Assessment of MALDI-TOF MS for arthropod identification based on exuviaes

doi:10.21203/rs.3.rs-4840478/v1

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Assessment of MALDI-TOF MS for arthropod identification based on exuviaes

https://doi.org/10.21203/rs.3.rs-4840478/v1

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Background.

MALDI-TOF MS is an innovative tool for identifying both hematophagous and non-hematophagous arthropods at various life stages. However, identification by MALDI-TOF MS requires currently, euthanizing of the specimen, hindering further phenotypic tests. All arthropods have a common factor which is the molting of their exoskeletons, called exuviae. This phenomenon is indispensable for their growth and metamorphosis, and can evidence past arthropod presence.

Objective.

This study assessed the performance of MALDI-TOF MS biotyping for arthropod identification using exuviae from nine distinct laboratory-reared species (Aedes aegypti, Anopheles coluzzii, Cimex lectularius, C. hemipterus, Pediculus humanus corporis, Triatoma infestans, Rhodnius prolixus, Supella longipalpa and Blatta germanica) and compared it efficiency with molecular biology approach.

Results.

Molecular analysis showed low DNA quantity in exuviae (n = 108) across species, resulting low success of COI, 16s, and 18s amplification (50.0%), depending on the species and sequencing (10.2%). The establishment of exuviae protocol for MS submission, yielded MS spectra of high reproducibility and specificity per species. After upgrading home made reference MS database with exuviae spectra, query with remaining spectra revealed that 100% of samples were correctly identified, with 85.8% (278/324) exceeding the threshold score value for reliable identification.

Conclusion.

MALDI-TOF MS shown it high efficiency to identify various arthropod species based on their exuviae. This approach is a groundbreaking development in the field of entomology underlining that MALDI-TOF outperformed traditional methods of exuviae identification, including morphological and molecular tools. It allows also to prevent specimen sacrifice which could be used for complementary analyses.

Exuviae

arthropods

species identification

MALDI-TOF MS

biotyping

Arthropods are the most diverse and populous phylum in the animal kingdom (1) ; (2) and they represent about 79% of the animal species (3). They encompass numerous classes, like the insects, which have the largest number of orders, the arachnids including ticks and scorpions, or the crustaceans comprising shrimps, crabs, lobster, as well as myriapods (4). Insects stand out as the most dominant group, accounting for over 80% of arthropod species, followed by mites, such as ticks and spiders (5).

Some of the arthropods are hematophagous, and a fraction of them act as vectors for the transmission of pathogenic agents responsible of human and animal diseases (6). Mosquitoes are the primary vectors of infectious diseases, transmitting arboviral (eg, dengue, chikungunya or zika viruses) (7), parasitic (eg, plasmodia or filarioid helminths) (8) and potentially bacterial (eg, Rickettsia felis) (9) pathogens. Ticks are considered as the second order of pathogenic agent vectors (eg, Borrelia burgdorferi, the agent of Lyme disease) (10). Lice and triatomine bugs are others examples of human disease vectors transmitting bacterial agents (eg, Borrelia recurrentis) and Chagas diseases (eg, Trypanosoma cruzi), respectively (11) ; (12). Some other arthropods require also attention because they are considered as pests like bed bugs or cockroaches (13) ; (14).

The identification of arthropods to distinguish vectors from non-vectors, remains the first step in the process of monitor and control of vector-borne diseases (VBDs). Currently, the specimen identification is frequently done by morphological tool (15) ; (16) ; (17). However, this method is time-consuming, depends on experienced entomologists and the availability of dichotomous keys (18). As specimen identification is based on morphological criteria, damages on these delicate arthropods could hamper their taxonomic classification (19). The decline of entomological expertize is another additional limiting factor (20). To overcome limitations of morphological identification, the molecular biology was largely developed for arthropod identification. This approach is widely recognized as an accurate and reliable identification method (21) ; (22). Moreover, molecular biology method is independent of the specimen developmental stage (23). However, the time require to obtain the results and the cost of the reagents which remains relatively high, continue to be a drawback of molecular identification (24) ; (25).

Since the early 2010’s, a rapid, accurate and inexpensive innovative proteomic approach was successfully applied for arthropod identification (26) ; (27). The principle of this proteomic approach was based on the submission to matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOF MS) a protein extract from arthropod samples. The resulting spectra are compared to a reference MS spectra DB for specimens identification at the species level (18). MALDI-TOF MS was demonstrated effective for the identification of a wide range of arthropod families, such as mosquitoes (28), ticks (29), lice (30), fleas (31), sandflies (32) ; (33), bedbugs (34) or triatomines (35), using specific body parts according to the arthropod family and its development stage (36) ; (37). It is interesting to note that, conversely to genome, the protein repertory from an arthropod change according to body part (37) ; (38). Moreover, as arthropods are metamorphic organisms (39)(40), the protein repertory could also change throughout it developmental cycle (41). In this way, the establishment of standardized protocols was compulsory for an efficient arthropod identification at distinct developmental stage by MALDI-TOF MS (42).

Until now, few arthropod families were submitted to MALDI-TOF MS analyses for their identification at different developmental stages (eg, mosquitoes (43), ticks (44) or phlebotomine sandflies (33). Mosquito is the unique family which was tested at pre-immature (ie, eggs) (45) ; (46), at immature (ie, aquatic stages) (47) and at imago (ie, flying adult) (28) developmental stages. Unfortunately, for identification of the arthropod by MALDI-TOF MS, the euthanasia of the specimen is necessary. In such condition complementary analysis on alive specimen was not possible, such as assessed their susceptibility to insecticide, their vector competence or their responses to environmental changes (48).

One alternative to sacrifice is to identify specimens based on their exuviae (48). Effectively, arthropods are characterized by a segmented body covered by rigid cuticle forming the external skeleton (49), secreted by epidermal cells. The skeleton is primarily composed of two layers, the epi-cuticle, which includes a layer of cement and a waxy layer, and the pro-cuticle, consisting of the exo-cuticle and endo-cuticle containing chitin embedded in a proteinaceous matrix (50). During their life cycle, the arthropods molt correspond to the shedding of their exoskeleton (4). This metamorphose is incomplete for heterometabolous species involving resemblance between immature and adult stages, corresponding to size variations (51). Whereas for holometabolous species, the metamorphose is complete involving morphological dissimilarity between immature and mature stages, like for mosquitoes. Their growth is not continuous and only occurs during molting, when a renewal of the rigid cuticle takes place, allowing the arthropod to increase in size and/or to transform. The discarded exoskeleton from the previous stage is referred as exuviae (52). More recently, the proof of concept was established to apply MALDI-TOF MS for the identification of two Aedes mosquito species using exuviates (48). Moreover, in the field, alive arthropods can be highly mobile (eg, flying insects) and can escape of sampling by going into hiding (eg, bugs), whereas exuviates which cannot evade to collection, represent an interesting alternative.

Then, the aims of the present study was to assess the feasibility to identify several arthropod species from distinct families based on their exuviae using MALDI-TOF MS biotyping, but also by conventional molecular biology tool. To establish this proof-of-concept, exuviae from 9 distinct arthropod species laboratory-reared were tested, including mosquitoes (n = 2), bedbugs (n = 2), lice (n = 1), triatomaes (n = 2) and cockroaches (n = 2). For these experiments, exuviae from pupal and 3th instar or upper stages were used for mosquitoes and the others arthropods, respectively. Whole exuviae were submitted to MS analysis, excepted for species (ie, triatomaes and cockroaches) generated exuviae of large size for which one body part was selected. Moreover, the assessment of intra-species MS spectra variations according to the compartment submitted was also tested.

2.1. Ethical considerations

No permits were required for the described study, because all arthropod specimens were laboratory reared (not collected in the field). Moreover, none of the species included in the present work were protected or considered as sensitive animals. Ethical approval for in vitro blood feeding of mosquitoes, triatomine bugs and bedbugs using human blood was obtained from the Laboratory Research Ethics Board for Molecular Hematology, of the French Blood Establishment (EFS). New Zealand white rabbits raised at IHU Méditerranée Infection and sourced from Charles River Laboratories were used to feed the lice. They were handled according to Decree No. 2013 − 118 of February 7, 2013, and in accordance with official experimental procedures (references APAFIS #42524-2023040414041603 v5). The methods were approved by the Ethics Committee "C2EA-14" of Aix-Marseille University, France, as well as by the French Ministry of National Education, Higher Education, and Research.

2.2. Arthropod rearing and exuviae collection

Nine arthropod species, laboratory reared, coming from eight genus and five families were selected. The exuviae collection was done immediately following arthropod molting. The modalities of management and storing were indicated for each arthropod family. For each collection of exuviae, at least two counterspecies fresh specimens were collected, frozenly sedated and were used as controls of molecular and MALDI-TOF MS analyses.

2.2.1. Mosquitoes. Two mosquito species, Anopheles coluzzi and Aedes aegypti (Bora strain) were reared in the laboratory, as previously described (48). The exuviae from pupal stage were collected daily and rinsed once with alcohol and twice with water prior to transfer them individually in an Eppendorf tube. The remaining water was removed and the exuviae were either immediately processed for molecular or MALDI-TOF MS analyses or frozen at − 20°C until future analysis.
2.2.2. Lices. Adult Pediculus humanus corporis lice were reared in a climatic chamber (temperature: 25°C; relative humidity: 80–90%) and successive generations were obtained by feeding lice on rabbits, as previously described (30). Exuviae from the third larval stages were collected twice a week. The exuviae were either immediately processed for molecular or MALDI-TOF MS analyses or frozen at − 20°C until future analysis.
2.2.3. Triatomine bugs and bedbugs. Two triatomine species, Triatoma infestant and Rhodnius prolixus, and two bedbug species, Cimex hemipterus and Cimex lectularius, were laboratory reared in jars and boxes in incubators. The Triatomine species required a constant temperature of 26°C, with a relative humidity of 70% and a day cycle of 12h light/12h dark (53). The Cimex species were maintained at a temperature of 26°C and constant humidity of 60% with a day cycle of 12h light/12h dark. Details of rearing for Triatomine and Cimex species are available in previous works (53) ; (54). Exuviae were recovered twice a week by simple sorting. The exuviae sizes recovered from the triatomines and bed bugs measured, respectively at least 7 and 3 millimeters in length, corresponding to the 4th or 5th developmental stages.
2.2.4. Cockroaches. Two cockroach species, Blatella germanica and Supella longipalpa, were raised in climatic chambers at 24°C and fed once a week with fish food (Fit + Fun Teichsticks, Krefeld, Germany) and jelly water (Delical Gelodiet, Torcé, France). The exuviae were collected after metamorphosis through a simple sorting process twice a week, and were immediately analyzed by molecular biology or MALDI-TOF or stored at − 20°C until future analysis. The exuviae recovered from the cockroaches measured at least 10 mm in length corresponding to 3th instar or above larval stage.

2.3. DNA extraction and quantitative analysis

Five specimens (n = 45) and twelve whole exuviae (n = 108) per arthropod species (n = 9) were used for DNA extraction using a NucleoSpin® 96 Tissue genomic DNA kit following the manufacturer’s instructions (MACHEREY-NAGEL, Düren, Germany). The compartment and stage selected for DNA extraction per species are indicated in the Table 1. Extracted DNA was quantified with Qubit® 2.0 Fluorometer (Life Technologies, ThermoFisher Scientific, USA) using the Qubit dsDNA Assay Kits (Thermo Fisher Scientific). Samples with too low DNA concentrations to be detected by the fluorometer (values below 0.50 ng/mL) were recorded as zero values for data analysis.

Table 1

Comparison of molecular analysis effectiveness on exuviae samples per arthropod species.
	Fresh specimens (n = 5)		Exuviae (n = 12)
Species	Stage	Body part^a	Stage	Body part^a	Number of PCR success (%)^b	Number of sequencing success (%)^b	Number of target sequence success (%)^b	Accession number (NCBi)
P. humanus corporis	Adults	Whole	3th instars	Whole	0 (-)	/	/	/
Ae. aegypti	Pupae	Whole	Pupae	Whole	0 (-)	/	/	/
An. coluzzii	Pupae	Whole	Pupae	Whole	5 (41.6%)	3 (25.0%)	3 (25%)	MT375223.1
B. germanica	Adults	One leg	≥ 3th instars	Whole	7 (58.3%)	3 (25.0%)	3 (25%)	MT467295.1
S. longipalpa	Adults	One leg	≥ 3th instars	Whole	2 (16.6%)	1 (8.3%)	1 (8.3%)	KU543635.1
Cx. lectularius	Adults	Half^c	4-5th instars	Whole	12 (100%)	3 (25.0%)	1 (8.3%)	MN088697.1
Cx. hemipterus	Adults	Half^c	4-5th instars	Whole	12 (100%)	2 (16.7%)	0 (-)	/
T. infestans	Adults	One leg	4-5th instars	Whole	9 (75.0%)	4 (33.3%)	3 (25.0%)	NC_035547.1
Rh. prolixus	Adults	One leg	4-5th instars	Whole	7 (58.3%)	0 (-)	/	/
Total, n (%)^d	45		108		54 (50.0%)	16 (14.8%)	11 (10,2%)
^aBody part used for DNA extraction. ^bAmong the 12 exuviae per species. ^cThe specimens were cut longitudinally, half was used for DNA extraction and the remaining part was stored at -20°C. ^dThe proportion among the total number tested, n = 108.

2.4. Molecular analyses of exuviae

PCR amplification was carried out using primer pairs of the mitochondrial cytochrome oxidase subunit I (COI), the ribosomal 16S or 18S subunit sequences according to the arthropod family targeted (see Table S1 for details). PCR amplification was performed in 45 µL Master Mix composed of 25 µL of Amplitaq gold TM 360 Master Mix, 1 µL per primer and 18 µL of sterile water. Five microliters of extracted DNA was added to this Master Mix. DNA amplification was conducted by standard PCR conditions (Table S1) using an Applied Biosystems™ Veriti™ Thermal Cycler, 96-Well (Applied Biosystems, 2720, Foster City, CA, USA). The DNA extracted from five fresh specimens per species were used as positive controls of molecular experiments (Table 1). For negative controls, DNA was replaced by sterile water (PCR-grade water). PCR products were verified by electrophoresis using 1.5% agarose gel stained with SYBER Safe™ DNA gel dye (Thermo Fisher Scientific), and visualized with the CheliDoc™ MP ultraviolet imager (Bio-Rad, Marnes-la-Coquette, France). The presence of a band at expected size for each exuviae sample per species was used to determine proportion of PCR success. The positive samples from PCR underwent purification and sequencing according to the previously procedures (55). Sequencing was performed with a Sanger Sequencing 3500 Series Genetic Analyzer (Applied Biosystems®). The quality of the obtained sequences was assessed using Geneious Prime 2024.0.5 (Dotmatics, Boston, USA). A sequencing was considered as success if a high-quality sequence was obtained according to geneious score (> 80%) (Dotmatics, Boston, USA). All obtained sequences were assembled and corrected on Geneious Prime 2024.0.5 (Technelysium Pty Ltd., Tewantin, Australia) and then were blasted against GenBank using the Basic Local Alignment Search Tool (BLAST) (www.ncbi.nlm.nih.gov/blast/Blast.cgi). Blast sequence query was considered as target sequence success if correct species identification was obtained with proportions of identity and coverage, both upper than 90%.

2.4. Optimization of sample preparation conditions for MALDI-TOF MS analysis

To ensure the reproducibility of MS spectra within a species and specificity between species, several parameters were tested such as the exuviae compartment (whole or a specific part), the volume of homogenization buffer added and the duration of sample homogenization. The selection of the exuviae compartment was based on the size of the exuvia for each species (see Table S2). Each exuviae sample was individually placed in 1,5 mL Eppendorf tubes and mixed with homogenization buffer composed of a 50/50 (v/v) mix of formic acid (70%v/v) (Sigma) plus acetonitrile (50% v/v) (Fluka, Buchs, Switzerland). An adjustment of homogenization buffer volume was established for each kind of sample. The exuviae were homogenized using Tissue Lyser (Qiagen, Germany) with a pinch of glass beads (Sigma, Lyon, France) as disruptor. The parameters for sample homogenization were a frequency of 30 Hertz per cycle of 180 s. The number of cycle were also adjusted for each kind of sample. After homogenization, a quick spin (30 s at 200g) was performed and 1 µL of supernatant was deposited in quadruplicate spots onto the MALDI-TOF MS target plate (Bruker). After drying, each spot was overlaid with 1 µL of CHCA matrix solution, composed of saturated α-cyano-4-hydroxycinnamic acid, 50% acetonitrile (v/v), 2.5% trifluoroacetic acid (v/v) and HPLC-grade water (21). The cephalothorax and legs of a fresh Ae. aegypti specimen were homogenized under the same conditions and used as quality control of sample preparation and MS spectra acquisition. Matrix solution was loaded in duplicate onto each MALDI-TOF target plate as negative controls.

2.5. MALDI-TOF MS parameters

Protein mass spectra were obtained using a MicroFlex LT MALDI-TOF mass spectrometer (Bruker Daltonics, Germany). The laser frequency was set at 50 Hz within a mass range of 2‒20 kDa, in a linear positive ion mode detection (56) ; (57). Each spectrum represented ions generated from 240 laser shots performed in six regions of the same spot and acquired automatically using the AutoXecute method in FlexControl v2.4 softwares (Bruker Daltonics).

2.6. Analysis of MS spectra

Flex Analysis v3.3 (Bruker Daltonics, Germany) was used to visualize and to compare spectra, which were then exported to ClinProTools v2.2 and MALDI-Biotyper v3.0 softwares (Bruker Daltonics, Germany) for data processing including smoothing, baseline subtraction, peak selection and evaluation of intra-species reproducibility and inter-species specificity [58, 59]. The reproducibility and specificity of the exuviae MS spectra were examined using unsupervised statistical analyses of average spectral profiles (MSP, Main Spectrum Profile) obtained from the four spots for each sample, like cluster analyses (MSP dendrogram) using MALDI-Biotyper v3.0 software, or using supervised statistical analyses assessing the distribution of exuviae MS spectra from different compartments (legs and cephalothorax) of triatomine and cockroache species, using Principal Component Analysis (PCA) from ClinProTools v2.2 software with default settings. Cluster analyses (MSP dendrogram) tool from MALDI-Biotyper v3.0. software was also used, to assess the spectral variations within and between each body parts per triatomine and cockroache species, as previously described (60) (38). CCI matrix was calculated using MALDI-Biotyper v3.0. software with default settings (mass range 3.0–12.0 kDa; resolution 4; 8 intervals; auto-correction off). Higher correlation values (expressed by mean ± standard deviation – SD) reflecting higher reproducibility for the MS spectra, were used to estimate MS spectra distance between body parts per species.

2.7. Reference database creation

After checking intra-species reproducibility and inter-species specificity of exuviae MS profiles, two exuviae spectra per species were added to the home-made reference database. The reference MS spectra were created with an unbiased algorithm using information on the peak position, intensity and frequency using the MALDI-Biotyper v3.0 software (Bruker Daltonics) (61). A total of 18 MSP from exuviae were included in the reference MS spectra DB (Table 2). The raw exuviae MS spectra added to the DB were deposited on the website of the University Hospital Institute (UHI) Méditerranée infection under the following web link (https://doi.org/10.35081/w84y-eg03), on September 2023.

Table 2

Exuviae submitted to MALDI-TOF MS analysis, including database creation, blind tests and results of correct and relevant identification for each species.
Species	Number tested	Good spectra (%)	Added to DB	Blind tests	Correct identification (%)	Range of LSVs	Correct identification with LSVs ≥ 1.8 (%)
Ae. aegypti	98	90 (91.8%)	2	88	88 (100%)	1.62–2.57	81 (92.0%)
An. colluzzi	18	18 (100%)	2	16	16 (100%)	1.99–2.45	16 (100%)
C. lectularius	51	39 (76.5%)	2	37	37 (100%)	1.87–2.44	37 (100%)
C. hemipterus	46	44 (95.6%)	2	42	42 (100%)	2.05–2.50	42 (100%)
P. humanus corporis	45	32 (71.1%)	2	30	30 (100%)	1.65–2.56	28 (96.7%)
Tr. infestans	18	18 (100%)	2	16	16 (100%)	1.99–2.53	16 (100%)
Rh. prolixus	18	18 (100%)	2	16	16 (100%)	1.47–2.49	15 (93.8%)
B. germanica	23	23 (100%)	2	21	21 (100%)	1.77–2.64	20 (95.2%)
S. longipalpa	25	25 (100%)	2	23	23 (100%)	2.14–2.47	23 (100%)
Total, n (%)^a	342	307	18	289	289 (89.2%)		278 (85.8%)
^aThe proportion among the total number submitted to MS after deduction of those included in the DB, n = 324 (ie, n = 342 − 18). DB, database; LSVs, log score values.

2.8. Blind tests and experiment validation

The MS spectra of the remaining exuviae were queried against our home-made arthropod reference spectra DB (43). The reliability of exuviae species identification was estimated using the log score values (LSVs) obtained from the MALDI Biotyper software v.3.0, which ranged from 0 to 3. LSVs greater than 1.8 were considered reliable for species identification, according to previous studies (57). Data were analyzed with Prism software v.7.00 (GraphPad, San Di-ego, CA, USA).

2.9. Statistical analysis

The comparison of DNA quantity or LSVs in each group (ie, body parts) per species were computed using Mann–Whitney or Wilcoxon matched-pairs signed-rank tests when appropriate with GraphPad Prism 7.0.0 (GraphPad Software, San Diego, CA, USA). All differences were considered significant at p < 0.05.

3.1. Collection of exuviae from various arthropod species

Exuviae of species selected for inclusion in this study, were collected exclusively from colonies reared within controlled laboratory environments. One exuviae per species was photographed and are presented in Fig. 1. The absence of dichotomy keys for exuviae did not allowed to validate their identity based on their morphological analysis. Exuviae from mosquitoes, bedbugs and lice are between three to ten-fold smaller than those from triatomines and cockroaches. To limit DNA and protein degradation, the exuviae were promptly frozen at -20°C after their collection.

3.2. Submission of arthropod exuviae to molecular biology analyses

As all the exuviae used for molecular analyses come from laboratory reared colonies, their identity at the species level was known. The effectiveness of molecular biology in identifying arthropods from exuviae has been assessed. Genomic DNA was extracted from the exuviae of the nine species (n = 108, 12 exuviae per species) and from paired fresh specimens (n = 45, five per species). DNA was quantified using Qubit dsDNA Assay Kits. As expected, the quantities of DNA extracted from fresh specimens (mean ± standard deviation (SD): 10.81 ± 10.61 ng/mL) were significantly higher (Mann–Whitney test, p < 0.0001) than those from the exuviae (mean ± SD: 0.22 ± 1.53 ng/mL, Fig. 2). To control whether the DNA extraction from exuviae was sufficient for molecular identification, the amplification of COI genes for mosquitoes, lice, and cockroaches, 16S for triatomines, and 18S for bedbugs, was successful for 100% (n = 45) of the fresh samples. In contrast, PCR success were obtained for 50% (n = 54/108) of the exuviae tested (Table 1). No PCR product was detected for exuviae from P. humanus corporis and Ae. aegypti. Among the 54 exuviae samples with PCR success, high quality sequence were obtained for 16 of them (Table 1). Among the 16 exuviae samples with sequencing success, 11 of them obtained matching to target taxonomic groups, representing solely 10,2% (n = 11/108) of the exuviae samples submitted to molecular identification. Conversely, the rate of sequencing success and target sequencing success were both of 100% for the DNA extracted from one fresh specimen per species used as positive controls (n = 9).

3.3. MALDI-TOF MS analyses

3.3.1. Optimization of sample preparation conditions

As the size of the exuviae differed according to arthropod families, two strategies were used. For mosquitoes, bedbugs and lice, whole exuviae were analyzed by MALDI-TOF MS, whereas for triatomines and cockroaches, leg and cephalothorax exuviae were submitted independently to MS to select the best body part. At the exception of Ae. aegypti species, it is the first time that these exuviae were submitted to MALDI-TOF MS (48). It was then necessary to establish the most appropriate protocols in order to obtain the best protein profiles. The criteria used to determine the better sample preparation condition were the reproducibility and the intensity of resulting MS spectra. The parameters tested were the body parts for large samples, the volume of homogenization mix buffer and the conditions of homogenization. For triatomines and cockroaches, leg exuviae were selected for MS submission. The optimal volume of mix buffer was adjusted from 15µL to 40µL according to the size of the exuviae samples (Table S2). The homogenization time was set at two cycles of three minutes at a frequency of 30 Hertz with the TissueLyser apparatus. These setting conditions were applied for future submission of exuviae samples to MS analysis.

3.3.2. Assessment of MS spectra reproducibility and specificity according to species

A total of 342 samples from exuviae were subjected to MALDI-TOF MS analysis (Table 2). Among them, MS spectra of high intensity were obtained for 89.8% (307/342) of the samples tested. The 35 MS spectra which did not reach the inclusion criteria (Intensity > 3000 a.u., background lower than 15 fold of the more intense peak), were considered as unconform and were then excluded of the analysis (Table 2). A visual comparison of the MS profiles indicated an intra-species reproducibility and inter-species specificity, respectively, between exuviae from the same species and between different species (Fig. 3(i)). These results were confirmed by the comparison of the MS profiles from nine exuviae per species using ClinProTools gel view (Fig. 3(ii)). To evaluate the reproducibility and specificity of the MS spectra of exuviae, a clustering analysis was conducted. A MSP dendrogram was performed with two MS spectra of each species. The spectra of exuviae of the same species were clustered in the same branch of the MSP dendrogram, confirming the reproducibility and specificity of protein profiles (Fig. 3(iii)). It is interesting to note that species from the same family were grouped on the same part of the dendrogram.

3.3.3. Evaluation of MALDI-TOF MS for arthropod species identification based on exuviae MS spectra

After checking the reproducibility and specificity of the MS spectra, a database was created using MALDI-Biotyper 3.0 with two representative MS spectra of high intensity and quality for each species. MS spectra of each species, at the exception of those added to the database (n = 18), were queried against our updated reference MS database. Of the 289 MS spectra queried against the database, 100% of them were correctly identified (Table 2). The LSVs ranged from 1.62 to 2.64 (mean ± SD: 2.25 ± 0.19). For relevant arthropod identification to species level, LSV should be upper than 1.8 (48). Here, more than 89.8% (307/342) of exuviae correctly classified reached this threshold, underlining the reproducibility and specificity of these exoskeleton arthropod spectra. Among the eleven spectra which did not reached the threshold, seven, two, one and one were originated from exuviae of Ae. aegypti, P. humanus corporis, Rh. prolixus and B. germanica, respectively (Fig. 4).

3.3.4. Similarity of exuviae MS spectra from distinct body parts of the same species

For species producing exuviae of large size, such as triatomines or cockroaches, two compartments were evaluated (ie, legs and cephalothorax) and the legs were selected for MS identification. However, as these samples are highly breakable, the loose of leg exuviae is possible compromising their identification. As arthropod exoskeletons are composed essentially of chitin and associated proteins (62) we investigated whether MS spectra from exuviae of the same species were similar independently of the body part tested. In this way, an evaluation of the reproducibility of the MS spectra between legs and cephalothorax per species from the two species of triatomines or cockroaches was done.

The visual comparison of the paired MS profiles between legs and cephalothorax per species, including B. germanica (n = 23), S. longipalpa (n = 25), Tr. Infestans (n = 8) and Rh. prolixus (n = 8) using Flex analysis software indicated a similarity of MS profiles per species (Fig. 5(i)). An accurate comparison of spectra from these two body parts per species using PCA, revealed a clustering of the spectra per body part (Fig. 5(ii)), suggesting the presence of a specific signature of the MS profiles associated with the compartments for each of the four species. However, despite the presence of specific peaks distinguishing exuviae body parts from the same species, numerous peaks were shared between leg and cephalothorax spectra, which was confirmed by the CCI analysis (Additional Figure S1). As expected, higher CCI values were obtained between spectra from the same body per species, ranging from 0.57 to 0.85. Nevertheless, the relative high CCI values obtained between leg and cephalothorax of paired species highlighted the proximity of the spectra.

To evaluate the similarity of spectra per species between body parts, blind tests were performed. In this way, cephalothorax MS spectra of exuviae from these four species were queried against our home made DB containing, among others, only reference MS spectra of exuviae legs from these four species (ie, no reference spectra from exuviae of cephalothorax). Despite, that legs LSVs were significantly (Wilcoxon matched-pairs signed-rank tests) higher than LSVs of cephalothoraxes for B. germanica (p < 0.001), S. longipalpa (p < 0.001), T. infestans (p < 0.01) and Rh. prolixus (p < 0.01), all the spectra (100%, n = 120) were correctly identified at the species level. Nevertheless, among the exuviae cephalothorax spectra, 20% (12/60) of them did not reach the threshold for relevant identification with LSVs ranging from 1.43 to 2.39 (mean ± SD: 1.94 ± 0.19). Only one leg spectra did not reach the threshold (LSV = 1.77 from B. germanica). Among the 12 cephalothorax exuviae spectra which did not reached the threshold, nine were originated from S. longipalpa (Fig. 5(iii)).

Unfortunately, the use of MALDI-TOF MS for arthropod identification requires the sacrifice of the specimen under study. This constraint preclude to carry out additional analyses on live specimens, which could provide essential information on various aspects of their biology and behavior. For instance, it is currently not possible to test susceptibility of arthropods to insecticides, or to investigate their vector competence on specimens collected in the field and submitted them concomitantly to MS identification (48). The analysis of their exuviae occurring during molting processes from these metamorphosis specimens appears as another option.

Currently, the identification of arthropods based on morphological analysis of exuviae remains scarcely applied and involves a meticulous analysis of their structures (63). The requirement of well-preserved breakable samples and experienced specialists to carry out relevant identification are factors limiting it widely use (20). Moreover, the availability of dichotomy keys and the rapid degradation of exuviae in the field, notably for species having aquatic stages (48), are supplementary drawbacks for it application (64). Here, the failing to find morphological identification keys for exuviae of the nine species included confirmed the limitations of this approach [64, 65] Effectively, arthropod identification based on the morphological analysis of their exoskeleton remains confidential and restricted to a limited numbers of species, such as dragonflies (65), chironomid (67) or cicadinae (68) ; (69).

DNA-based identification methods offer a promising avenue for addressing the challenges associated with the identification of sister species, variations in traits within the same species that cannot be morphologically identified, and with samples that have undergone alterations during their field collection (48). Previous evidence has established that exuviae can be subjected to molecular biology techniques for the identification of specimens (70) ; (66). Nonetheless, molecular identification of exuviae appeared challenging. Here, solely half of the exuviae tested yielded positive PCR products and less than 11% were successfully sequenced with correct target taxonomy. The low rate of sequencing success revealed that this approach is not a reliable means of identifying species from their exuviae. The very low quantity of DNA detected could explain the failure of amplification. The quantity of DNA extracted in the exuviae was ten-fold to few hundreds less abundant than in fresh specimens from the same species. These results corroborate previous work done on arthropod exuviae (70). For instance, chironomid pupal exuviae, a bioindicator of water quality, collected in Norway lakes were submitted to molecular identification (71). Although the proportion of positive COI amplification product overtook 82% in chironomid pupal exuviae, the rate of correct sequencing was below than 20% (71). In a recent study, the application of molecular biology for the identification of the mosquito species from pupal exuviae succeeded to distinguish two Aedes sibling species (ie, Ae. coluzzii from Ae. detritus) (72). However, the rate of correct sequencing success reached nearly 60%, which could be problematic to monitor closely-related species. The low amount of DNA in the exuvia of arthropod, which is mainly constitute of non-cellular epicuticle and exocuticle, explains the high difficulty of their identification molecularly (72). The failure of amplification could be attributed to the rapid degradation of the exuviae and the attached epithelial cells, which may also not be present in sufficient quantity for successful amplification (70). It is also possible that method used for DNA extraction was not ideal and an optimization DNA extraction method could improve amplification success rate (71).

The use of mass spectrometry for the identification of arthropod exuviae will enable to overcome the limitations of morphological and molecular identification methods. The application of MALDI-TOF MS to a range of arthropod exuviae famillies was essential to validate it use as an innovative and alternative method for arthropod identification. Until now, a limited number of arthropod families have been subjected to MALDI-TOF MS analyses for their identification at various developmental stages (43) ; (44) ; (33). For heterometabolous species, the same body part could be used for specimen identification by MS at various developmental stages (44). Whereas, for holometabolous species, the complete metamorphose occurring at one step of the cycle, leads to morphological changes inducing evolution protein repertory of the specimen’s tissues (1, 43). These metamorphic species require the creation of specific reference MS spectra according to stages and body parts (73) ; (28). Interestingly, the analysis of the immature aquatic stages of few Culicidae using MALDI-TOF MS revealed that the success of species identification was better for late instar larvae than the pupal stage (47). Effectively, at the pupal stage, mosquito metamorphosis occurred conducting to important changes in protein composition during this step. These protein repertory changes altered the identification rate of these pupal stages in mosquito species collected in the field, hampering the application of this tool to monitor to last aquatic stage of mosquitoes (41).

In contrast, pupal exuviae, corresponding to the exoskeleton, are composed essentially of chitin and cuticle proteins, which are then stable within a species (74). Until now, only one study demonstrated the suitability of MALDI-TOF MS for the identification of two mosquito species, Ae. albopictus and Ae. aegypti using their exuviae at the fourth instar larval and pupal stages (48). The protein signatures were different between the two species, as well as between larval and pupal stages within the same species, demonstrating the specificity of the MS spectra and the distinction of representative peaks of cuticular proteins compared to those obtained on specimens of respective stage and species. The profiles of the larval exuviae exhibited limited diversity, and their handling proved challenging due to their fragility (48). Here, for species with aquatic immature stages (mosquitos), it was opted to analyze uniquely pupal exuviae. MS submission revealed reproducible MS spectra for mosquito exuviae pupal stage per species (Ae. aegypti and An. coluzzii) confirming the application of this tool for their identification. In the future, the enlargement to other culicid species will be necessary to comfort it application.

The present study revealed that MALDI-TOF MS biotyping could be applied to identify a whole range of arthropod families, including mosquitoes, bedbugs, lice, triatomines and cockroaches, based on their exuviae. Although around 10% of the sample were excluded because their spectra were classified as non-compliant, the analysis of remaining protein profiles from exuviae revealed inter-species specificity and intra-species reproducibility. The creation of an exuviae database allowed to identify correctly 100% of the samples, among which 85.8% (278/324) exceeded the threshold value (LSV > 1.8), ensuring reliable identification as previously established (75) ; (58). As it can be challenging to directly identify the specimen, the collection of exuviae could be a pertinent alternative to evidence an arthropod infestation (76) ; (77). The advantages of identifying arthropods from their exuviae are particularly relevant when the arthropod is in a state of active dispersal, moving locally or attempting to escape (76). The search of arthropod traces, such as exuviae, can be more easily found.

The large size of cockroach and triatomine exuviae led us to select their legs for MS identification. However, as exuviae can be damaged during their collection or storing due to their extreme fragility, and conduct to the target body part lost, another body part was tested for sample identification without upgrading the reference MS database. The comparison of leg and cephalothorax exuviae MS spectra within triatomine and cockroach species revealed intra-species specificity between the compartments, even though numerous peaks were shared among compartments. Collectively, these results revealed a sharing of exuviae MS spectra between body parts per species. However, despite the reduction in LSVs, which could compromise the acquisition of relevant identification scores for some samples, correct species identification was achieved. Nonetheless, the lower LSVs of exivuae from cephalothorax compared to legs does not rule out the risk of misidentification that could occur during this cross-body part query. Conversely to fresh specimens for which highly specific MS spectra per body part were obtained such as for tick (44) or for mosquito (38) specimens, for exuviae, cephalothorax profiles from triatomine and cockroach species remained sufficiently similar with their respective legs for a correct species identification. As the profiles are not radically different, in case of damaged samples, an exchange of the body part tested could be done. The results obtained show that, despite some specificity between the compartments of exuviae, they remain similar enough to allow a correct species identification. This consistency is explained by the composition of the exuviae which is closely related whatever the body part (50), (78).

Identifying arthropods from their exuviae using MALDI-TOF mass spectrometry is an innovative and promising approach for entomological research, monitoring, and managing arthropod disease vectors and pests. This study demonstrates that MALDI-TOF biotyping overcomes the limitations of morphological identification of exuviae, which cannot be used due to the unavailability of appropriate keys for numerous arthropod families. Additionally, molecular biology has limitations due to the low quantity of DNA extracted in exuviae. This study marks the first attempt to establish a broader and more diversified reference database for the identification of arthropods from their exuviae. In addition, the assessment of this tool on exuviae collected in the field, with the risk of MS profile alteration due to protein degradation appears compulsory to determine whether it could become a powerful technique and a promising method for the reliable identification of arthropod exuviae.

CCI: Composite Correlation Index; CHCA: Cyano, Hydroxy, Cinnamic, Acid (α-cyano-4-hydroxycinnamic acid); COI: cytochrome oxidase subunit I; DB: Database; DNA: Deoxyribonucleic Acid; HPLC: High-Performance Liquid Chromatography; LSV: log score values; MALDI-TOF MS: Matrix Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry; PCA: Principal Component Analysis; PCR: Polymerase Chain Reaction;

Availability of data and materials

The MS reference spectra included in the database of this study are freely accessible and can be downloaded via the provided DOI (https://doi.org/10.35081/w84y-eg03).

Conflict of interest

The authors declare that there are no conflicts of interest.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Funding

This work has been supported by the Délégation Générale pour l’Armement (DGA, MSProfileR project, Grant no PDH-2-NBC 2-B-2201). This work was supported by the University Hospital Institute (IHU) Méditerranée Infection. RB received a doctoral scholarship from the IHU Méditerranée Infection. The funding had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Authors' contributions

Conceived and designed the experiments: LA. Performed the experiments: RB, RA, LA. Analyzed the data: RB, AZD, LA. Contributed reagents/materials/analysis tools: RB, AZD, RA, JMB, AB, PP. Investigation: RB, AZD, JMB. Drafted the paper: RB, LA. Revised critically the paper: all the authors.

Acknowledgments

We would like to thank Nicolas Gomez for his technical expertise during the experimental execution of this study.

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Assessment of MALDI-TOF MS for arthropod identification based on exuviaes

Status:

Version 1

Abstract

Background.

Objective.

Results.

Conclusion.

Figures

1. Introduction

2. Materials and methods

2.1. Ethical considerations

2.2. Arthropod rearing and exuviae collection

2.3. DNA extraction and quantitative analysis

2.4. Molecular analyses of exuviae

2.4. Optimization of sample preparation conditions for MALDI-TOF MS analysis

2.5. MALDI-TOF MS parameters

2.6. Analysis of MS spectra

2.8. Blind tests and experiment validation

2.9. Statistical analysis

3. Results

3.1. Collection of exuviae from various arthropod species

3.2. Submission of arthropod exuviae to molecular biology analyses

3.3. MALDI-TOF MS analyses

3.3.1. Optimization of sample preparation conditions

3.3.2. Assessment of MS spectra reproducibility and specificity according to species

3.3.3. Evaluation of MALDI-TOF MS for arthropod species identification based on exuviae MS spectra

3.3.4. Similarity of exuviae MS spectra from distinct body parts of the same species

4. Discussion

Conclusion

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1