; (ii) the mating-induced rapid blood uptake (“big-sip”) occurs 12 to 24 h before detachment from the host, during which the female imbibes an amount of blood equivalent to approximately two-thirds of the total blood meal (2, 3, 4). Most of the ingested blood proteins are digested in the preoviposition period after feeding (2–3 weeks) and used to synthesize yolk proteins (vitellogenins), which are transported to the tick's ovaries to produce eggs. After oviposition, the female ticks die (4). During feeding, the process of intestinal blood digestion in ticks, in contrast to blood-sucking insects, takes place within the endolysosomal system of the digestive cells of the tick's intestine (midgut [MG]). The highly distensible inner lumen of this large and branched organ serves primarily as a spacious food reservoir. The stored blood proteins are then slowly uptaken by the intestinal cells and processed intracellularly in the acidic environment of the endolysosomes of the tick intestinal digestive cells in a process known as heterophagy (3, 4). Digestion of the blood meal in the tick MG was systematically studied in partially engorged I. ricinus females that fed for 6 days just prior to entering the rapid engorgement phase. The multienzymatic proteolytic network includes the initial endopeptidolytic activity of the aspartic protease cathepsin D—IrCD (5, 6) and cysteine protease cathepsin L—IrCL (7), and asparaginyl endopeptidase (legumain)—IrAE (8). The large protein fragments are further cleaved by IrCL and endopeptidase activity of cysteine cathepsin B—IrCB, followed by the exopeptidase activities of IrCB and cysteine cathepsin C—IrCC, which cleave the short fragments into the dipeptides. The process is completed by the formation of free amino acids by the serine carboxypeptidase and leucine aminopeptidase activities (3, 9). This proteolytic system is most likely also involved in blood meal processing during the development of the nymphal stage of I. ricinus, as recently demonstrated by dynamic proteomic analyses of MGs dissected from unfed (UF) nymphs up to the premolt stage (10). However, for the fully fed (FF) adult females that fall off the host, several questions remain to be answered: (i) Is the same proteolytic system based on a network of aspartic and cysteine cathepsins involved in detached ticks during the preoviposition period? (ii) What is the contribution of serine proteases in the processing of the blood meal? (iii) Does the digestion of blood remain within the epithelial cells of the MG, or does it take place (at least partially) in the MG lumen? (iv) How is the proteolytic system in the tick MG regulated by protease inhibitors? To address these knowledge gaps, we conducted a label-free proteomic analysis of MGs across various feeding and postfeeding stages of I. ricinus adult females, focusing on proteolytic enzymes and protease inhibitors, and complemented this with enzyme-specific activity profiling.

Experimental Procedures

Ticks and Animals

Adult ticks of I. ricinus were collected by flagging in the area of České Budějovice (Czech Republic) and stored in the animal-rearing facility of the Institute of Parasitology. Ticks were maintained in vials with a relative humidity of about 95%, a temperature of 24 °C, and a day/night period set to 15/9 h. For all experiments described later, 25 adult females were fed naturally on laboratory guinea pigs with an equal number of adult males. All laboratory animals were treated in accordance with the Animal Protection Law of the Czech Republic no.: 246/1992 Sb., approved by the ethics committee of the Institute of Parasitology, Biology Centre CAS, the State Veterinary Administration, and the Central Commission, ethics approval no.: 98/2020.

Tissue Dissection

MGs from different feeding timepoints of adults (UF; 1, 3, 5 days of feeding—1D, 3D, 5D; FF; 4, 6, 11 days after detachment—4AD, 6AD, and 11AD) were dissected under a drop of ice-cold PBS, transferred to a sterile Petri dish filled with PBS, and washed extensively to remove the MG contents containing the host blood. The FF stage was defined as the point at which a tick naturally detaches from the host without external interference (i.e., not forcibly removed). Subsequently, MG tissues were transferred to a microtube and stored at −80 °C until further use. All investigated timepoints were collected in triplicates with different numbers of MGs per sample (described later).

For proteomic analysis and activity profiling of MG contents from FF, 4AD, 6AD, and 11AD ticks, whole MGs were dissected and transferred to the Petri dish. MG tissues were opened with fine scissors, washed with 600 μl of precooled PBS, and the diluted contents were transferred to the microtubes and stored at −80 °C until further use. The remaining washed MG tissues were stored at −80 °C for subsequent preparation of soluble protein extract and enzyme activity assays. This analysis was performed in quadruplicate, while one individual tick was used per one replicate.

Experimental Design and Statistical Rationale

All eight adult female stages (UF, feeding, and postfeeding) were dissected and collected in triplicates with different numbers of individuals. Specifically, 10, 10, 10, 5, 3, 4, 3, and 3 females were used for UF, 1D, 3D, 5D, FF, 4AD, 6AD, and 11AD, respectively. A multiplicate nanoLC–MS/MS run in data-dependent acquisition mode was performed for each sample. The multiplicated runs were then combined in MaxQuant as described later. Three technical replicates were performed for each of the three biological replicates of the eight different experimental conditions (for more details, see raw data on PRIDE). In total, 83 LC–MS/MS analyses were carried out.

For activity profiling, technical duplicates were performed for each of the four biological replicates of the four different experimental conditions (FF, 4AD, 6AD, and 11AD). One individual tick was used per one biological replicate.

Protein Extraction and In-Solution Digestion

Samples were processed as previously described (10). MG tissues were homogenized in 200 μl of 50 mM sodium phosphate buffer, pH 7.5, containing 7 M urea, 2 M thiourea, 2% CHAPS, and Halt protease inhibitors (Thermo Fisher Scientific; 78439). Samples were further sonicated (5 cycles, 15 s of sonication, 50% amplitude) using Ultrasonic processor UP100H (Hielscher Ultrasonics), and the cell debris was removed by centrifugation at 15,000g for 15 min at 4 °C. Proteins were subjected to acetone precipitation overnight at −20 °C, and the suspension was clarified by centrifugation at 15,000g for 15 min at 4 °C. Protein pellets were redissolved by shaking in 100 mM ammonium bicarbonate supplemented with 8 M urea for 30 min at room temperature. Samples were consequently diluted with 100 mM ammonium bicarbonate to a final concentration of 2 M urea and shaken at 37 °C for 30 min. The BCA Protein Assay Kit (Thermo Fisher Scientific; 23250) was used to determine protein concentration. Ten micrograms of protein were subjected to in-solution digestion. Proteins were reduced with 10 mM DTT at 56 °C for 45 min and alkylated with 55 mM iodoacetamide at room temperature in the dark for 20 min. Subsequently, the alkylation was quenched with 50 mM DTT. Proteins were digested using trypsin (Pierce Trypsin Protease, mass spectrometry [MS] grade; Thermo Fisher Scientific, 90057) as described (10). Peptides were purified using StageTips solid-phase C18 disc (Empore; 66883) according to the described protocol (11). In addition, pellets undissolved in 8 M urea (PU) were suspended in 50 μl of ammonium bicarbonate and subjected to in-solution digestion.

For proteomic analysis of MG contents, samples containing 50 μg of proteins were subjected to reduction, alkylation, and trypsin digestion and desalted as described previously. Data were analyzed separately and independently from the MG tissue extracts.

NanoLC electrospray ionization–MS/MS and Data Analysis

Peptides were dissolved in 30 μl of 3% acetonitrile/0.1% formic acid and subjected to MS analysis as previously described (10). Peptide separation was performed using an UltiMate 3000 RSLCnano System (Thermo Fisher Scientific) on-line coupled to a timsTOF Pro mass spectrometer (Bruker Daltonics). The peptide solution was injected onto an Acclaim PepMap 100 C18 trapping column (300 μm i.d., 5 mm length, particle size 5 μm, pore size 100 Å; Thermo Fisher Scientific) at a flow rate of 2.5 μl/min with 2% acetonitrile/0.1% formic acid for 2 min. Peptides were then eluted from the trapping column onto an Acclaim PepMap 100 C18 analytical column (75 μm i.d., 150 mm length, particle size 2 μm, pore size 100 Å; Thermo Fisher Scientific) and separated using a 48-min linear gradient of 5% to 35% acetonitrile/0.1% formic acid at a constant flow rate of 0.3 μl/min. The column oven was maintained at 35 °C throughout the process. Data acquisition was performed in parallel accumulation–serial fragmentation scan mode with positive polarity. Electrospray ionization was achieved using a CaptiveSpray (Bruker Daltonics) source with a capillary voltage of 1500 V, a dry gas flow rate of 3 l/min, and a dry temperature of 180 °C. The ion mobility range (1/K0) was set from 0.6 to 1.6 Vs/cm², and mass spectra were collected over an m/z range of 100 to 1700. Polygon filtering was applied to exclude low m/z values of singly charged ions. The target intensity for parallel accumulation–serial fragmentation was set at 20,000, ensuring repetitive precursor selection for MS/MS analysis. Collision energies ranged from 20 to 59 eV, adjusted in five equal steps across the 1/K0 ion mobility range of 0.6 to 1.6 Vs/cm². Protein analysis was carried out using MaxQuant software (version 1.6.14, Computational Systems Biochemistry) in the case of MG tissue extracts and version 2.4.0 in the case of MG contents) with the integrated Andromeda search engine (12). Proteins from the soluble fraction and digested proteins from the PU pellet were set as fractions 1 and 2, respectively. The I. ricinus database, available in UniProt (June 8, 2021; 64,414 entries), supplemented with sequences from our previously published I. ricinus MG transcriptomes (13, 14) (bioproject nos.: PRJNA217984, PRJNA311553, and PRJNA685402) with 16,002, 7215, and 17,185 entries, respectively, and the contaminant database included in the MaxQuant software to identify proteins, were used. In addition, the guinea pig Cavia porcellus protein database available in UniProt (July 28, 2021; 25,660 entries) was used for the identification of proteins of host origin. Trypsin/P was specified as the enzyme, allowing up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification, whereas N-terminal protein acetylation and methionine oxidation were specified as variable modifications. Precursor ion tolerance was set to 20 ppm for the first search and 10 ppm for the main search, whereas the mass tolerance for MS/MS fragment ions was set to 40 ppm. Peptide spectrum matches and protein identifications were filtered using a target-decoy approach with a false discovery rate of 1%. Data were further analyzed as previously described (10) using Perseus software (version 1.6.14.0, Computational Systems Biochemistry) (15). Filtering steps included the removal of hits to the reverse database, contaminants, and proteins identified only with modified peptides. Proteins identified by a single peptide with a score lower than 40 were excluded from further analysis. In addition, only proteins detected in at least two samples within a single biological replicate group were considered for downstream processing. Missing values were imputed for each column independently from a normal distribution, using a width parameter of 0.3 and a downshift of 1.8. Multiple-sample tests were performed using a permutation-based false discovery rate of 0.05 with 250 randomizations, followed by a post hoc Tukey’s honestly significant difference test, using algorithms integrated into Perseus.

Functional Annotation

For functional annotation of I. ricinus proteins identified via LC–MS/MS analysis, we initially employed BLASTp and rpsBLAST against various databases. These included a subset of the National Center for Biotechnology Information nonredundant database, transcriptome shotgun assembly, UNIPROTKB, Refseq-invertebrate, Refseq-vertebrate, Refseq-virus, EC, MEROPS, PFAM, CDD, and Smart. Subsequently, we utilized an in-house program that scans a vocabulary of approximately 450 words, and their order of appearance in proteins matches the BLASTp and rpsBLAST results, along with their percent identities and coverage, attributing each putative protein to a specific functional class. The annotated protein sequences were exported as a Windows-compatible hyperlinked Excel file that is available for download (see Data availability section).

Preparation of Tick Midgut Tissue Extracts, Midgut Contents, and Guinea Pig Serum for Activity Profiling

Soluble protein extracts of dissected MG tissue were prepared by homogenization in 50 mM sodium acetate buffer, pH 4.5, containing 1% (w/v) CHAPS on ice. The extracts were cleared by centrifugation (16,000g for 10 min at 4 °C), ultrafiltered using a 0.22 μm Ultrafree-MC device (Millipore), and stored at −80 °C. MG contents were cleared by centrifugation (16,000g for 10 min at 4 °C). Guinea pig blood was collected from the front leg vein, kept at room temperature for 1 h, and then left to fully clot at 4 °C overnight. Serum was obtained by centrifugation at 5000g for 15 min at 4 °C.

Protease Activity Assays

Proteolytic activities in MG tissue extracts and MG contents were measured in a continuous kinetic assay using peptidyl fluorogenic substrates (Bachem) or internally quenched FRET substrates (IOCB) as previously described (9, 16). The following substrates were used at 50 μM concentration: Abz-Lys-Pro-Ala-Glu-Phe-Nph-Ala-Leu (Abz, aminobenzoic acid; Nph, 4-nitrophenylalanine) for cathepsin D, Cbz-Phe-Arg-Amc (Cbz, benzyloxycarbonyl; Amc, aminomethylcoumarin) for cathepsins B/L, measured as one activity, and trypsin, Gly-Arg-amc for cathepsin C, Cbz-Ala-Ala-Asn-Amc for asparaginyl endopeptidase, Leu-Amc for leucyl aminopeptidase, and Suc-Ala-Ala-Phe-Amc (Suc, succinyl) for chymotrypsin. Measurements were performed at 35 °C in 96-well microplates in a total volume of 100 μl. MG tissue extracts, MG contents, or guinea pig serum (0.05–5 μl) were used. For comparison of activities in MG tissue extracts and MG contents, the volumes applied in the assay were normalized per tick. For the comparison between host serum and MG contents, the samples used in the assay were normalized to protein concentration. All samples were preincubated for 15 min in 80 μl of 100 mM sodium acetate, pH 4.0, containing 10 μM cysteine protease inhibitor E-64 (L-trans-3-carboxyoxiran-2-carbonyl-L-leucylagmatine) (for cathepsin D), 100 mM sodium acetate, pH 5.0, containing 10 μM E-64 and 2.5 mM DTT (for asparaginyl endopeptidase), 100 mM sodium acetate, pH 5.5, containing 2.5 mM DTT and 1 mM serine protease inhibitor AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride) (for cathepsins B/L, including 25 mM NaCl for cathepsin C), 100 mM Mes, pH 6.5 containing 10 μM E-64 and 1 mM AEBSF (for leucine aminopeptidase), 100 mM Tris–HCl, pH 8.0, containing 10 μM E-64 (for trypsin and chymotrypsin). After the addition of substrate (20 μl in the same buffer), substrate hydrolysis was measured continuously using an Infinite M200 or M1000 microplate reader (Tecan) at excitation and emission wavelengths of 360/465 nm for Amc substrates and 320/420 nm for FRET substrates. Cathepsin B/L and trypsin activities were compared by measurement with Cbz-Phe-Arg-Amc substrate in 100 mM sodium acetate, pH 5.5, containing 2.5 mM DTT (MG tissue extracts) or 100 mM Mes, pH 6.6, containing 2.5 mM DTT (MG contents) in the presence of 10 μM E-64 (trypsin activities) or 1 mM AEBSF (cathepsin B/L activities). Serine carboxypeptidase activity was assayed with 1 mM Cbz-Phe-Leu substrate in 50 mM sodium acetate, pH 5.0, containing 10 μM E-64. Aliquots were withdrawn at defined time intervals, and the product release was monitored after derivatization with 0.03% fluorescamine in 100 mM sodium borate, pH 9.0 at 365 nm excitation and 470 nm emission wavelengths (17). Samples taken immediately after the substrate addition were used as a blank for the reaction. The pH value of MG contents was determined as a mean of pH values measured by microelectrode of dissected MG contents diluted in preboiled distilled water.

Statistical Analysis

All statistics were performed with GraphPad Prism (version 6.00 for Windows; GraphPad Software, Inc).

Results

Determination of Sample Collection Timepoints

We first determined the timepoints for the collection of MG tissue samples during the feeding and off-host digestion periods of adult I. ricinus females (Fig. 1). We assumed that feeding on the host lasts between 6 and 8 days and can be divided into two phases (3, 4). Most critical for the females is the phase of rapid engorgement, which takes place during the last 12 to 24 h of feeding on the host. During this time, the mated females ingest up to 100 times their original body weight of host blood and store it in their MG lumen. After detachment from the host, the preoviposition period begins, during which the female ticks process a considerable amount of the host’s blood proteins to produce clutches of thousands of eggs. At this time, the body size of the female tick, including the MG tissue, decreases slightly because of the onset of egg laying (Fig. 1). The first eggs are laid 11 days postdetachment, and females die shortly after oviposition is completed. Based on this chronology, we collected MG samples during feeding at UF, 1D, 3D, 5D, and FF and during off-host digestion at 4AD, 6AD, and 11AD and used these for subsequent analyses.

Fig. 1.

Ixodes ricinus adult females and their midguts. Photos of adult I. ricinus females at all stages of examination. In addition, one female is shown at the end of oviposition (6 weeks after detachment). The photos were taken with the Leica Z16 APO macroscope. For microscopic visualization, the unwashed midgut of each timepoint was used. Semithin sections were stained with toluidine blue as described (10). 5AD females represent the 4AD and 6AD timepoints used for proteomic analysis as there are no visual differences in their appearance. 1D, 3D, 5D, fed for 1, 3, and 5 days, respectively; 5AD, 11AD, 5 and 11 days after detachment, respectively; FF, fully fed; UF, unfed.

Overall Midgut Proteomic Analysis

The comparative label-free quantitative (LFQ) proteomic analysis was performed on MG tissues from adult I. ricinus females. To minimize the presence of host proteins, MG tissues were opened, and MG contents were washed out (Fig. 2). A total of 4061 tick and host proteins were identified, and after filtering out proteins according to the parameters described previously, 1917 ticks and host proteins were subjected for further analysis (supplemental Table S1, MG tissue extract proteome sheet). The ratio of host proteins to total proteins ranged from 1 to 23% with the highest number of host proteins detected in the FF stage (Fig. 3A). A significant proportion of host proteins was also observed after detachment, indicating the uptake of host blood by MG digestive cells or remnants after incomplete washing. The highest number of identified proteins was detected in the 5D stage (1158 tick and host proteins), whereas the highest number of tick timepoint-specific proteins was observed in the UF stage (159 proteins) (Fig. 3, A and B). The UpSet plot (Fig. 3B) showed that 324 tick proteins were detected in all investigated stages and represent the backbone proteome. It was shown that 65 proteins are shared between UF and all feeding stages but are absent in FF and all timepoints following detachment, indicating that these proteins may be essential for tick feeding. Principal component analysis showed clear clustering of samples from the UF stage. Feeding stages clustered together and differed markedly from the post-rapid engorgement FF and off-host stages, which clustered close to each other (Fig. 3C).

Fig. 2.

Experimental workflow. Adult females were fed on guinea pigs, midgut (MG) tissues were dissected and washed to remove MG contents, and samples were subjected to proteomic analysis. Data were processed using MaxQuant and Perseus programs, and comparative and functional analyses were performed. In addition, activities of the main digestive proteases in MG tissue extracts and contents were profiled in activity assays.

Fig. 3.

Overall characterization of the Ixodes ricinus midgut proteome.A, number of tick proteins, feeding-stage specific, and host proteins identified in all investigated timepoints. B, the upset plot of tick proteins. Groups with less than 10 proteins were excluded. C, principal component analysis (PCA) of individual timepoints.

Functional Classification

For the functional annotation, we conducted a detailed annotation of the identified proteins, with a specific focus on tick proteins (1690) rather than host proteins. Our functional classification approach categorized I. ricinus proteins into 26 functional classes (see the Data availability section and column O in the hyperlinked Excel table).

It is noteworthy that our in-house approach (10) includes an “unknown” functional class. Proteins in this functional group cannot be properly classified within the other categories and mainly consist of proteins with sequences that bear similarities to proteins with unknown functions. In our dataset, the “unknown” functional class was consistently the most prevalent across all timepoints, with average LFQ values ranging from 14.6% to 17.3%, peaking in the UF stage (Fig. 4), followed by the “protein synthesis” class, with LFQ values ranging from 12.8% to 9.1%. Last, the third most abundant class varied as feeding progressed: in UF females and the initial feeding stage (1D and 3D ticks), it was the “Energetic metabolism—met/Energy” class; in FF ticks, it was the “cytoskeletal” class; after detachment 4AD and 6AD, it was the “secreted” class (8.8% ± 0.5%); that reverted to the “Met/Energy” class prior to oviposition (11 AD). Furthermore, analysis of the average LFQ values for the 26 functional classes at each feeding stage (Fig. 4) offers insights into the temporal distribution of major physiological processes within the MG of adult female I. ricinus ticks as feeding progresses. Notably, certain functional classes exhibited distinct patterns. For example, the “cytoskeletal” group, which comprises proteins such as actin, tubulin, tropomyosin, myosin, and dynactin, showed consistently similar LFQ values across all feeding stages, except for a notable spike in FF ticks.

Fig. 4.

Functional classification of Ixodes ricinus proteins identified by LC–MS/MS analysis. Relative quantification of the 25 functional classes observed in I. ricinus adult females at different feeding stages. The average LFQ (%) was used to estimate the overall abundance of each class. The error bars represent the standard error of the mean. 1D, 3D, 5D, 1 day, 3 days, and 5 days of feeding, respectively; 4AD, 6AD, and 11AD, 4 days, 5 days, and 11 days after detachment, respectively; FF, fully fed; LFQ, label-free quantitation; UF, unfed.

Proteases and Protease Inhibitors in the Midgut of I. ricinus Females

In this work, we focus on the functional classes “protease” and “protease inhibitor” present in the tick MG. Relative to all identified proteins, both groups showed overall low abundances during the UF and initial feeding stages, which increased significantly after the ticks were fully engorged (Fig. 4). However, while the “protease” group in the FF samples showed a surge increase followed by a decrease after detachment, LFQ levels for the “protease inhibitor” group increased steadily and peaked 6 days after detachment (6AD).

Within the “protease” group (Fig. 5A), we identified 43 proteins that were classified into five classes: aspartic, cysteine, metallo-, serine, or threonine proteases, which were further subdivided into protease families based on the MEROPS database classification (18) (Table 1). The cysteine protease class stood out as the most represented, comprising a total of 17 proteins with the highest sum of mean LFQ values (Fig. 5A), mainly from the C01 family, including cathepsins B, L, and C, along with additional representatives from the C12, C13, C19, C69, and C111 families. In addition, we found 12 and 10 sequences belonging to the metallo- and serine protease classes, respectively. Among metalloproteases, members from the M03, M17, M20, M24, and M49 families were identified. The majority of serine proteases determined from our dataset were chymotrypsin-like proteases belonging to the S01 family. The aspartic class was represented by three proteases. As the proteolytic subunits of the proteasome were classified under the proteasome rather than the protease functional class, the threonine protease class contains only a single protease.

Fig. 5.

Overview of proteomically identified protease classes and protease inhibitor families in the Ixodes ricinus midgut.A, the stacked bars represent the sum of mean LFQ values of identified proteases derived from Table 1. B, the sum of mean LFQ values of identified protease inhibitors derived from Table 2. LFQ, label-free quantitation.

Table 1.

Proteases

Protein annotation	Family	Protein id	Accession no. (major protein IDs)	National Center for Biotechnology Information accession no.	Razor + unique peptides	Unique + razor sequence coverage (%)	Mean transformed LFQ values (± SD)
							UF	1D	3D	5D	FF	4AD	6AD	11AD
Aspartic proteases
Cathepsin D1 (IrCD1)	A01	2545	A4GTA5	ABO26561	11	30.6		18.59 (0.19)	18.03 (0.42)	18.02 (0.63)	19.28 (0.22)	18.36 (0.46)	18.07 (0.27)	18.43 (0.75)
Cathepsin D2 (IrCD2)	A01	2570	E7E820	ADU03674	8	40.3								19.11 (0.58)
DNA-damage inducible protein 1 (Ddi1)	A28	1652	V5HYX6	JAP72895	9	38.1			17.38 (0.11)	17.54 (0.72)
Cysteine proteases
Cathepsin B1 isoform 1 (IrCB1.1)	C01	3783	V5I2X9	ABO26563	27	71.2	21.90 (0.04)	22.47 (0.33)	23.32 (0.26)	23.49 (0.05)	22.92 (0.12)	22.42 (0.18)	22.11 (0.24)	22.82 (0.33)
Cathepsin B1 isoform 2 (IrCB1.2)	C01	2546	A4GTA7	ABO26563	4	20.8	18.39 (1.13)	19.59 (0.45)	19.73 (0.68)	20.13 (0.42)	19.35 (0.74)	18.85 (0.14)	17.86 (0.07)	19.92 (0.67)
Cathepsin B1 isoform 3 (IrCB1.3)	C01	3605	V5HDT5	JAB75624	9	30.6	18.64 (0.11)	19.59 (0.54)	20.95 (0.91)	20.47 (0.97)	19.87 (0.69)	19.95 (0.19)	18.90 (0.60)	19.43 (1.03)
Cathepsin B1 isoform 4 (IrCB1.4)	C01	3132	Irseq_780802		8	22.9	19.66 (0.11)	20.45 (0.41)	21.00 (0.57)	21.67 (0.63)	20.74 (0.10)	20.65 (0.32)	20.30 (0.86)	21.34 (0.17)
Cathepsin B2 (IrCB2)	C01	3399	V5H0E3	JAB70284	5	17.2			18.53 (0.14)
Cathepsin B-like nonpeptidase homolog	C01	104	A0A090XD06	JAC94489	5	10.9	18.40 (0.39)	19.01 (0.49)	20.11 (0.63)	20.24 (0.31)	19.89 (0.34)	19.44 (0.83)	18.67 (0.72)	19.05 (0.65)
Cathepsin B/L-like nonpeptidase homolog (long propeptide)	C01	2040	A0A147BI21	JAR90436	22	51.4	22.18 (0.19)	21.75 (0.20)	20.20 (0.57)	20.21 (0.62)	18.40 (0.95)	18.58 (0.51)	18.53 (1.43)	20.66 (0.23)
Cathepsin B3 (longipain)	C01	3952	A0A0K8RDY9	MBK3725830	11	37.8	19.31 (0.05)	18.09 (0.01)	17.83 (0.35)				18.91 (0.93)	18.43 (0.42)
Cathepsin C (IrCC)	C01	2549	A8D0K2	ABV29335	18	45.1	22.91 (0.30)	22.77 (0.09)	23.13 (0.37)	22.94 (0.30)	21.75 (0.27)	21.35 (0.05)	22.15 (0.38)	22.88 (0.08)
Cathepsin L1 (IrCL1)	C01	541	A0A0K8REA0	ABO26562	28	60.3	24.88 (0.39)	25.66 (0.20)	25.68 (0.24)	24.51 (0.15)	23.25 (0.16)	22.79 (0.11)	23.65 (0.05)	24.04 (0.45)
Cathepsin L3 (IrCL3)	C01	2380	V5HGD6		7	45.3						17.90 (0.03)	18.13 (0.15)	19.86 (0.20)
Cathepsin long (Ir26/29)	C01	3410	V5GJA7	JAB70370	29	54.1	22.41 (0.21)	22.27 (0.21)	22.59 (0.21)	22.26 (0.33)	21.78 (0.30)	21.60 (0.32)	22.25 (0.17)	22.85 (0.39)
Ubiquitinyl hydrolase 1	C12	873	A0A131Y8Y7	JAP75317	10	59.9	19.57 (0.42)	18.39 (0.57)	17.84 (0.13)	18.66 (0.23)				18.01 (0.05)
Asparaginyl endopeptidase 1 (Legumain 1) (IrAE1)	C13	1549	A0A131Y148	AAS94231	18	55.5	19.08 (0.21)	18.71 (0.94)	20.48 (0.26)	20.98 (0.06)	20.00 (0.16)	19.35 (0.50)	18.79 (0.22)	20.07 (0.09)
Ubiquitinyl-specific protease 10	C19	3038	A0A131Y644	JAP73526	9	15.3				18.06 (0.24)	17.89 (1.16)	17.83 (0.35)		16.81 (0.49)
Dipeptidase A (Secernin)	C69	48	A0A131Y431		7	22.5	18.06 (0.32)
Protein-glutamine gamma-glutamyltransferase 2	C111	2132	A0A131Y2F0	JAP73419	19	43.3	18.00 (0.05)		17.77 (0.47)	18.13 (0.36)	18.20 (0.78)	17.90 (0.38)		19.12 (1.24)
Metalloproteases
Thimet oligopeptidase isoform 1	M03	365	A0A0K8R9F9	JAA67513	16	30.5	19.13 (0.17)	18.84 (0.56)	19.62 (0.24)	19.56 (0.90)	17.87 (0.35)	18.69 (0.31)	18.98 (0.76)	19.63 (0.65)
Thimet oligopeptidase isoform 2	M03	1155	A0A131XTW4	JAP69096	3	7.2								17.53 (0.83)
Mitochondrial processing peptidase β-subunit	M16	1040	A0A131XNF3	JAP68974	29	56.1	22.40 (0.53)	22.23 (0.15)	22.14 (0.12)	21.56 (0.25)	19.76 (0.37)	19.93 (0.36)	20.03 (0.53)	20.79 (0.33)
Leucine aminopeptidase (IrLAP)	M17	1044	A0A131XNV2	JAP69104	40	79.2	19.74 (0.30)	19.10 (0.29)	19.42 (0.47)	19.79 (1.48)	20.86 (0.29)	21.77 (0.30)	20.72 (0.17)	22.10 (0.53)
Leucine aminopeptidase 2 (IrLAP2, Fasciola type)	M17	2924	Irseq_1238865	MBK3727385	46	92.1	21.64 (0.11)	21.08 (0.39)	21.19 (0.16)	22.24 (0.92)	22.46 (0.25)	21.83 (0.34)	21.16 (0.23)	22.76 (0.52)
Carnosine dipeptidase II	M20	187	A0A0K8R3X9	JAA65860	36	81.1	20.47 (0.51)	19.17 (0.19)	20.01 (0.45)	20.25 (0.44)		18.55 (1.18)		21.11 (0.56)
Xaa-Arg Dipeptidase 1	M20	1212	A0A131XVD3	JAP70165	21	73.2	21.28 (0.51)	20.37 (0.30)	19.45 (0.35)	17.88 (0.54)				19.45 (0.47)
Methioninyl aminopeptidase 1	M24	504	A0A0K8RD90	JAA69107	2	7.9	18.39 (0.14)		18.10 (0.58)
Mitochondrial methioninyl aminopeptidase	M24	2436	V5H804	JAB68943	4	24.8	17.87 (0.04)
Methioninyl aminopeptidase 2	M24	440	A0A0K8RBC0	JAA68467	9	38.2	18.18 (0.12)	17.54 (0.54)
Xaa-Pro dipeptidase (Prolidase)	M24	3697	V5HJJ8	JAB77794	14	29.4	18.96 (0.34)		18.85 (0.27)	18.53 (0.92)				18.82 (0.81)
Dipeptidyl peptidase III	M49	1116	A0A131XSR9	JAP69255	19	37.6				18.54 (0.74)				17.79 (0.74)
Serine proteases
Chymotrypsin	S01	1921	A0A131Y9D4	JAP75487	5	20.7			17.91 (0.24)	18.03 (0.20)
Chymotrypsin-like nonpeptidase homolog (HD rich)	S01	1389	A0A131XYL3	JAP70806	3	13	21.08 (0.87)			21.70 (0.60)	21.87 (0.50)	21.20 (0.98)	19.65 (0.41)	20.56 (0.85)
Chymotrypsin-like nonpeptidase homolo (HD rich)	S01	3402	V5HAY7	JAB70298	31	69.6	23.57 (0.64)	20.75 (0.90)	21.39 (0.23)	22.22 (0.95)	23.39 (0.17)	22.95 (0.37)	22.46 (0.19)	22.72 (0.49)
Chymotrypsin-like nonpeptidase homolog (HD rich)	S01	3524	V5HFD4	JAB72178	11	43	21.48 (0.99)		18.06 (0.47)	19.05 (1.59)	20.28 (0.28)	19.35 (1.32)
Cubilin-related serine protease 2	S01	2467	A0A6B0VD03	JAB71130	13	28.9		19.88 (0.66)	21.35 (0.30)	20.10 (0.73)
Furin	S08	2533	A0A6B0VHQ1	JAB71353	25	30.5	18.65 (0.06)	19.64 (0.27)	20.03 (0.29)	18.85 (0.44)	18.55 (0.89)	19.10 (0.53)	19.27 (0.54)	19.35 (0.21)
Prolyl oligopeptidase	S09	417	A0A131XSG7	JAP69282	10	18.7	18.81 (0.62)	19.16 (0.21)	18.74 (0.29)	18.79 (0.22)
Serine carboxypeptidase	S10	2887	A0A131XXT5	MXU99612	4	17.3			18.49 (0.68)	18.63 (0.81)
Lysosomal Pro-Xaa carboxypeptidase (IrPRCP)	S28	3627	V5HKM6	JAR91867	8	36	18.48 (0.10)	18.36 (0.12)	19.03 (0.56)	19.01 (0.15)				18.52 (0.07)
Dipeptidyl peptidase II (IrDPPII)	S28	3814	V5IJN1	JAB83321	23	55.6	19.70 (0.26)	19.55 (1.10)	22.30 (0.09)	22.25 (0.19)	20.68 (0.22)	18.97 (0.70)	19.40 (0.38)	20.64 (1.00)
Threonine protease
Asparaginase	T02	3387	V5H9H0		14	49.2				17.65 (0.46)	17.79 (0.63)	18.77 (0.93)	20.27 (0.30)	20.14 (0.95)

Within the “protease inhibitor” group (Fig. 5B), 28 proteins were identified and categorized. The broad-spectrum pan-protease inhibitor α2-macroglobulin (specifically IrA₂M-2), detected exclusively in 11AD ticks, might be a contaminant from the tick hemolymph (19). The other identified inhibitors specifically target serine and cysteine proteases (Table 2). Among these, serine protease inhibitors were the most frequently identified. Specifically, we found 22 serine protease inhibitors distributed across various families. The predominant family consisted of inhibitors featuring the trypsin inhibitor–like cysteine-rich (TIL) domain (MEROPS database I08 family), comprising 11 proteins containing a TIL domain. In addition, we also identified nine Kunitz-type serine protease inhibitors (MEROPS I02 family) that contained 1, 2, 4, 5, or 10 tandem Kunitz domains and other inhibitors of serine proteases, namely one serpin and one Kazal-type inhibitor (Table 2). Proteomic analysis also revealed five cysteine protease inhibitors from the cystatin family (MEROPS I25 family), including one type-1 cystatin (stefin) and 4 from the type-2 subfamily (“true cystatins”) (Table 2).

Table 2.

Protease inhibitors

Family	Protein annotation	Prot. Id	Accession no. (major protein IDs)	National Center for Biotechnology Information accession no.	Razor + unique peptides	Unique + razor sequence coverage (%)	Mean LFQ (SD)
							UF	1D	3D	5D	FF	4AD	6AD	11AD
I25—Cystatin	Stefin	3347	A0A131Y5V6	JAP73872	15	99	20.65 (0.53)	20.18 (0.21)	20.53 (0.75)	20.36 (0.04)	19.09 (0.50)	20.06 (0.33)	20.89 (0.57)	22.36 (0.17)
	Mialostatin	2882	V5H924	JAB71122	10	76.4	17.78 (0.47)	17.77 (0.24)	17.62 (0.52)	18.47 (1.81)	18.31 (0.60)	18.51 (0.05)	18.24 (0.39)	17.73 (0.10)
	Iristatin	2379	A0A3B6UEB6	ARQ18757	6	52.5						18.62 (0.66)	19.07 (0.44)	18.76 (0.89)
	Cystatin 1	3884	A0A131YB86	JAP75211	7	69.9							18.09 (0.17)	18.66 (0.72)
	Cystatin 2	1868	A0A131Y7Z0	JAP74937	14	72.9	21.13 (0.41)					17.16 (0.40)	20.22 (1.10)	19.82 (1.28)
I04—Serpins	Serpin	3208	A0A131Y8E0	JAB70658	5	21.4								17.63 (0.89)
I08—TIL domain	TIL-domain inhibitor	875	A0A0K8RMJ2	JAA72280	2	27.1	17.57 (0.28)	17.92 (0.54)				17.88 (0.23)
	TIL-domain inhibitor	3375	V5H4F1	JAB69137	4	51.9	18.53 (0.22)						19.35 (0.65)	17.97 (1.18)
	TIL-domain inhibitor	924	A0A0K8RNV1		4	61.7	18.69 (0.54)	18.69 (0.01)			19.94 (0.97)	20.55 (0.77)	20.06 (0.25)
	TIL-domain inhibitor	1037	A0A131XMQ1	JAP68754	3	30				17.82 (0.93)	17.37 (0.36)	18.21 (0.57)	19.14 (0.69)
	TIL-domain inhibitor	1817	A0A131Y6K8	JAP74447	6	84.1					21.08 (0.61)	22.93 (1.37)	23.14 (0.53)	20.97 (1.09)
	TIL-domain inhibitor	1825	A0A131Y6Y7	JAP74597	11	80.9				18.52 (0.41)	20.05 (0.56)	19.82 (0.56)	19.93 (0.31)	19.96 (0.48)
	TIL-domain inhibitor	1962	V5IDG5	JAB72491	4	43.4							17.56 (0.33)
	TIL-domain inhibitor	215	A0A131Y8L3	JAP75157	3	37.6							18.36 (0.40)
	TIL-domain inhibitor	3530	V5IDG7	JAB72501	12	67.3					22.04 (0.89)	24.91 (0.49)	24.86 (0.40)	24.27 (0.70)
	TIL-domain inhibitor	3961	seqSigP-1033419		6	56.8					19.64 (0.27)	19.69 (0.14)	19.85 (0.60)	20.70 (0.42)
	TIL-domain inhibitor	3708	A0A131Y789	JAP74697	5	50.5				18.84 (1.31)				19.30 (1.48)
I02—Kunitz	Single Kunitz–domain inhibitor	4002	seqSigP-680411	MBK3723990	6	59.8	17.67 (0.34)					17.83 (0.43)
	Single Kunitz–domain inhibitor	519	V5H5D1	JAB78230	3	39.4	18.45 (0.01)	20.29 (0.74)	19.68 (1.62)	22.15 (0.68)	25.10 (1.22)	26.17 (0.97)	26.65 (0.52)	24.98 (0.86)
	Multi Kunitz–domain (2×) inhibitor	3970	seqSigP-1091517	MBK3720611	16	59.9	21.00 (0.33)	21.12 (0.25)	20.18 (0.69)	21.13 (1.61)	21.50 (1.40)	21.70 (0.61)	21.41 (0.55)	19.46 (1.08)
	Multi Kunitz–domain (2×) inhibitor	2416	A0A6B0V2R9		11	48.5						21.31 (0.33)	21.95 (0.36)	21.69 (0.28)
	Multi Kunitz–domain (4×) inhibitor	1770	A0A131Y5D8	JAP73590	20	75.9						22.44 (0.91)	23.54 (0.80)	21.74 (0.43)
	Multi Kunitz–domain (5×) inhibitor	3107	Irseq_751628		5	20.1	17.78 (0.38)	17.62 (0.45)	17.23 (0.17)
	Multi Kunitz–domain (5×) inhibitor	3850	V5I5H0	JAB84103	11	23.3				18.56 (0.34)				18.59 (0.12)
	Multi Kunitz–domain (10×) inhibitor	2128	A0A147BW46	JAR95007	46	33.3	22.05 (0.28)	21.12 (0.13)	18.28 (1.80)					17.61 (1.26)
	Single Kunitz–domain inhibitor (noninhibitory fragment)	3507	V5H472	JAB71794	3	40.8					16.87 (0.51)
I01—Kazal	Kazal serine protease inhibitor domain protein	3521	V5HAZ7	JAB72027	8	58.9			18.00 (0.49)	19.71 (0.37)
I39—α2M	α2-Macroglobulin 2	3427	V5HBQ6	QOJ54011	12	13.9								18.46 (0.23)

Longitudinal Analysis of Proteases Involved in Blood-Meal Digestion

Several digestive isoenzymes have been identified in accordance with the previously described multienzyme network consisting mainly of cysteine and aspartic proteases (3, 9, 16). First, cathepsin D1 (IrCD1, A4GTA5) was found by proteomic analysis to be almost equally present in MG tissues of all stages except UF and the last examined stage (11AD), in which it is replaced by the “late” cathepsin D, termed as cathepsin D2 (IrCD2, E7E820) (Table 1). From the cysteine proteases, the asparagine endopeptidase legumain (IrAE1, A0A131Y148) was detected in all examined stages with relatively stable LFQ. The papain-family cathepsin L1 (IrCL1, A0A0K8REA0) represented the most abundant protease over the monitored course of female feeding and off-host digestion (Table 1). Another distinct form of cathepsin L, termed IrCL3 (V5HGD6), was detected only in the MG tissues of females that dropped off the host (4AD, 6AD, and 11AD). In contrast, relatively high and stable LFQ values in all timepoints were detected for cathepsin L-like protease designated cathepsin long (Ir26/29, V5GJA7). Another papain family cysteine protease, cathepsin B, was detected in three forms from the I. ricinus MG tissue. The most abundant was the cathepsin B1 (IrCB1, V5I2X9) represented by four different allelic isoforms (supplemental Fig. S1), most likely encoded by the same gene (20) (Table 1). The presence of cathepsin B2 (IrCB2, V5H0E3), which is encoded by a different gene (20), was marginal and detected only in the 3D feeding stage. Similarly, cathepsin B3 (IrCB3, A0A0K8RDY9), the homolog of the Haemaphysalis longicornis protease named longipain (21), was detected in UF, 1D, 3D MG tissues and also after detachment 6AD and 11AD (Table 1). The last cysteine protease of the papain family, cathepsin C (IrCC, A8D0K2), also called dipeptidyl aminopeptidase I (3, 9), was abundant at all stages with relatively stable LFQ values (Table 1).

Two leucine aminopeptidases (IrLAP, A0A131XNV2 and IrLAP2, Irseq_1238865) from the M17 family of metalloproteases were detected in all investigated stages. In contrast, the serine carboxypeptidase (IrSCP, A0A131XXT5) from the S10 family of serine proteases was abundant only at 3D, 5D, and 11AD timepoints.

Serine proteases from the S01 family, which comprises the (chymo)trypsin-like endoproteases, are moderately represented in the I. ricinus MG tissue proteome. However, the only typical chymotrypsin-like enzyme (A0A131Y9D4) with the conserved catalytic triad H/D/S was detected with relatively low LFQ values and shown to be present solely in MG tissues from partially engorged (5D) females. Apparently, the major contributors to the LFQ-detected S01 family of serine proteases are the inactive chymotrypsin-like homologs possessing K/N/L residues at the position of the conserved H/D/S catalytic triad and containing histidine- and asparagine-rich central insertions (A0A131XYL3, V5HAY7, and V5HFD4) (supplemental Fig. S2). These inactive pseudoproteases are relatively highly abundant in the MG tissue homogenates across most analyzed timepoints. Another S01 family protease found at the early stages of female feeding (1D, 3D, and 5D) is an active serine protease tagged as cubilin-related serine protease (A0A6B0VD03), which has an extended N-terminal part containing a cubilin-like domain followed by a low-density lipoprotein receptor class A domain.

We also identified two lysosomal serine postproline-cleaving exopeptidases of the S28 family: the lysosomal Pro-Xaa carboxypeptidase (IrPRCP, V5HKM6), detected in UF, 1D, 3D, 5D, and 11AD stages, and the omnipresent dipeptidyl peptidase II (IrDPPII, V5IJN1).

Nondigestive Midgut-Associated Proteases

In addition to the lysosomal digestive proteases, proteomic analysis of tick MG tissue identified several biochemically uncharacterized proteases whose classification and functions can only be deduced on the basis of their homology to mammalian enzymes (Table 1).

Three enzymes involved in the ubiquitin system, in particular the ubiquitin-dependent aspartic protease annotated as DNA-damage inducible protein 1 (Ddi1, V5HYX6) and the deubiquitinating enzymes, ubiquitinyl hydrolase 1 (A0A131Y8Y7) and ubiquitinyl-specific protease 10 (A0A131Y644), were detected during the on-host feeding stage but also in the detached females.

Several metalloproteases, including oligopeptidases, dipeptidases, and monopeptidases, were detected. Some were present in most or all the recorded timepoints, whereas others were identified as either “early” or “late” enzymes based on their temporal expression patterns (Table 1). Early enzymes include methioninylaminopeptidase 1, 2 (A0A0K8RD90, A0A0K8RBC0) and Xaa-Arg dipeptidase 1 (A0A131XVD3). Late metallopeptidases are represented by thimet oligopeptidase isoform 2 (A0A131XTW4) and dipeptidyl peptidase III (A0A131XSR9), which are only expressed at the last timepoint examined (before oviposition). A member of the proprotein convertase family of serine proteases, furin (A0A6B0VHQ1), was detected at all collected stages.

Protease Inhibitors

The most represented family of protease inhibitors in the MG are the TIL domain inhibitors, which were detected in all analyzed stages, except the 3D MG tissue. Their number increased after detachment and reached a maximum at the 6AD stage (Table 2). The two closely related TIL domain inhibitors, V5IDG7 and A0A131Y6K8, were found to be the most abundant (Table 2). The multiple sequence alignment of TIL-domain inhibitors shows the presence of two major types with Lys or Ala at the P1 position (supplemental Fig. S3A), which plays a key role in defining inhibitor activity and specificity (22).

Kunitz-type inhibitors, which generally target the S01 serine protease family, were differentially expressed throughout the feeding and off-host digestion. Some are ubiquitously expressed at all timepoints examined (single and double domain inhibitors, V5H5D1, MBK372061), some in the early phase of feeding (Irseq_751628), and others only after detachment (A0A6B0V2R9 and A0A131Y5D8) (Table 2). The 30 identified Kunitz domains were aligned with the bovine pancreatic trypsin inhibitor, a prototype of Kunitz-type protease inhibitors (supplemental Fig. S3B). This alignment classifies the inhibitors into three major types based on the amino acid at the P1 position: typical trypsin inhibitors with basic amino acids, chymotrypsin/elastase inhibitors with nonpolar amino acids, and inhibitors with an aspartic acid targeting unknown proteases (22).

Regarding cysteine protease inhibitors, two molecules were identified as consistently present at all examined timepoints of feeding and off-host digestion: the intracellular stefin (A0A131Y5V6) and mialostatin (V5H924) (23). Other MG cysteine protease inhibitors iristatin (A0A3B6UEB6) (24) and two yet uncharacterized cystatins 1 and 2 (A0A131YB86 and A0A131Y7Z0) are exclusively expressed during off-host digestion, indicating specific role in protease regulation in the fully engorged gut of I. ricinus females.

Proteases and Protease Inhibitors in the Midgut Lumen

To answer the fundamental question of whether protein digestion in the tick remains intracellular after detachment from the host or takes place in the MG lumen, we performed an additional proteomic analysis of MG contents at four intervals after rapid engorgement: FF, 4AD, 6AD, and 11AD. Proteomic analysis of luminal contents is complicated by the high abundance of host blood proteins, for example, Hb or SA, whose intensity exceeds that of tick-specific proteins by three to four orders of magnitude (Supplemental Table S1, MG contents proteome sheet). Despite this challenge, we identified a total of 106 I. ricinus-specific proteins in addition to 87 proteins from the guinea pig (Supplemental Table S1, MG contents proteome sheet). Of the digestive cathepsins, only the papain-type cysteine proteases, IrCL1, Ir26/29, IrCB1, and IrCC (A0A0K8REA0, V5GJA7, V5HDT5, and A8D0K2, respectively) were detected in the MG contents at all timepoints after detachment, except in FF ticks (Table 3). Among the serine proteases present in the MG contents, the three inactive HD-rich chymotrypsin-like homologs (A0A131XYL3, V5HAY7, and V5HFD4) and also the cubilin-related serine protease 2 (A0A6B0VD03) dominated in most timepoints examined (Table 3). An active trypsin-like serine protease (V5H4M9) was only detected in 11AD, which was not found in MG extracts (Table 3).

Table 3.

Proteases and protease inhibitors identified in I. ricinus Midgut contents

Protein annotation	Protein ID	Accession no. (major protein IDs)	National Center for Biotechnology Information accession no.	Razor + unique peptides	Unique + razor sequence coverage (%)	Mean transformed LFQ values (±SD)				Tissue
						FF	4AD	6AD	11AD
Proteases
Cathepsin L1 (IrCL1)	59	A0A0K8REA0	ABO26562	9	39.4			18.08 (0.20)	17.71 (0.87)	Yes
Cathepsin long (Ir26/29)	371	V5GJA7	JAB70370	4	19			16.61 (0.05)	16.71 (0.18)	Yes
Cathepsin B1 isoform 3 (IrCB1.3)	400	V5HDT5	JAB75624	7	37.4		17.88 (0.39)	18.07 (0.73)	17.71 (0.80)	Yes
Cathepsin C (IrCC)	266	A8D0K2	ABV29335	6	21.7		16.59 (0.39)	16.62 (0.85)	16.71 (0.34)	Yes
Chymotrypsin-like nonpeptidase homolog (HD rich)	127	A0A131XYL3	JAP70806	3	13		18.15 (0.27)	18.60 (0.43)	19.37 (0.78)	Yes
Chymotrypsin-like nonpeptidase homolo (HD rich)	396	V5HAY7	JAB70298	19	61.1	17.97 (0.73)	20.29 (1.12)	19.12 (0.54)	20.75 (0.28)	Yes
Chymotrypsin-like nonpeptidase homolog (HD rich)	402	V5HFD4	JAB72178	5	25.6		19.56 (1.23)	17.60 (1.53)	19.20 (0.58)	Yes
Cubilin-related serine protease 2	257	A0A6B0VD03	JAB71130	3	9.5	16.36 (0.13)	17.01 (0.22)	17.17 (0.71)		Yes
Trypsin-like serine protease	388	V5H4M9		3	17.5				16.66 (0.85)	No
Protease inhibitors
Stefin	150	A0A131Y5V6	JAP73872	2	33.3				14.99 (0.12)	Yes
Iristatin	252	A0A3B6UEB6	ARQ18757	5	43.3		18.02 (0.45)	18.26 (0.53)	17.22 (0.54)	Yes
Cystatin 1	360	A0A131YB86	JAP75211	6	69.9		18.77 (2.07)	19.78 (0.66)	19.26 (0.63)	Yes
Cystatin 2	165	A0A131Y7Z0	JAP74937	9	57.1		18.29 (0.95)	17.89 (1.67)		Yes
TIL-domain inhibitor	97	A0A0K8RNV1		4	53.3	18.36 (0.49)	19.07 (0.47)	20.15 (0.55)	16.97 (0.52)	Yes
TIL-domain inhibitor	108	A0A131XMQ1	JAP68754	6	43.3	17.80 (1.61)	19.65 (0.90)	18.52 (0.68)	16.94 (2.00)	Yes
TIL-domain inhibitor	156	A0A131Y6Y7		9	57.4	16.28 (1.34)	19.48 (0.74)	17.39 (1.65)	14.50 (1.62)	Yes
TIL-domain inhibitor	389	A0A131Y789	JAP74697	5	49.5		18.77 (1.67)	18.68 (1.25)	19.45 (0.39)	Yes
TIL-domain inhibitor	414	V5IDG7	JAB72501	8	66.4		19.16 (1.16)	21.17 (1.04)	20.88 (0.40)	Yes
TIL-domain inhibitor	29	A0A131Y8L3	JAP75157	4	48.4		16.02 (0.50)	17.11 (2.76)	16.76 (0.29)	Yes
TIL-domain inhibitor	154	A0A131Y6K8	JAP74447	4	75		19.52 (0.82)	21.17 (0.96)	19.04 (1.96)	Yes
TIL-domain inhibitor	427	W8JJG5		1	15.3		18.00 (0.71)	17.47 (0.96)		No
Single Kunitz–domain inhibitor	54	V5H5D1	JAB78230	3	39.4	19.91 (0.39)	20.25 (0.12)	22.30 (0.92)	22.53 (0.72)	Yes
Multi Kunitz–domain (4×) inhibitor	147	A0A131Y5D8	JAP73590	3	27.1			16.39 (1.06)		Yes

Inhibitors of cysteine proteases stefin (A0A131Y5V6) and cystatins 1 and 2 (A0A131YB86 and A0A131Y7Z0) were identified in the MG lumen along with their target enzymes (cysteine cathepsins) in the later stages of off-host digestion (Table 3).

Besides cysteine protease inhibitors, the MG lumen contained multiple serine protease inhibitors of the TIL-domain family. These inhibitors were also identified in MG tissue extracts, with the exception of the TIL-domain protein (W8JJG5) (Table 2). The TIL-domain inhibitors were secreted into the female lumen, mainly in 4AD, 6D, and 11AD stages (Table 3). The most abundant serine protease inhibitor detected in MG contents of all postfeeding stages was the single Kunitz-domain inhibitor (V5H5D1), whereas the presence of the multi (4×) Kunitz domain (A0A131Y5D8) was only marginal at a single timepoint (6AD) (Table 3).

Profiling of Proteolytic Activities in Tick Midgut after Feeding

To investigate the enzyme activities of proteases detected by proteomic analysis in and outside the gut digestive cells—specifically in MG tissue extracts and MG contents—samples of ticks from four timepoints after feeding, namely FF and 4AD, 6AD and 11 AD, were analyzed. Kinetic assays with diagnostic peptidyl substrates, as previously used to study the activities in the MG of partially engorged I. ricinus females (9, 16), were used for comparison.

The proteolytic enzymes examined included a variety of protease classes: aspartic protease (cathepsin D), cysteine proteases (asparaginyl endopeptidase/legumain and cathepsins B/L and C), serine proteases (trypsin, chymotrypsin, and serine carboxypeptidase), and metalloprotease (leucine aminopeptidase). The overlapping activities of cathepsins B and L were assayed together. Figure 6 shows that protease activities in the tissue extract peaked in FF ticks and then gradually decreased with time after detachment. By 11 days after detachment (11AD), enzyme activities had dropped to levels between 1.5 and 9 times lower than their peak in FF. This trend was observed for all measured enzymes, except for chymotrypsin, whose activity was not detectable in the MG extracts. A reverse trend was observed in MG contents for the activities of cathepsins B/L, C, and D and serine carboxypeptidase, which were lowest in FF ticks and increased after detachment. Specifically, cathepsin C reached its highest activity at 6AD and cathepsins B/L, D, and serine carboxypeptidase peaked at 11AD. The peak activities of these enzymes were three to eight times higher than in FF ticks, indicating increased secretion of these enzymes into the MG lumen upon tick detachment from the host. Conversely, the activities of asparaginyl endopeptidase, trypsin, and chymotrypsin in MG contents showed pronounced dynamics, peaking at 4AD, whereas their activities outside this timepoint were not notably pronounced. Leucine aminopeptidase levels remained constantly high in the MG contents at all stages. Overall, the activities of the individual enzymes were at similar levels in both the MG tissue extracts and the MG contents. However, there are exceptions—chymotrypsin activity was not detected at all in the MG tissue extract, whereas the activities of serine carboxypeptidase and leucine aminopeptidase were up to 10-fold higher in the MG contents than in the tissue extract. To assess whether the observed activity in the MG contents originated from the host's ingested blood or was secreted by the MG tissue, the host's serum was collected from the guinea pig and analyzed for the activity of the selected proteases. These activities were then compared with those present in the MG contents of FF ticks. The comparative analysis showed that the activities of cathepsins B/L, D, and asparaginyl endopeptidase in the host serum were minimal in proportion to the substantial activities in the MG contents of FF ticks. This finding indicates that these enzymes are predominantly secreted from the MG tissue into the lumen of the MG and do not originate from the ingested blood of the host. The activities of chymotrypsin, serine carboxypeptidase, and cathepsin C in the host serum reached about 10%, 16%, and 30%, respectively, of the levels detected in the tick MG contents (Supplemental Fig. S4). This suggests that these activities may be derived from both the tick tissue and host blood. In contrast, leucine aminopeptidase and trypsin-like proteases showed higher activities in the host serum, whereas only small amounts were detected in the MG tissue.

Fig. 6.

Activity profiling of cysteine, aspartic, serine, and metalloproteases in the gut tissue extracts and the gut contents of FF, 4AD, 6AD, and 11AD ticks. Activities were measured with indicated substrate and are expressed in relative fluorescence units per minute. Means ± SEM are given. Measured in triplicates in four independent midgut tissue extracts and midgut contents. For details on activity assays, see the Experimental procedures section. 4AD, 6AD, 11AD, 4, 6, and 11 days after detachment, respectively; FF, fully fed.

Discussion

Our initial approach to studying tick proteases in the MG focused on a specific developmental stage defined as partially engorged I. ricinus females (prior to rapid engorgement). The choice of a defined feeding timepoint was driven primarily by the scarcity and inconsistency of information on digestive enzymes across different tick species at the time. Using biochemical assays and molecular cloning, we elucidated the multienzyme network of cysteine and aspartic proteases and developed a model for hemoglobin and albumin digestion in tick digestive cells at this feeding stage (3, 9). Recently, our understanding of tissue-specific processes has been greatly enhanced by the rapidly improving methods of MG tissue processing in late and off-host stages of female ticks. This is coupled with the availability of large-scale systems biology studies that allow us to longitudinally examine the proteolytic system of the MG of nonfed, FF, and off-host digesting female ticks (25, 26). Previous work has used our model tick I. ricinus to study the genes expressed in the salivary glands (13, 27, 28, 29) and MG (14, 29), as well as during different developmental stages (30, 31). While these studies reveal changes in gene expression, modern proteomics using shotgun MS provides deeper insights into the actual protein profiles. Therefore, LFQ-based proteomics, as recently used to study protein expression dynamics in the nymphal stage of I. ricinus (10), was used in this work to identify and quantify proteases and protease inhibitors in the adult MG of the same species.

Our data show that key components of the gut-associated proteolytic network of partially engorged females (3, 9) remain steadily abundant throughout the full-time course of female feeding and off-host digestion. The longitudinal LFQ-based profiles of digestive proteases and their inhibitors throughout ontogeny from UF I. ricinus nymphs (10) to herein profiled adult females are shown in the supplemental Fig. S5. However, further insights into the isoenzymes of each protease class are needed, as their different expression patterns indicate potentially distinct roles.

Within the proteolytic network in the digestive tract, the proteases cathepsin B, cathepsin L, and cathepsin D stand out. Cathepsin D forms in the tick gut are represented by the previously characterized isoenzyme IrCD1 and its late-expressed isoenzyme IrCD2 (6). While IrCD1 is expressed during feeding and shortly after detachment of the female from the host, IrCD2 appears to be upregulated in fertilized females at the later stage before oviposition. Its role could therefore be related to late digestion, or it could be secreted into the intestinal lumen, where it could fulfill different roles—for example, in the formation of antimicrobial peptides from the host's hemoglobin, as previously described in the cattle tick Rhipicephalus microplus (32).

Cathepsin B, which is represented by its isoenzyme IrCB1 in several allelic variants, is surprisingly consistently present in the MG proteome at all observed timepoints of female feeding and off-host digestion. Its isoenzyme IrCB2, on the other hand, is only detectable on the third day of female feeding, suggesting a very specific role—most likely related to the necessary remodeling and preparation of tissue prior to rapid engorgement. IrCB3, which is clearly distinct from the other IrCB isoforms (supplemental Fig. S1), is the direct I. ricinus analog of H. longicornis longipain, which has been reported to promote digestion of host blood while reducing the survival of Babesia parasites (21). However, in I. ricinus, this isoform has only been detected in UF and 3-day-fed (3D) female ticks, as well as in UF nymphs (10). Therefore, its role in digestion is questionable. In addition to endopeptidase activity, cathepsin B (IrCB) exhibits exopeptidase activity as a peptidyl dipeptidase, cleaving C-terminal dipeptides from predigested protein fragments (9). Conversely, cathepsin C (IrCC) exhibits dipeptidyl aminopeptidase activity, sequentially removing dipeptides from the N terminus of processed proteins. IrCC exists as a single, consistently present isoenzyme (3, 9).

Cathepsin L is represented by two gut-associated isoenzymes: IrCL1 and IrCL3. IrCL1, which was first identified as multitissue cathepsin L (7, 33), shows stable expression in the gut over time, with a slight decrease in FF females. In contrast, IrCL3 appears only during the off-host digestion phase. This suggests distinct functions for the two isoenzymes. IrCL3 colocalizes with mialostatin in large vesicles of intestinal cells on day 11 postdetachment (23), suggesting that mialostatin primarily targets IrCL3. Mialostatin is also stored in these vesicles during tick feeding. The formation of an inhibitory complex between mialostatin and IrCL3 could play a crucial role in the intracellular trafficking of inactive IrCL3. Since mialostatin was not detected in the gut contents (Table 3), the inhibitory complex is likely terminated before IrCL3 is secreted into the MG lumen, where active IrCL3 may contribute to anticoagulation and the generation of antimicrobial hemocidins, similarly to its homolog BmCL1 in R. microplus (32, 34). The cathepsin L-like protease Ir26/29, containing an elongated propeptide, has not yet been functionally and biochemically characterized, so its potential role in blood meal processing remains to be elucidated.

Another aspect we wanted to address in this study is an insight into the hitherto unclear contribution of serine proteases to the intestinal processing of blood in ticks. Previous work on the related tick species Ixodes scapularis has proposed a model of intestinal digestion in which intracellular digestion occurs during the slow feeding phase prior to rapid engorgement and later shifts to extracellular digestion by serine proteases in the intestinal lumen (35). This would resemble the situation in the MG of blood-sucking insects, which preferentially use luminal serine proteases to digest host blood proteins extracellularly (36) with the major exception of triatomines (37). However, our results do not support this hypothesis. Apart from a scarcely present chymotrypsin-like enzyme, most of the highly represented serine proteases of the S01 family—which comprises the (chymo)trypsin-like endoproteases—are inactive chymotrypsin-like homologs. These inactive pseudoproteases with as yet unknown function are relatively abundant in the MG tissue extracts at most of the timepoints studied in this work as well as in the MG of nymphs (10) (supplemental Fig. S5B). Moreover, the total trypsin-like activities of serine proteases measured in the MG tissue extracts or MG content samples using specific substrates were several orders of magnitude lower than the activity of cysteine cathepsins B and L (supplemental Fig. S6). This suggests that the S01 family serine proteases do not contribute significantly to blood digestion and may play a different role in the physiology of the tick MG. An enzyme that appears to play a role in the proteolytic digestive machinery of female ticks is the S10 family serine carboxypeptidase, which releases C-terminal amino acids from peptides at the boundary between lysosomes and the cytoplasm of intestinal digestive cells. In agreement with previous reports (3, 9, 16), this enzyme is detected in partially engorged ticks and, interestingly, also at the latest measured point of off-host digestion, prior to oviposition. The members of the serine carboxypeptidase S10 family were the only serine proteases reported to be involved in the luminal digestion of replete I. scapularis (35). The second example is the two potentially digestive post–proline-cleaving exopeptidases, which differ in their expression patterns and mechanism of action: IrPRCP functions as a carboxypeptidase that hydrolyzes peptide bonds at the C-terminal end of proline residues, whereas the ubiquitous IrDPPII cleaves dipeptides from the N-terminal end of polypeptides.

The cubilin-related serine protease is probably not directly involved in the digestion of blood proteins, but previous reports from other tick species indicated the role of this enzyme in red blood cell hemolysis in the early stages of female feeding (38, 39). Among the nondigestive serine proteases, the ubiquitous subtilisin-like proprotein convertase furin is of particular interest. As the major processing enzyme of the trans-Golgi network, which plays a central role in the processing and activation of a wide range of precursor proteins involved in physiological processes, such as defensins active in MG immunity (40). It is noteworthy that, in contrast to cysteine and aspartic proteases, serine proteases have also been detected in the host blood serum from the guinea pigs (supplemental Fig. S4). Trypsin-like proteases showed even higher activity in this host serum, whereas only low levels were detected in the contents of the MG of female ticks. This suggests that host-derived trypsin-like proteases may contribute to the measured activity, which is also supported by proteomic detection (Supplementary Table S1).

Metalloproteases identified in tick MG extract include oligopeptidases and dipeptidases, which were present at various stages of feeding and off-host digestion. Since these enzymes were not detected in the MG contents, we hypothesize that they are mainly cytoplasmic enzymes involved in the final processing of oligopeptides and dipeptides released from the late endolysosomes of digestive gut cells. Resulting free amino acids are utilized for de novo protein synthesis, which is important for the massive egg production by hard tick females. The most important finding seems to be the identification of the two M17 family leucine aminopeptidases, IrLAP and IrLAP2, which were detected at all timepoints measured. Leucine aminopeptidase removes N-terminal amino acids from proteins and peptides, thereby contributing to the formation of a pool of free amino acids. Parasite M17-LAPs play a crucial role in natural host nutrition, migration, and invasion, making them potential therapeutic targets in various parasitic diseases, such as malaria, schistosomiasis, fasciolosis, or leishmaniasis (41, 42, 43, 44, 45, 46). An M17 LAP enzyme was previously identified from the hard tick H. longicornis (HlLAP) and partially characterized as a recombinantly expressed enzyme with a neutral or slightly basic pH optimum (47), which was also confirmed by the measured pH optimum of LAP in the MG extract of I. ricinus (9). It was later shown that this enzyme is expressed in both the MG and ovaries and plays an important role in oocytogenesis (48). Therefore, the potential of tick M17-LAPs for the investigation of novel strategies for tick control should be further investigated.

Proteolytic activities in tick tissues are regulated by protease inhibitors of proteinaceous character (proteinase inhibitors [PIs]). Ticks express a wide variety of specific PIs from different families targeting individual protease classes. The PIs present in tick saliva have been intensively studied, as they play a role in regulating hemostasis and modulating host immune responses and thus have enormous pharmaceutical potential (review in Ref. (49)). As previous transcriptome analyses of the tick (14, 25) and another recent omics study (26) show, the gut tissue of ticks apparently also expresses a variety of PIs that probably exert different physiological functions than those at the tick–host interface.

Our dynamic LFQ data show that certain cysteine protease inhibitors (stefin, mialostatin) and some Kunitz inhibitors of serine proteases are present in intestinal tissue during all measured timepoints, whereas other PIs of cysteine and serine proteases—such as iristatin, cystatins 1 and 2, some TIL domain inhibitors, and additional Kunitz inhibitors—tend to be upregulated during the rapid engorgement phase, with their expression increasing toward the end of the off-host digestion. Furthermore, two multidomain Kunitz inhibitors are expressed only in UF and 1D females. This suggests fundamental differences in the functional role and targets of these inhibitors. The parallel presence of digestive proteases and their potential inhibitors in MG tissue extracts and MG contents raises the question of the nature of the resulting enzymatic activity: Is it primarily intracellular or secreted? Based on the data presented in Table 1, Table 2, Table 3, we hypothesize that MG iristatin, cystatins 1 and 2, apparently serve as regulators of the undesired proteolytic activity of cysteine cathepsins secreted simultaneously in the MG lumen toward the end of off-host digestion. On the other hand, the constantly expressed PIs, such as mialostatin, which were not detected in the MG contents, probably regulate their target protease(s) intracellularly throughout the feeding and off-host digestion periods (Table 3).

In addition, we hypothesize that the multiple TIL-domain serine protease inhibitors and a single Kunitz domain inhibitor secreted into the MG lumen during the off-host digestive phase (Table 3) help females overcome two critical challenges to accomplish their mission—to lay thousands of eggs before dying (4): (i) the prevention of blood clotting, which depends on the cascade of host serine proteases (50) and (ii) the preservation of ingested blood from microbial decay. Indirect evidence for the role of serine protease inhibitors secreted into the MG contents to inhibit trypsin-like serine proteases of host origin is given in supplemental Fig. S4, showing that host serum but not MG tissue is the major source of trypsin-like proteases. The potential antibacterial role of TIL domain inhibitors in the tick MG is derived from two characterized cysteine-rich proteins, named Ixodidin (51) and BmSI-7 (52), which were isolated from hemocytes and eggs of R. microplus, respectively. Ixodidin is a specific inhibitor of chymotrypsin and elastase and inhibits the growth of the Gram-positive bacterium Micrococcus luteus at micromolar concentrations, whereas BmSI-7 inhibits the activity of subtilisin A and Pr1 proteases of the entomopathogenic fungi (Metarhizium anisopliae). Thus, inhibition of extracellular proteases secreted by ingested microbes by TIL domain inhibitors present in MGs of fully engorged females may represent another branch of MG immunity in addition to the abovementioned antimicrobial activity of hemoglobin fragments.

To uncover a possible shift from intracellular to luminal digestion in fully engorged and detached females, we matched our LFQ proteome data obtained from MG tissue extracts and MG contents with activity measurements of proteases involved in the multienzymatic digestive network (3, 9). The activity measurements in both compartments confirmed that proteolytic activities were mainly dependent on cathepsins B, L, C, and D and not on serine proteases (Fig. 6). The general trend of enzymatic activities was a slow decrease in the MG tissue extract, whereas the activities in the lumen increased toward the oviposition. These results should be interpreted with caution, as the intestinal wall is very inconsistent in engorged females, and although the sample preparation was carefully performed, the release of cell contents into the lumen cannot be completely avoided. A detailed analysis of the proteins identified in the MG lumen also revealed the presence of tick housekeeping, intracellular proteins, such as elongation factor 1-alpha (A0A7D5BM75) and ferritin 1 (A0A0K8RA47). This observation suggests that disruption of the fragile MG tissue may have occurred during sample preparation, leading to the unintended release of intracellular contents into the lumen. The contribution of individual enzyme isoforms and the opposing action of present protease inhibitors have to be taken into account as well. Furthermore, host blood contains enzymes like trypsin, cathepsin C, and leucine aminopeptidases (supplemental Fig. S4), which hinder the analysis of endogenous enzymatic activities in the MG lumen. Although a quantitative comparison of protein abundance ratios between lumen and tissue could potentially offer additional insights, the complexity introduced by highly abundant host proteins and variability in sample composition made such an analysis challenging within the current scope. Therefore, the question of whether digestion of the blood meal occurs, at least partially, in the MG lumen of fully engorged females cannot be answered conclusively or unambiguously and will require further experimental investigation.

In summary, dynamic LFQ proteomic profiling of the female tick gut during feeding and off-host digestion has provided new insights into the mechanisms and regulation of tick MG-associated proteolysis. The model proposed for partially engorged females is now updated with an overall view of protease dynamics, the intriguing role of protease isoenzymes, and regulation of proteolysis in gut tissue and luminal contents by specific protease inhibitors. As indicated in the text, the knowledge gained after experimental validation will further enhance our ability to exploit the proteolytic interactions in the tick gut to control ticks and tick-borne diseases—either by specific inhibition or by using individual components as recombinant vaccines.

Data Availability

The Windows-compatible hyperlinked Excel file and the associated files can be downloaded as a single .zip file from: https://proj-bip-prod-publicread.s3.amazonaws.com/transcriptome/Iricinus/AdultDynamicsProteome/IrAdultProteomeDynamics.zip.

The MS proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository (53) with the dataset identifier PXD057920. All MS/MS spectra have been generated in MS-Viewer (54) and can be accessed using the unique key vlenx8huer and kmppu1kv4a.

Supplemental Data

This article contains supplemental data (10, 22).

Conflict of Interest

The authors declare no competing interests.