Blechomonas campbelli from Ctenocephalides felis: in vitro development, thermotolerance, and phylogenetic insights

Abstract

BACKGROUND  Blechomonas spp. are flea-specific monoxenous trypanosomatids, whose biology, host range, and geographic distribution remain poorly understood, even for species inhabiting ubiquitous cat and dog fleas.

OBJECTIVES  To isolate and characterise blechomonads from fleas of domestic dogs in Brazil, focusing on culture growth, morphology, and phylogenetic relationships.

METHODS  Fleas collected from dogs in Sabará, Brazil, were morphologically identified and screened by cultivation. The isolate FL-1 was analysed for temperature-dependent growth, morphology, and phylogeny using 18S rRNA and gGAPDH sequences, including those mined from public metagenomic datasets.

FINDINGS  Of 340 fleas (four per pool), only one culture tested positive. Growth was optimal at 25ºC, reduced at 30ºC, and absent at 35ºC. Four cell types were revealed, including a predominant “uromonad” stage specialised for attachment and aggregation. Phylogenetic analyses placed the isolate within Blechomonas campbelli and indicated that this species and Blechomonas lauriereadi occur in Ctenocephalides spp. from multiple continents.

MAIN CONCLUSIONS  Blechomonas campbelli exhibits developmental adaptations to the flea gut and limited tolerance to elevated temperatures, constraining its potential to persist in warm-blooded hosts. These findings highlight the value of combining culture-based and in silico approaches to investigate flea-associated trypanosomatids.

Key words:
trypanosomatids; fleas; thermotolerance; morphology; phylogeny; host range

Fleas (Insecta: Siphonaptera) are obligate haematophagous insects that parasitise a wide range of mammals, especially rodents in sylvatic environments, but also domestic animals, such as dogs and cats.¹ The importance of fleas in animal and human health stems primarily from their role as vectors of epidemiologically significant pathogens, including Yersinia pestis, Rickettsia spp., Bartonella spp., and several haemoplasmas.²

Among microorganisms inhabiting fleas, monoxenous trypanosomatids of the genus Blechomonas Votýpka and Suková, 2013 are particularly notable, as they are strictly associated with these insects.³ The genus was formally described a little over a decade ago, when integrative morphological and phylogenetic analyses revealed that several flea-associated flagellate species form a distinct, deeply branching clade within the family Trypanosomatidae.³ Such a phylogenetic position justified assigning this lineage to a separate subfamily, Blechomonadinae Votýpka and Suková, 2013.³ It represents the third earliest-branching group among trypanosomatids, following the subfamilies Paratrypanosomatinae (Paratrypanosoma spp.) and Trypanosomatinae (Trypanosoma spp.), as confirmed by phylogenomic analyses.^4,5

Despite recent advances, including the discovery of RNA viruses in some Blechomonas spp.⁶ and the genomic characterisation of B. ayalai,⁷ the life cycle, host range, and geographic distribution of these trypanosomatids are still poorly understood. Moreover, the number and geographic coverage of available isolates remain limited, although fleas are abundant, diverse, and cosmopolitan insects.⁸ Yet, Blechomonas represents a highly unusual lineage: unlike most monoxenous trypanosomatids that parasitise dipterans or true bugs, it is strictly associated with fleas. Along with its deep phylogenetic position, this suggests that its evolution was shaped by distinctive pressures and adaptations warranting detailed study.

While monoxenous trypanosomatids are arguably of lesser practical importance than dixenous ones, their complex evolutionary history, remarkable taxonomic diversity, and striking adaptive traits have attracted increasing attention in recent decades.^9,10,11 In addition, monoxenous trypanosomatids have repeatedly been detected in vertebrate hosts, including humans, by polymerase chain reaction (PCR) or in vitro cultivation.¹² In an early report, a parasite causing leishmaniasis-like symptoms in a human immunodeficiency virus (HIV)-infected patient showed kinetoplast DNA cross-hybridisation with a monoxenous species now placed in the genus Blechomonas,^3,13 although the significance of this finding remains uncertain. Theoretically, blechomonads may be predisposed to colonise vertebrate hosts, given their specific association with fleas, haematophagous insects that transmit rodent trypanosomes of the subgenus Herpetosoma.¹⁴

In this study, we characterise the first Neotropical isolate of Blechomonas campbelli from fleas collected in south-eastern Brazil. Previously, another species of the same genus, Blechomonas lauriereadi, was reported from Ctenocephalides felis in localities of the Neotropical-Nearctic transition in Mexico.¹⁵ However, that study relied exclusively on molecular detection and did not establish parasite cultures suitable for experimental research. By combining in vitro cultivation, morphological analyses, and phylogenetic inference, we provide novel insights into the diversity, distribution, and biology of flea-associated trypanosomatids. In addition, we establish a cultured reference isolate to support future comparative and experimental studies.

MATERIALS AND METHODS

Sample collection and cultivation - A total of 340 fleas were collected from 85 domestic dogs over an eight-month period (March to October 2019) in the municipality of Sabará, an endemic area for visceral leishmaniasis (VL) in the State of Minas Gerais, Brazil.¹⁶ Visual inspection of insects at the time of collection revealed no signs of recent blood feeding (e.g., abdominal distension or visible blood); therefore, all specimens were considered unfed.

After collection, fleas were maintained in transport containers under controlled conditions (approximately 25ºC and 70-80% relative humidity) and processed on the same day at Fiocruz Minas (Belo Horizonte, Brazil). Prior to processing, specimens were briefly maintained in saline solution supplemented with penicillin (5,000 U/mL), streptomycin (500 μg/mL), and 5-fluorocytosine (50 μg/mL) to reduce microbial load.

Fleas were identified under stereomicroscopy using a taxonomic key for Brazilian species,¹⁷ based on diagnostic morphological characters including the shape of the frons, the relative length of the first spine of the genal comb, and the number of setae on the metepisternum (one-two setae in C. felis and three in C. canis). Fleas were then grouped into 85 pools (four individuals from the same host per pool) and mechanically macerated using sterile pestles for culture inoculation.

Primary cultures were established based on previously described protocols for the isolation of trypanosomatids,^18,19 with minor modifications. Flea pools were incubated for 48 h at 4-8ºC in 1.5 mL tubes containing 1× phosphate-buffered saline (PBS) supplemented with antibiotics and antifungal agents to suppress microbial growth associated with the flea microbiota.²⁰ Subsequently, samples were inoculated into biphasic Novy-MacNeal-Nicolle/Liver Infusion Tryptose (NNN/LIT) medium with the same antimicrobial supplementation and incubated at 25ºC, with weekly monitoring for the presence of flagellates. If trypanosomatids were detected, sub-culturing in the same medium was performed once per week.

For growth curve analysis, we adapted a previously described procedure for the thermotolerant trypanosomatid Leptomonas seymouri.²¹ Specifically, 5×10⁶ trypanosomatid cells were inoculated into 5 mL of biphasic medium and incubated for 14 days at three temperatures (25ºC, 30ºC, and 35ºC). Parasite concentrations were determined daily by diluting culture aliquots in 1× PBS containing 2% paraformaldehyde for fixation and counting cells in a Neubauer chamber, using technical triplicates and biological duplicates. Growth curves were compared in GraphPad Prism 8.0 (GraphPad Software, Boston, USA) using multiple t-tests with Holm-Šidák correction. The overall comparison of the growth curves was performed by calculating the area under the curve (AUC) using Simpson’s rule implemented in the SciPy library for Python. The differences between the conditions were estimated by the two-tailed Welch t-test.

Morphological analysis - Cultured cells were washed with 1× PBS, smeared onto glass slides, fixed in methanol for 30 min, and stained with Giemsa. The smears were examined under a light microscope with a 100× oil immersion objective, and images were captured using an AxioCam MRc colour camera (Zeiss, Jena, Germany). Photographed cells were measured using Fiji v.1.54j,²² estimating six standard morphometric parameters (when available): cell length (excluding flagellum), cell width, nucleus length, distances from the anterior end to the nucleus and kinetoplast, and free flagellum length.

To assess whether the visually defined cell categories could be discriminated based on morphometric features, we applied a Linear Discriminant Analysis (LDA) using the scikit-learn v.0.21.3 library in Python.²³ Prior to analysis, missing values were imputed with the mean value within each cell category, while free flagellum length was set to zero for cells lacking a typical whip-like flagellum. All features were then standardised using the RobustScaler method to achieve zero median and unit variance. Since there were four cell types, up to k - 1 = 3 linear discriminants were retained. The model was trained on the full dataset without dimensionality reduction, and results were visualised using all pairwise combinations of the three linear discriminant axes (LD1-LD3) with the Python seaborn library v.0.9.0.²⁴ Feature importance for each linear discriminant (defined as the absolute value of the corresponding LDA coefficient) was used to estimate the relative contribution of each morphometric measurement to cell type discrimination.

DNA isolation, PCR, and sequencing - Cultured cells were washed three times with 10 mL of 1× PBS by centrifugation at 3,000 rpm for 10 min at 4ºC. The resulting pellets were then used for DNA extraction with the PureLink^® Genomic DNA Mini Kit (Thermo Fisher Scientific, Waltham, USA).

The full-length (~2,100 bp) 18S rRNA gene was amplified using primers S762 (5’-GACTTTTGCTTCCTCTAWTG-3’) and S763 (5’-CATATGCTTGTTTCAAGGAC-3’),²⁵ and subsequently sequenced with primers 883F (5′-GACTTGAATTAGMAAGCATGGGA-3′), 907R (5′-TCCCATGCTTKCTAATTCAAGTC-3′), A757 (5′-GCGAACGTACTCCCCCCTGA-3′), and S757 (5′-TCAGGGGGGAGTACGTTCGC-3′).²⁶ Partial gGAPDH gene (~750-800 bp) was amplified and sequenced using the primers GAPtryF (5′-GGBCGCATGGTSTTCCAG-3′) and GAPtryR (5′-CCCCACTCGTTRTCRTACC-3′).²⁷ PCR products were purified with the QIAquick^® PCR Purification Kit (Qiagen, Hilden, Germany) and sequenced by the Sanger method at the René Rachou Institute Sequencing Platform. The obtained sequences were deposited in GenBank under the accession numbers PX442322 and PX569296.

Phylogenetic analyses - The sequences generated in this study were combined with those available in the core nucleotide database of GenBank for Blechomonas spp. and four outgroup taxa. Additional sequences were obtained as follows: (i) sequence read archives (SRAs) of Ctenocephalides spp. containing trypanosomatid signatures were identified by BLASTn using the 18S rRNA sequence of B. campbelli (KF054134) as a query; (ii) reads from positive SRAs were assembled into the 18S rRNA and gGAPDH gene sequences in Geneious Prime 2024.0 as described previously.²⁸ For both genes, sequences were aligned using MAFFT v.7.490 with the E-INS-i algorithm.²⁹

The alignment of the 18S rRNA gene was trimmed twice: (i) low-quality ends of short sequences from GenBank identified by significant divergence from the conserved consensus for Blechomonas spp. were manually removed; (ii) the entire alignment was processed with trimAl v.1.5 using a gap threshold of 0.5.³⁰ Maximum likelihood (ML) analysis was conducted in IQ-TREE v.2.3.6³¹ under the automatically selected TNe+I+G4 model with edge support assessed by both ultrafast and standard bootstrap methods (1,000 replicates each). Bayesian inference was performed in MrBayes v.3.2.7 under the GTR + I + G model, with 1,000,000 generations, sampling frequency of 100 and other parameters set to default.³²

The analysis of the gGAPDH gene was performed similarly, but the alignment was not trimmed and partitioned by codon position as described previously.³³ Briefly, analyses were conducted under partitioned models with linked branch lengths and unlinked evolutionary rates: TIM+F+G4/TPM3+G4/GTR+F+G4 in IQ-TREE and GTR+G+I in MrBayes.

Ethics - The collection of biological material from ectoparasites was conducted in accordance with Brazilian regulations. The study was approved by the Animal Use Ethics Committee (CEUA) of the René Rachou Institute - FIOCRUZ Minas under license number LW-14/17 and complies with Federal Law No. 11.794/2008 and the principles of the Brazilian Society of Laboratory Animal Science (SBCAL).

RESULTS AND DISCUSSION

All collected fleas were identified as C. felis (Fig. 1). Only one pooled sample (containing four fleas from a single dog) tested positive by cultivation. The obtained culture (isolate FL-1) was stable, which allowed us to study cell morphology.

Fig. 1:
representative specimens of Ctenocephalides felis: female (A) and male (B). Diagnostic morphological characters include an elongated and anteriorly inclined frons, the genal comb with the first spine of similar length to the others (arrow), and the number of setae on the metepisternum (1-2 setae; red circle). Total magnification 350×.

Analysis of parasite’s thermotolerance - The growth of isolate FL-1 was temperature-dependent. At 25ºC, a brief lag (days 1-3) was followed by exponential growth until day 7, a plateau at ~5.8× 10⁶ parasites/ml (days 8-10), and a gradual decline. Growth at 30ºC was slower, with significant differences from day 5 onward, ~1.7-fold lower peak densities, and a shorter stationary phase, indicating moderate thermal stress (Fig. 2). The overall comparison of the curves using the AUC analysis showed their highly significant difference (p = 2×10⁻⁸) in growth dynamics between 25 and 30°C [Supplementary data (Table)]. At 35ºC, no proliferation was observed, and all cells died after three days. Together with other lines of evidence, including the absence of recent blood feeding at the time of collection and the known association of other Blechomonas spp. with fleas, this supports the interpretation that the isolate FL-1 is a genuine flea parasite rather than one derived from a transient blood meal.

Fig. 2:
growth kinetics of the parasite in NNN/LIT biphasic medium at 25ºC and 30ºC. The grey shaded area marks time points with statistically significant differences in parasite concentrations between the two conditions (p < 0.05, multiple t-tests with Holm-Šidák correction).

These results are consistent with observations in other trypanosomatids, where temperatures slightly above the optimum trigger stress responses, increased cell death,³⁴ or abrupt reductions in parasite load.³⁵ Thermotolerance in monoxenous species has been associated with the upregulation of protective genes (e.g., desaturases, chaperones, flagellar proteins) at elevated temperatures.^21,36 However, addressing the molecular basis of the observed temperature-dependent growth patterns in the studied strain is beyond the scope of the current work.

Morphological analysis - Only a small proportion of cells in the culture swam freely, whereas the vast majority adhered to the plastic bottom, gradually forming large aggregates, which is a common strategy for many trypanosomatid species.⁹ In older cultures, these aggregates could detach from the substrate and float in the medium. The cells exhibited considerable variability in both shape and size (Table, Fig. 3). We tentatively classified them into four types: tailed cells (uromonads), choanomastigotes, promastigotes, and amastigotes.

TABLE
Morphometry of the four cell types observed in the culture

	Cell length	Cell width	Nucleus	A-N	A-K	Free flagellum	N
Amastigotes	2.5 ± 0.2 (2.2 - 2.9)	1.6 ± 0.2 (1.2 - 2.0)	1.2 ± 0.2 (0.9 - 1.6)	1.2 ± 0.2 (0.9 - 1.7)	0.7 ± 0.2 (0.3 - 1.1)	-	25
Choanomastigotes	4.5 ± 0.9 (2.7 - 6.1)	3.1 ± 0.7 (1.6 - 4.4)	1.9 ± 0.3 (1.4 - 2.6)	1.9 ± 0.6 (0.8 - 3.6)	1.3 ± 0.5 (0.4 - 2.6)	2.6 ± 0.8 (1.0 - 4.5)	50
Uromonads	10.4 ± 1.6 (7.2 - 14.5)	3.1 ± 0.6 (1.7 - 4.4)	2.1 ± 0.4 (1.6 - 3.4)	3.1 ± 0.5 (2.1 - 4.6)	2.1 ± 0.4 (1.0 - 3.0)	-	60
Promastigotes	16.1 ± 4.1 (10.8 - 23.8)	1.7 ± 0.3 (1.0 - 2.3)	2.1 ± 0.6 (1.2 - 4.2)	4.5 ± 1.3 (2.7 - 8.7)	1.8 ± 0.3 (1.7 - 3.3)	9.1 ± 2.3 (5.5 - 14.2)	30

• A-N is the distance from the anterior end of the cell to the nucleus; A-K is the distance from the anterior end of the cell to the kinetoplast. All measurements are in μm [M ± SD (min-max)].

Fig. 3:
morphology of cells in the culture. (A-G) single uromonads; (H) early division of uromonad (two flagella and widened kinetoplast are visible in the cell); (I) asymmetric division of uromonad-like cell; (J) heterogeneous rosette of uromonads and promastigote-like cells; (K) uromonads attached to a matrix-like substance; (L) single choanomastigote; (M-N) dividing choanomastigotes; (O-P) rosettes of choanomastigotes of different sizes and shapes; (Q) pair of joined choanomastigotes (left) and short promastigote (right); (R-T) promastigotes; (U-V) amastigotes; (W) narrow uromonad (left) and group of amastigotes (right). Scale bars: 5 μm (A-I and K-W) and 10 μm (J).

The LDA revealed clear separation among the four cell categories in the three-dimensional discriminant space, achieving classification accuracy of 98% ± 1% across folds, which indicates strong discriminatory power based on the morphometric features. In particular, LD1 and LD2 accounted for the majority of between-class variance and effectively discriminated uromonads and promastigotes, while choanomastigotes and amastigotes were separated when using projections with LD3 [Supplementary data (Figure A)]. The free flagellum length, distance from the anterior end to the kinetoplast, and the cell width were the most important features for the cell discrimination, contributing to LD1, LD2 and LD3, respectively [Supplementary data (Figure B)]. Of note, the culture also contained relatively rare intermediate forms, which were excluded from the analysis.

The predominant cell type was the tailed form, for which we propose the term “uromonad” (from Ancient Greek οὐρά — tail, and μονάς — unit). The name is reminiscent of terms such as necto- and haptomonad, which are used for developmental stages of Leishmania spp. in the sand fly gut.³⁷ These cells were characterised by an elongated body with a clear contrast between its main swollen portion and a significantly narrower “tail”, creating a tadpole-like appearance (Fig. 3A-K). The free flagellum was either not detectable or appeared as a short, wide, and blurry structure, suggesting that it might be transformed into an attachment organelle characteristic of various trypanosomatids.⁹ The cells were proliferating at this stage (Fig. 3H-I). The presence of uromonads with two nuclei but no signs of duplication of other organelles suggests that cell division may be prone to aberrations. Although their flagellum was reduced, these attached cells were still capable of slight waggling movements. When aggregated, they could produce an amorphous matrix that covered the anterior portion of the cell (Fig. 3K), a feature especially apparent in old cultures. This trait is reminiscent of the behaviour observed in some other trypanosomatids, such as Trypanosoma theileri and Lotmaria passim, which secrete protective biofilms when attaching to the insect hindgut.^38,39

Choanomastigotes were shorter than uromonads and when their posterior end was pointed, it was not distinctly separated from the main part of the cell. As a result, they generally appeared drop-shaped (Fig. 3L-P). Oval cells lacking posterior tapering appeared smaller (Fig. 3Q). Choanomastigotes possessed a flagellum that was typically shorter than the cell body. Nevertheless, it appeared to facilitate aggregation of cells into rosettes (Fig. 3O-Q). This cell type was also proliferative (Fig. 3M-N) and the presence of intermediate forms (Fig. 3I) suggests a developmental transition between choanomastigotes and uromonads.

Promastigotes represented another type of flagellated cells, ranging from relatively short, thick, club-shaped forms (Fig. 3Q-R) to very long cells with the body twisted along its axis and both ends tapered (Fig. 3S-T). In the latter case, they resembled the characteristic haemocoelic forms of Phytomonas spp.^40,41 The flagellum in these cells varied in length but was always shorter than the cell body. Despite their evident motility, these cells were also observed in rosettes, where they appeared to originate from elongated uromonads (Fig. 3J).

Amastigotes were the typical oval to rounded aflagellate cells, either comparable in size to oval choanomastigotes or smaller (Fig. 3U-W). We hypothesise that amastigotes and oval choanomastigotes may transform into each other. No intracellular portion of the flagellum could be observed in light microscopy images; therefore, we refer to these cells here as amastigotes rather than endomastigotes, which are known from the life cycles of various monoxenous trypanosomatids and phytomonads and primarily serve for transmission.⁹

The observed diversity of cell types suggests a relatively complex life cycle that appears to be reproduced in culture. The ability of most cell types to attach, especially pronounced in uromonads, is likely crucial for survival in fleas, which can pump blood through their intestine in volumes exceeding 15 times their body volume per day.⁴² The fibrous matrix produced by these cells apparently helps them to hold on the hindgut surface, a site from which the majority of species were reported.³ However, the anatomical localisation of the studied parasite within the flea was not assessed in this study (as isolation was performed from whole-body macerates), nor in the original description of the species,³ to which it belongs according to phylogenetic analysis (see below). Therefore, the proposed association of this trypanosomatid with the hindgut and the functions of the observed cell types should be considered inferential and warrant confirmation through future in situ investigations.

Amastigotes, the only cell type that is unable to attach, are likely to be effectively shed with faeces and serve to infect other individuals, presumably larvae, which readily feed on the adult flea droppings.⁴³ The rapid drying of these droppings (known as flea dirt) likely forces parasites to develop elevated resistance to adverse environmental conditions, analogous to compact aflagellate stages of other trypanosomatids — endomastigotes and cyst-like amastigotes.⁹ Motile choanomastigotes and promastigotes are likely involved in spreading within the host. The reason for the coexistence of two such morphotypes is unclear, although the non-proliferating nature of promastigotes suggests that their formation may be triggered by specific conditions, such as host starvation, which is a known factor for cell differentiation in various trypanosomatids.^44,45

Although our morphological analysis was not intended for taxonomic identification or revision, it appeared natural to compare obtained our data with those available in the literature. The original description of B. campbelli (to which the FL-1 strain was eventually assigned, see below) is extremely brief, reporting “bended” promastigotes (subdivided into short- and long-flagellated forms) as the main morphotype, with only three measurements, and rare (unmeasured) amastigotes.³ Given the limited information and the absence of illustrations, a reliable morphological comparison was not possible. Therefore, our taxonomic assignment of the FL-1 strain was based exclusively on molecular data. We, nevertheless, speculate that promastigotes with short flagella may correspond to uromonads, whereas those with long flagella likely correspond to the promastigotes identified in our study. The reported size ranges considerably overlap with our measurements.

Interestingly, an earlier study of trypanosomatids isolated into culture from the gut of a cat flea provided a more detailed morphometric analysis, distinguishing four cell morphotypes with measurements largely overlapping those of strain FL-1, although also without illustrations, which complicates direct comparison.⁴⁶ The main differences from our data concern the upper limits of promastigote width (6.2 vs 2.3 μm) and flagellum length (24.9 vs 14.2 μm). Although the maximal length and width of “cystic-like bodies” are bigger than those of FL-1 amastigotes (6.4 vs 2.9 μm and 3.8 vs 2.0 μm, respectively), this may reflect inclusion of choanomastigotes, which fall within these size ranges (referred to as “rounded amastigotes” in the cited study). We cannot determine whether the same or a different species was examined in that study, as the observed differences may reflect variation in culture conditions or strain-specific traits. The only molecular analysis reported in that paper excluded affiliation of the trypanosomatid with Leishmania, but did not resolve its taxonomic identity.⁴⁶

Phylogenetic analyses - The phylogenetic trees reconstructed from the 18S rRNA and gGAPDH gene sequences displayed different relationships among species (Fig. 4). Notably, the topology based on the latter gene was consistent with the tree inferred from the concatenated dataset of both genes, which was published in the study establishing the genus and describing most of its species.³ This suggests that in the concatenated dataset the overall topology was mainly determined by the gGAPDH gene. In our case, using the two genes separately was justified since the number of available sequences for them was significantly different and, moreover, they showed different variability levels.

The sequences of both genes from the studied isolate reliably affiliated it with B. campbelli (Fig. 4). While for the 18S rRNA gene there were no differences from the previously published sequences of that species, gGAPDH exhibited 10 substitutions at the 3’ end of the gene, yet all these were synonymous, i.e., not reflected in the amino acid sequences. In sum, the sequence data support the assignment of the new isolate to B. campbelli.

Fig. 4:
maximum likelihood phylogenetic trees of Blechomonas spp. based on two different genes. (A) 18S rRNA. (B) gGAPDH. Both trees are rooted using complex outgroups, with trypanosomes representing the earliest branch according to the phylogenomic inference.⁽⁴⁾ The isolate obtained in this study is highlighted in black. Geographic and host origins are indicated for Blechomonas spp. from cat and dog fleas. Numbers at branches are Bayesian posterior probabilities, as well as ultrafast and standard bootstrap supports (values below 0.5 or 50% are replaced with dashes or omitted). Black circles indicate maximum supports for all three methods (1.0/100/100). The triple-crossed branches were rescaled to one-third of their original length. The scale bar denotes the number of substitutions per site.

Our analysis of SRAs from Ctenocephalides spp. revealed trypanosomatid reads in a metagenomic dataset of C. felis from Guangxi, China (GenBank accession number SRR23478486). We successfully assembled sequences of both genes despite the low read coverage for gGAPDH (4×). The sequences unambiguously clustered with B. lauriereadi (Fig. 4) and, relative to the previously analysed isolates of this species, differed by only 0-2 substitutions in the 18S rRNA gene and 16 substitutions (+1 N) in the gGAPDH gene, corresponding to a single amino acid change. These findings highlight the value of publicly available datasets for parasite surveillance, enabling detection of cryptic infections and expanding knowledge of parasite distribution and host associations with minimal additional sampling.

In total, the inferred 18S rRNA gene tree comprised eight sequences of flagellates from Ctenocephalides spp.: three belonged to B. campbelli and the other five to B. lauriereadi (Fig. 4A). Both species have been found in C. felis, but only a single isolate of the latter was from C. canis. Such a situation is likely explained by the fact that the first flea species is quite common, while the second is relatively rare.⁴⁷ The clades of both trypanosomatid species included sequences from the New World (Brazil or Mexico) and Old World (Czechia and/or China). Therefore, although the currently available data remain limited, the results suggest that the same blechomonads can occur in different host species and geographic regions, indicating a lack of strict host or geographic association. This is apparently determined by the very wide specificity (euryxeny) of C. felis, which, despite its common name (cat flea), infests a broad range of unrelated hosts, including carnivores, ungulates, rodents, lagomorphs and marsupials.⁴³ Ease of host switching, high mobility, and association with domestic animals in the era of globalisation likely facilitate genetic homogenisation of parasite populations across continents and host taxa. Although less is known about the dog fleas, both species definitely can coexist on the same host and, therefore, exchange parasites.⁴⁸

Previous studies of trypanosomatids in fleas from Brazil, including those conducted in Minas Gerais, have reported the presence of trypanosomatids in Ctenocephalides spp. in the context of canine visceral leishmaniasis surveys.^46,49 However, these studies lacked detailed morphological characterisation and molecular identification, limiting taxonomic resolution and comparability at the species level. In this context, the present study complements earlier findings by providing an integrative approach based on cultivation, microscopy, and sequence-based identification.

In conclusion - This study provides the first detailed analysis of the flea-associated trypanosomatid B. campbelli, the original description of which was limited to a brief diagnosis and its phylogenetic placement.³ The studied isolate displayed temperature-dependent growth: cells survived and proliferated at lower temperatures, but all died at 35ºC, which prevents sustained development in warm-blooded vertebrate hosts. Morphological analyses revealed a remarkable diversity of proliferative and non-proliferative cell types, including a predominant “uromonad” stage specialised for attachment and aggregate formation, likely representing an adaptation to survival in the flea gut. Phylogenetic analyses corroborated the identification and, together with mining publicly available sequencing datasets, expanded the known distribution of trypanosomatids associated with Ctenocephalides spp. (B. campbelli and B. lauriereadi) across continents and host species. These findings demonstrate that blechomonads are neither host- nor region-restricted, a pattern likely facilitated by the broad host range and global spread of C. felis. Our work highlights the value of combining culture-based studies with in silico surveillance for uncovering the diversity, biology, and epidemiology of flea-associated trypanosomatids.

SUPPLEMENTARY MATERIALS

Supplementary material

Supplementary material

ACKNOWLEDGEMENTS

To the Fiocruz Network of Technological Platforms at Instituto René Rachou (Fiocruz Minas) for DNA sequencing services.

DATA AVAILABILITY

The contents underlying the research text are included in the manuscript.

• Financial support: This study was supported by the European Union (EU) Operational Programme Just Transition (LERCO CZ.10.03.01/00/22_003/0000003 to VY) and by the State Assignment for the Zoological Institute RAS No. 125012800894-6.
• How to cite:
  Estevam LGTM, Kostygov AY, Dutra-Rêgo F, Martins ALM, Freire MA, Lima ACVMR, et al. Blechomonas campbelli from Ctenocephalides felis: in vitro development, thermotolerance, and phylogenetic insights. Mem Inst Oswaldo Cruz. 2026; 121: e250286.

REFERENCES

• ¹ Bitam I, Dittmar K, Parola P, Whiting MF, Raoult D. Fleas and flea-borne diseases. Int J Infect Dis. 2010; 14(8): e667-76.
• ² Eisen RJ, Gage KL. Transmission of flea-borne zoonotic agents. Annu Rev Entomol. 2012; 57: 61-82.
• ³ Votýpka J, Suková E, Kraeva N, Ishemgulova A, Duží I, Lukeš J, et al. Diversity of trypanosomatids (Kinetoplastea: Trypanosomatidae) parasitizing fleas (Insecta: Siphonaptera) and description of a new genus Blechomonas gen n. Protist. 2013; 164(6): 763-81.
• ⁴ Kostygov AY, Albanaz ATS, Butenko A, Gerasimov ES, Lukeš J, Yurchenko V. Phylogenetic framework to explore trait evolution in Trypanosomatidae. Trends Parasitol. 2024; 40(2): 96-9.
• ⁵ Skalický T, Dobáková E, Wheeler RJ, Tesařová M, Flegontov P, Jirsová D, et al. Extensive flagellar remodeling during the complex life cycle of Paratrypanosoma, an early-branching trypanosomatid. Proc Natl Acad Sci USA. 2017; 114(44): 11757-62.
• ⁶ Grybchuk D, Kostygov AY, Macedo DH, Votýpka J, Lukeš J, Yurchenko V. RNA viruses in Blechomonas (Trypanosomatidae) and evolution of Leishmaniavirus. mBio. 2018; 9(5): e01932-18.
• ⁷ Opperdoes FR, Butenko A, Flegontov P, Yurchenko V, Lukeš J. Comparative metabolism of free-living Bodo saltans and parasitic trypanosomatids. J Eukaryot Microbiol. 2016; 63(5): 657-78.
• ⁸ Medvedev SG. Geographical distribution of families of fleas (Siphonaptera). Entomol Rev. 1996; 76(8): 978-92.
• ⁹ Frolov AO, Kostygov AY, Yurchenko V. Development of monoxenous trypanosomatids and phytomonads in insects. Trends Parasitol. 2021; 37(6): 538-51.
• ¹⁰ Lukeš J, Butenko A, Hashimi H, Maslov DA, Votýpka J, Yurchenko V. Trypanosomatids are much more than just trypanosomes: clues from the expanded family tree. Trends Parasitol. 2018; 34(6): 466-80.
• ¹¹ Maslov DA, Opperdoes FR, Kostygov AY, Hashimi H, Lukeš J, Yurchenko V. Recent advances in trypanosomatid research: genome organization, expression, metabolism, taxonomy and evolution. Parasitology. 2019; 146(1): 1-27.
• ¹² Boucinha C, Andrade-Neto VV, Ennes-Vidal V, Branquinha MH, Dos Santos ALS, Torres-Santos EC, et al. A stroll through the history of monoxenous trypanosomatids infection in vertebrate hosts. Front Cell Infect Microbiol. 2022; 12: 804707.
• ¹³ Pacheco RS, Marzochi MCA, Pires MQ, Brito CMM, Madeira MF, Barbosa-Santos EGO. Parasite genotypically related to a Monoxenous Trypanosomatid of dog’s flea causing opportunistic infection in an HIV positive patient. Mem Inst Oswaldo Cruz. 1998; 93(4): 531-7.
• ¹⁴ Kostygov AY, Karnkowska A, Votýpka J, Tashyreva D, Maciszewski K, Yurchenko V, et al. Euglenozoa: taxonomy, diversity and ecology, symbioses and viruses. Open Biol. 2021; 11(3): 200407.
• ¹⁵ Zazueta-Islas HM, Salceda-Sánchez B, Huerta-Jiménez H, Miranda-Caballero CI, Solis-Cortés M, de la Cruz-Pacheco Y, et al. Molecular detection of kinetoplastid species in ticks and fleas associated with dogs and humans in Mexico. Pathogens. 2024; 13(12): 1072.
• ¹⁶ Lopes JV, Michalsky EM, Lara Silva FO, Lima A, de Avelar DM, da Costa AAJ, et al. Seroprevalence and molecular characterization of Leishmania in dogs from an endemic area of zoonotic visceral leishmaniasis in Brazil. Int J Vet Sci Med. 2017; 5(1): 70-4.
• ¹⁷ Linardi PM, Guimarães LR. Sifonápteros do Brasil. São Paulo: Museu de Zoologia da Universidade de São Paulo/FAPESP; 2000. 291 pp.
• ¹⁸ Lukeš J, Votýpka J. Field isolation and cultivation of trypanosomatids from insects. Methods Mol Biol. 2020; 2116: 3-21.
• ¹⁹ Pereira AAS, Ferreira EC, Lima A, Tonelli GB, Rêgo FD, Paglia AP, et al. Detection of Leishmania spp in silvatic mammals and isolation of Leishmania (Viannia) braziliensis from Rattus rattus in an endemic area for leishmaniasis in Minas Gerais State, Brazil. PLoS One. 2017; 12(11): e0187704.
• ²⁰ Wu YL, Hu SF, Zhang XL, Wang HM, Pan HY, Liu GH, et al. Complete bacterial profile and potential pathogens of cat fleas Ctenocephalides felis Acta Trop. 2023; 243: 106923.
• ²¹ Kraeva N, Butenko A, Hlaváčová J, Kostygov A, Myšková J, Grybchuk D, et al. Leptomonas seymouri: adaptations to the dixenous life cycle analyzed by genome sequencing, transcriptome profiling and co-infection with Leishmania donovani PLoS Pathog. 2015; 11(8): e1005127.
• ²² Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012; 9(7): 676-82.
• ²³ Pedregosa F, Varoquaux G, Gramfort A, Michel V, Thirion B, Grisel O, et al. Scikit-learn: machine learning in Python. J Mach Learn Res. 2011; 12: 2825-30.
• ²⁴ Waskom M. Seaborn: statistical data visualization. J Open Source Softw. 2021; 6(60): 3021.
• ²⁵ Maslov DA, Lukeš J, Jirků M, Simpson L. Phylogeny of trypanosomes as inferred from the small and large subunit rRNAs: implications for the evolution of parasitism in the trypanosomatid protozoa. Mol Biochem Parasitol. 1996; 75(2): 197-205.
• ²⁶ Gerasimov ES, Kostygov AY, Yan S, Kolesnikov AA. From cryptogene to gene? ND8 editing domain reduction in insect trypanosomatids. Eur J Protistol. 2012; 48(3): 185-93.
• ²⁷ Teixeira MM, Borghesan TC, Ferreira RC, Santos MA, Takata CS, Campaner M, et al. Phylogenetic validation of the genera Angomonas and Strigomonas of trypanosomatids harboring bacterial endosymbionts with the description of new species of trypanosomatids and of proteobacterial symbionts. Protist. 2011; 162(3): 503-24.
• ²⁸ Klocek D, Grybchuk D, Macedo DH, Galan A, Votýpka J, Schmid-Hempel R, et al. RNA viruses of Crithidia bombi, a parasite of bumblebees. J Invertebr Pathol. 2023; 201: 107991.
• ²⁹ Katoh K, Standley DM. MAFFT multiple sequence alignment software version 7: improvements in performance and usability. Mol Biol Evol. 2013; 30(4): 772-80.
• ³⁰ Capella-Gutiérrez S, Silla-Martinez JM, Gabaldon T. trimAl: a tool for automated alignment trimming in large-scale phylogenetic analyses. Bioinformatics. 2009; 25(15): 1972-3.
• ³¹ Minh BQ, Schmidt HA, Chernomor O, Schrempf D, Woodhams MD, von Haeseler A, et al. IQ-TREE 2: new models and efficient methods for phylogenetic inference in the genomic era. Mol Biol Evol. 2020; 37(5): 1530-4.
• ³² Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Hohna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol. 2012; 61(3): 539-42.
• ³³ Spodareva VV, Grybchuk-Ieremenko A, Losev A, Votýpka J, Lukeš J, Yurchenko V, et al. Diversity and evolution of anuran trypanosomes: insights from the study of European species. Parasit Vectors. 2018; 11(1): 447.
• ³⁴ Cruz-Saavedra L, Muñoz M, Patiño LH, Vallejo GA, Guhl F, Ramírez JD. Slight temperature changes cause rapid transcriptomic responses in Trypanosoma cruzi metacyclic trypomastigotes. Parasit Vectors. 2020; 13(1): 255.
• ³⁵ González-Rete B, Gutiérrez-Cabrera AE, de Fuentes-Vicente JA, Salazar-Schettino PM, Cabrera-Bravo M, Córdoba-Aguilar A. Higher temperatures reduce the number of Trypanosoma cruzi parasites in the vector Triatoma pallidipennis Parasit Vectors. 2021; 14(1): 385.
• ³⁶ Ishemgulova A, Butenko A, Kortisova L, Boucinha C, Grybchuk-Ieremenko A, Morelli KA, et al. Molecular mechanisms of thermal resistance of the insect trypanosomatid Crithidia thermophila PLoS One. 2017; 12(3): e0174165.
• ³⁷ Dostálová A, Volf P. Leishmania development in sand flies: parasite-vector interactions overview. Parasit Vectors. 2012; 5: 276.
• ³⁸ Kostygov AY, Frolov AO, Malysheva MN, Ganyukova AI, Drachko D, Yurchenko V, et al. Development of two species of the Trypanosoma theileri complex in tabanids. Parasit Vectors. 2022; 15(1): 95.
• ³⁹ Carreira de Paula J, García Olmedo P, Gómez-Moracho T, Buendía-Abad M, Higes M, Martín-Hernández R, et al. Promastigote EPS secretion and haptomonad biofilm formation as evolutionary adaptations of trypanosomatid parasites for colonizing honeybee hosts. NPJ Biofilms Microbiomes. 2024; 10(1): 27.
• ⁴⁰ Frolov AO, Malysheva MN, Yurchenko V, Kostygov AY. Back to monoxeny: Phytomonas nordicus descended from dixenous plant parasites. Eur J Protistol. 2016; 52: 1-10.
• ⁴¹ Ganyukova AI, Frolov AO, Malysheva MN, Spodareva VV, Yurchenko V, Kostygov AY. A novel endosymbiont-containing trypanosomatid Phytomonas borealis sp n from the predatory bug Picromerus bidens (Heteroptera: Pentatomidae). Folia Parasitol. 2020; 67: 004.
• ⁴² Dryden MW, Gaafar SM. Blood consumption by the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae). J Med Entomol. 1991; 28(3): 394-400.
• ⁴³ Rust MK. The biology and ecology of cat fleas and advancements in their pest management: a review. Insects. 2017; 8(4): 118.
• ⁴⁴ Kollien AH, Schaub GA. The development of Trypanosoma cruzi (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of starvation. J Eukaryot Microbiol. 1998; 45(1): 59-63.
• ⁴⁵ Kollien AH, Schaub GA. The development of Blastocrithidia triatomae (Trypanosomatidae) in the reduviid bug Triatoma infestans (Insecta): influence of starvation. Parasitol Res. 2002; 88(9): 804-9.
• ⁴⁶ de Avelar DM, Melo MN, Linardi PM. Morphology and growth characteristics of cultured Leptomonas ctenocephali from Ctenocephalides felis felis (Siphonaptera: Pulicidae) of dogs in Brazil. Vet Parasitol. 2011; 180(3-4): 394-8.
• ⁴⁷ Garcia-Sánchez AM, Zurita A, Cutillas C. Morphometrics as a complementary tool in the differentiation of two cosmopolitan flea species: Ctenocephalides felis and Ctenocephalides canis Insects. 2022; 13(8): 778.
• ⁴⁸ Hernandez-Valdivia E, Cruz-Vazquez C, Ortiz-Martinez R, Valdivia-Flores A, Quintero-Martinez MT. Presence of Ctenocephalides canis (Curtis) and Ctenocephalides felis (Bouche) infesting dogs in the city of Aguascalientes, Mexico. J Parasitol. 2011; 97(6): 1017-9.
• ⁴⁹ de Avelar DM, Bussolotti AS, do Carmo ARM, Linardi PM. Endosymbionts of Ctenocephalides felis felis (Siphonaptera: Pulicidae) obtained from dogs captured in Belo Horizonte, Minas Gerais, Brazil. J Invertebr Pathol. 2007; 94(2): 149-52.

Edited by

• Handling editor:
  Geovane Dias-Lopes | https://orcid.org/0000-0003-4448-1398

FIRST REVIEW ROUND - REVIEWERS COMMENTS

About the reviewer

REVIEWER #1

This study provides a valuable characterization of Blechomonas campbelli, combining morphological, thermotolerance, and phylogenetic analyses to expand knowledge of flea-associated trypanosomatids. However, several aspects of the methodology and data interpretation require clarification and cautious revision.

Comments:

1. The Introduction is concise, clearly written, and effectively establishes fleas as relevant vectors. The novelty of reporting the first Neotropical isolate is clearly stated, but the justification for its broader biological or epidemiological importance could be expanded. It could benefit from a slightly deeper discussion in the final paragraph to more explicitly define the research gap and the significance of obtaining a Neotropical isolate.

2. The sampling is limited to one municipality and one host (domestic dogs) only, so interpretations about “Neotropical isolates” should be cautious.

3. It is important to indicate that whether the fleas collected were fed or unfed, as this can strongly influence the understanding if the isolates were presence in blood meal (from the host) vs. true infection in the fleas. If both fed and unfed fleas were included, it would be important to indicate how pools were composed and whether feeding status was recorded or if there are any specific controls being included.

4. The authors employed 48 h incubation of flea pools at 4–8 °C prior to homogenization as a contamination control step. Although this may reduce contamination by other microbes, prolonged cold storage, however, also affect Blechomonas viability or it will allow the selection bias for more cold- or thermotolerant strains. Given that the later experiments were to assess the Blechomonas growth at different temperatures, this initial prolonged low-temperature incubation step may not fully represent the natural diversity of Blechomonas in the sample, and subsequent growth-temperature experiments could underestimate the species’ overall thermotolerance range. Please explained whether this protocol was adapted from a previous reference or if it has been validated to ensure parasite recovery and diversity were not compromised.

5. Please ensure that Genbank Accession Numbers are completed (Line 124: XXXXXX)

6. Line 158-159: Additional clarification for “No growth occurred at 35 °C” would be useful. Please specify if this is referred to absence of proliferation or cell death.

7. Line 163-164: The statement “the studied trypanosomatid may lack such mechanisms” appears to be speculative as there is no transcriptomic or genomic data were presented to assess gene presence or regulation. This inference should be removed or rephrased unless the authors provide molecular evidence to support this.

8. The conclusion that “limited thermotolerance excludes colonization of warm-blooded vertebrates” should therefore be stated more cautiously. This is because the 48-h cold incubation step before culture could have introduced selection bias before the isolation toward more cold-tolerant or less heat-tolerant strains. Unless additional data are available to demonstrate that other Blechomonas isolates similarly fail to grow at 35 °C or that cold incubation does not affect viability or diversity, this interpretation should be revised or rephrased to reflect the limitations of the current dataset.

REVIEWER #2

Dear authors,

The manuscript provides valuable information on the association of Blechomonas with C. felis. It is written in a clear and fluid manner and contains all the essential elements expected for a study of this scope. However, several points remain overly general and would benefit from further clarification. Below are my detailed comments:

Line 43 – Such as?

Line 45 – Missing author and year.

Lines 48–49 – According to whom?

Line 52 – The phrasing here seems unnecessary. You may consider removing this sentence and introducing the findings in a different way.

Line 55 – Could you clarify what you meant by this?

Lines 78–86 – Is this protocol your own, or is it taken from the literature?

Lines 87–91 – Same question as above.

Line 124 – One sequence appears to be missing. Does this mean there was a single sequence for each gene?

Line 126 – Does this indicate something specific?

REVIEWER #3

The abstract is well-written and summarizes the study’s objectives, methodology, and main findings. However, it would benefit from explicitly acknowledging key limitations, such as the low number of fleas analyzed per host and the identification of only one positive pooled sample as Blechomonas campbelli. Additionally, the abstract should address potential issues related to culture contamination, especially given the use of NNN/LIT medium. Including information on the health status of the sampled dogs would also enhance the contextual interpretation of the findings.

b) **Originality and Importance of the Contribution to the Field**

The manuscript tackles a relevant topic within vector biology, parasite surveillance, and biodiversity studies. The detection of a trypanosomatid in *Ctenocephalides felis* may be a significant observation. However, the originality and overall impact of the contribution are limited by the identification of only a single positive culture and by the absence of host-related contextual data, such as the clinical or health status of the dogs and the measurement of the parasite load in the infected flea. Without this information, the relevance of the findings for animal or public health remains uncertain.

c) **Relevance and Adequacy of Methodology, Results, and Discussion**

The methodology is clearly described and technically appropriate. However, the number of samples should be included in the methodology section rather than the results section. Flea collection, morphological identification, cultivation using NNN/LIT medium, and DNA analyses are all standard procedures. Nevertheless, analyzing only four fleas per dog, grouped into pooled samples, significantly limits the analysis of parasite’ thermotolerance, phylogenetic analyses, and for estimating infection frequency.

Moreover, the manuscript does not address the potential for contamination during culture, despite the known susceptibility of NNN/LIT medium to bacterial or fungal overgrowth, even when antifungal agents and antibiotics are used. The lack of information on contamination control measures and culture monitoring weakens the interpretation of the single positive result. Molecular confirmation through the amplification of trypanosomatid DNA from flea samples and of cultures is strongly recommended.

Additionally, the health status of the dogs (e.g., asymptomatic, clinically ill, or showing signs compatible with trypanosomatid infection, as well as serological results) is not reported. This information is essential for contextualizing the epidemiological and biological relevance of the findings and should be included and discussed, particularly regarding seroprevalence and the molecular characterization of *Leishmania* in dogs in the study area.

d) **References**

The references are mostly appropriate and generally up to date. They adequately support the study’s background; however, the authors may want to consider adding references on contamination culture risks in culture-based trypanosomatid studies and on methods for molecular confirmation.

e) **Figures and Tables**

The figures and tables are clear, well-organized, and properly labeled. They accurately reflect the sampling strategy and results. However, given the descriptive nature of the data, care should be taken to ensure that visual elements do not imply broader epidemiological conclusions.

AUTHORS' RESPONSE TO THE REVIEWERS

January 29, 2026

Manuscript ID: MIOC-2025-0286

Blechomonas campbelli from Ctenocephalides felis - in vitro development, thermotolerance, and phylogenetic insights

Dear Dr. Dias-Lopes,

We sincerely thank you and the reviewers for the thorough and constructive evaluation of our manuscript. We carefully considered all comments and incorporated the suggested revisions to improve clarity, methodological transparency, and the overall scientific quality of the work. In several instances, the reviewers raised important conceptual and methodological points that helped us refine the presentation of our results and strengthen key interpretations.

Below, we provide a detailed, point-by-point response to all comments. Reviewer remarks are presented in italics, followed by our responses. For clarity, we refer to page and line numbers in the tracked-changes version of the manuscript. Whenever we slightly disagreed with a suggestion, we explain our reasoning while ensuring that the revised version adequately addresses the reviewer’s concern.

We hope that the revised manuscript meets the expectations of the editorial board and reviewers, and we remain at your disposal for any further adjustments if needed.

Sincerely,

Felipe Dutra Rêgo

Instituto René Rachou - Oswaldo Cruz Foundation

Reviewer 1

Reviewer #1: This study provides a valuable characterization of Blechomonas campbelli, combining morphological, thermotolerance, and phylogenetic analyses to expand knowledge of flea-associated trypanosomatids. However, several aspects of the methodology and data interpretation require clarification and cautious revision.

1- The Introduction is concise, clearly written, and effectively establishes fleas as relevant vectors. The novelty of reporting the first Neotropical isolate is clearly stated, but the justification for its broader biological or epidemiological importance could be expanded. It could benefit from a slightly deeper discussion in the final paragraph to more explicitly define the research gap and the significance of obtaining a Neotropical isolate.

Authors’ comment: We expanded the final paragraph of the Introduction to better define the research gap and highlight the biological significance of obtaining a Neotropical isolate. Specifically, we now acknowledge previous molecular detections of Blechomonas in the region (i.e., B. lauriereadi in Ctenocephalides felis from Mexico), while emphasizing that this was not accompanied by isolation of cultures (lines 76–84).

2- The sampling is limited to one municipality and one host (domestic dogs) only, so interpretations about “Neotropical isolates” should be cautious.

Authors’ comment: We clarify that our statement refers specifically to B. campbelli, for which no previous isolates have been reported from the Neotropical region. Throughout the manuscript, we explicitly restrict our interpretation to fleas collected from domestic dogs in a single municipality and do not imply that this isolate is representative of the Neotropics as a whole.

3- It is important to indicate that whether the fleas collected were fed or unfed, as this can strongly influence the understanding if the isolates were presence in blood meal (from the host) vs. true infection in the fleas. If both fed and unfed fleas were included, it would be important to indicate how pools were composed and whether feeding status was recorded or if there are any specific controls being included.

Authors’ comment: None of the collected fleas showed visible signs of recent blood feeding at the time of sampling, and this information has been added to the Methods section (lines 89–91). Importantly, C. felis feeds frequently in small blood meals and rapidly defecates; therefore, the absence of visible blood does not necessarily indicate lack of recent feeding but rather reflects the feeding behavior of this species.

4- The authors employed 48 h incubation of flea pools at 4–8 °C prior to homogenization as a contamination control step. Although this may reduce contamination by other microbes, prolonged cold storage, however, also affect Blechomonas viability or it will allow the selection bias for more cold- or thermotolerant strains. Given that the later experiments were to assess the Blechomonas growth at different temperatures, this initial prolonged low-temperature incubation step may not fully represent the natural diversity of Blechomonas in the sample, and subsequent growth-temperature experiments could underestimate the species’ overall thermotolerance range. Please explained whether this protocol was adapted from a previous reference or if it has been validated to ensure parasite recovery and diversity were not compromised.

Authors’ comment: We thank the reviewer for this important methodological comment. The 48 h incubation at 4–8°C in antibiotic/antifungal-supplemented PBS was adopted as a contamination control step, given the well-documented richness and diversity of microbiota in C. felis. This rationale, along with the relevant references was added to the Methods section (lines 93–99).

Our previous experiments have shown that such incubation does not affect the viability of Leishmania. Moreover, this approach has been employed in published studies (e.g. Pereira et al., 2017, cited in the manuscript). Recommendations to maintain trypanosomatids at lowered temperatures (around 4°C or slightly higher) to avoid heat stress and/or suppress contaminant overgrowth are also included in the guidelines for “Field isolation and cultivation of trypanosomatids from insects” (cited in the manuscript) as well as in the WHO “Handbook on isolation, characterization and cryopreservation of Leishmania” (https://iris.who.int/handle/10665/60795). Taken together, these considerations indicate that the applied low-temperature incubation is a standard precautionary measure and is unlikely to have a significant impact on the intrinsic thermotolerance of tyrpanosomatids.

5- Please ensure that Genbank Accession Numbers are completed (Line 124: XXXXXX).

Authors’ comment: The actual GenBank accession numbers are now included in the manuscript.

6- Line 158-159: Additional clarification for “No growth occurred at 35 °C” would be useful. Please specify if this is referred to absence of proliferation or cell death.

Authors’ comment: We clarified this point in the Results section (line 180): “At 35°C, no proliferation was observed, and all cells died after three days”.

7- Line 163-164: The statement “the studied trypanosomatid may lack such mechanisms” appears to be speculative as there is no transcriptomic or genomic data were presented to assess gene presence or regulation. This inference should be removed or rephrased unless the authors provide molecular evidence to support this.

Authors’ comment: We rephrased this sentence as follows:

Whether these mechanisms in the studied trypanosomatid are absent or have limited efficiency is to be determined experimentally (lines 189–191).

8- The conclusion that “limited thermotolerance excludes colonization of warm-blooded vertebrates” should therefore be stated more cautiously. This is because the 48-h cold incubation step before culture could have introduced selection bias before the isolation toward more cold-tolerant or less heat-tolerant strains. Unless additional data are available to demonstrate that other Blechomonas isolates similarly fail to grow at 35 °C or that cold incubation does not affect viability or diversity, this interpretation should be revised or rephrased to reflect the limitations of the current dataset.

Authors’ comment: We respectfully maintain that the lack of parasite survival at 35°C provides strong biological evidence against adaptation to vertebrate body temperatures. However, to address the reviewer’s concern regarding potential selection bias during isolation, we rephrased the conclusion to avoid categorical wording. The revised text now refers only to the obtained isolate, rather than uncompromisingly “excluding” this possibility for any trypanosomatids inhabiting fleas (lines 294–297).

Reviewer 2

Reviewer #2: The manuscript provides valuable information on the association of Blechomonas with C. felis. It is written in a clear and fluid manner and contains all the essential elements expected for a study of this scope. However, several points remain overly general and would benefit from further clarification.

1- Line 43 – Such as?

Authors’ comment: We specified examples of epidemiologically relevant pathogens transmitted by fleas (e.g., Yersinia pestis, Rickettsia spp., Bartonella spp.).

2- Line 45 – Missing author and year.

Authors’ comment: We added the taxon authorship information along with the appropriate reference (now citing reference 3; line 48).

3- Lines 48–49 – According to whom?

Authors’ comment: We added the taxon authorship information and the appropriate reference (lines 51-52).

4- Line 52 – The phrasing here seems unnecessary. You may consider removing this sentence and introducing the findings in a different way.

5- Line 55 – Could you clarify what you meant by this?

Authors’ comment: We rephrased the paragraph to improve clarity and flow (lines 55–66).

6- Lines 78–86 – Is this protocol your own, or is it taken from the literature?

7- Lines 87–91 – Same question as above.

Authors’ comment: The protocols were adapted from previous publications. We clarified this in the Methods and added supporting references (lines 93–105).

8- Line 124 – One sequence appears to be missing. Does this mean there was a single sequence for each gene?

Authors’ comment: We added the missing GenBank accession number.

9- Line 126 – Does this indicate something specific?

Authors’ comment:

The sentence was revised: “The sequences generated in this study were combined with those available in the core nucleotide database of GenBank...” (lines147-148).

Reviewer 3

Reviewer #3:

1- Adequacy of the Abstract: The abstract is well-written and summarizes the study’s objectives, methodology, and main findings. However, it would benefit from explicitly acknowledging key limitations, such as the low number of fleas analyzed per host and the identification of only one positive pooled sample as Blechomonas campbelli. Additionally, the abstract should address potential issues related to culture contamination, especially given the use of NNN/LIT medium. Including information on the health status of the sampled dogs would also enhance the contextual interpretation of the findings.

Authors’ comment: We revised the Abstract to acknowledge key limitations, including the low number of fleas per pool and the recovery of a single positive culture. These points are now stated in the Findings and Main Conclusions sections. No issues related to culture contamination were detected, as no contaminating microorganisms were observed in any of the cultured samples. The single positive culture contained a trypanosomatid, whose presence is well supported by previous knowledge of flea-associated flagellates. The comment regarding the health status of dogs is addressed below.

2- Originality and Importance of the Contribution to the Field: The manuscript tackles a relevant topic within vector biology, parasite surveillance, and biodiversity studies. The detection of a trypanosomatid in Ctenocephalides felis may be a significant observation. However, the originality and overall impact of the contribution are limited by the identification of only a single positive culture and by the absence of host-related contextual data, such as the clinical or health status of the dogs and the measurement of the parasite load in the infected flea. Without this information, the relevance of the findings for animal or public health remains uncertain.

Authors’ comment: We acknowledge the limitations raised. However, this study was not designed as an epidemiological investigation. Its primary contribution consists in the isolation and biological characterization of B. campbelli, representing the first Neotropical isolate of this species and providing novel experimental data on its phenotype and phylogenetic placement. Importantly, in the context of this study, fleas - not dogs - are the hosts of the investigated trypanosomatids. We therefore consider the scope of the study and the conclusions drawn to be appropriate and believe that no changes are required.

3- Relevance and Adequacy of Methodology, Results, and Discussion:

a. The methodology is clearly described and technically appropriate. However, the number of samples should be included in the methodology section rather than the results section. Flea collection, morphological identification, cultivation using NNN/LIT medium, and DNA analyses are all standard procedures. Nevertheless, analyzing only four fleas per dog, grouped into pooled samples, significantly limits the analysis of parasite’ thermotolerance, phylogenetic analyses, and for estimating infection frequency.

Authors’ comment: We moved the sample size information (number of dogs and fleas) from Results to Methods (line 87).

b. Moreover, the manuscript does not address the potential for contamination during culture, despite the known susceptibility of NNN/LIT medium to bacterial or fungal overgrowth, even when antifungal agents and antibiotics are used. The lack of information on contamination control measures and culture monitoring weakens the interpretation of the single positive result. Molecular confirmation through the amplification of trypanosomatid DNA from flea samples and of cultures is strongly recommended.

Authors’ comment: Contamination control measures and culture monitoring are now explicitly described in the Methods. In addition, the isolate identity was molecularly confirmed using two independent markers (18S rRNA and gGAPDH genes). Direct molecular screening of flea pools was beyond the scope of this study and could not be reliably performed in parallel with culture-based detection due to the small body size of fleas.

c. Additionally, the health status of the dogs (e.g., asymptomatic, clinically ill, or showing signs compatible with trypanosomatid infection, as well as serological results) is not reported. This information is essential for contextualizing the epidemiological and biological relevance of the findings and should be included and discussed, particularly regarding seroprevalence and the molecular characterization of Leishmania in dogs in the study area.

Authors’ comment: Host clinical data were not collected, as sampling was designed exclusively for ectoparasites. This study did not aim to assess canine infection or public health relevance but was restricted to the characterization of flea-associated flagellates. Such data are therefore beyond the scope of the present work and are not required to support our conclusions.

4- References: The references are mostly appropriate and generally up to date. They adequately support the study’s background; however, the authors may want to consider adding references on contamination culture risks in culture-based trypanosomatid studies and on methods for molecular confirmation.

Authors’ comment: In response to a similar comment by another reviewer, we included two additional references supporting the culture procedure and contamination control measures. (see Methods).

5- Figures and Tables: The figures and tables are clear, well-organized, and properly labeled. They accurately reflect the sampling strategy and results. However, given the descriptive nature of the data, care should be taken to ensure that visual elements do not imply epidemiological conclusions.

Authors’ comment: We reviewed all figures and captions and confirm that they are strictly descriptive and do not imply epidemiological conclusions. No changes were required.

• peer review recommendation: accept

History

• Received
  14 Oct 2025
• Accepted
  11 May 2026

REVIEWERS COMMENTS

About the reviewer

REVIEWER #1

All suggestions have been accepted; therefore, I have no further comments, and the manuscript can be accepted in its current form.

REVIEWER #2

Below are my comments in English, structured according to the requested evaluation criteria.

a) Adequacy of the Abstract – Adequate but Needs Greater Precision

The abstract is generally clear and aligned with the proposed objectives. It adequately summarizes the isolation, in vitro characterization, thermotolerance observations, and phylogenetic positioning of Blechomonas campbelli from Ctenocephalides felis.

The abstract would benefit from:

• Clearer specification of the methodological approaches used for host identification and parasite confirmation.

• More precise wording regarding the strength of the phylogenetic support.

• Avoiding overstatement of novelty without robust comparative evidence.

b) Originality and Importance of the Contribution

The isolation of Blechomonas campbelli from Ctenocephalides felis in a Neotropical region is potentially relevant, as flea-associated monoxenic trypanosomatids remain underexplored. Expanding geographic records contributes to understanding parasite diversity, distribution, and host associations.

However, the originality claim is weakened by:

• Insufficient comparison with previously described isolates.

• Lack of detailed morphometric comparison with the original description by Votypka & Suková (2013).

• Limited discussion of prior regional reports of monoxenic trypanosomatids in fleas (e.g., reports from Minas Gerais).

The study has potential importance for the field, but the current manuscript does not convincingly demonstrate a significant conceptual or methodological advance. Its contribution appears incremental rather than transformative.

c) Methodology, Results, and Discussion – Major Weaknesses

1. Host Identification – Methodological Deficiency

The identification of Ctenocephalides felis is not sufficiently documented.

• No detailed morphological description is provided.

• No indication of chemical clearing procedures for proper visualization of cephalic and comb structures.

• No molecular confirmation (e.g., COI barcoding) is reported.

• No explicit differentiation from Ctenocephalides canis or other related taxa.

Accurate host identification is essential in studies of monoxenic trypanosomatids. The lack of transparency compromises reproducibility and weakens ecological interpretation.

2. Isolation Procedures and Parasite Detection – Insufficient Detail

The manuscript does not provide adequate methodological details regarding:

• Time and conditions between flea capture and processing.

• Dissection procedures.

• Culture medium composition.

• Temperature conditions.

• Subculturing frequency.

• Contamination control measures.

Without these details, reproducibility is compromised. Isolation protocols should be described comprehensively, particularly in studies claiming a new geographic record.

3. Anatomical Localization – Missing Critical Information

The anatomical localization of the parasite within the flea is not clearly described.

Given that Blechomonas species are commonly associated with the hindgut surface, and the authors hypothesize the presence of a “uromonad” stage involved in attachment, the study should:

• Specify where parasites were observed.

• Provide microscopy images supporting localization.

• Compare findings with previous descriptions.

Localization is biologically relevant and cannot be omitted.

4. Morphological Characterization and Taxonomic Context – Incomplete Comparative Analysis

Although morphometric measurements were performed, there is no structured comparison with the original description of Blechomonas campbelli.

This is particularly important because:

• Species in this genus exhibit overlapping morphological traits.

• Morphology alone is insufficient for reliable identification.

• Previous cases (e.g., reclassification of Leptomonas pulexsimulantis to Blechomonas) demonstrate the limitations of morphology-based taxonomy.

A side-by-side morphometric comparison table would significantly strengthen the manuscript.

5. Phylogenetic Analyses – Limited Taxon Sampling

The phylogenetic analysis would benefit from:

• Broader inclusion of representative isolates from multiple trypanosomatid genera.

Restricted sampling can artificially inflate bootstrap or posterior probability values due to reduced topological alternatives. Strong support values under limited sampling do not necessarily reflect true phylogenetic stability.Broader sampling would improve clade stability and strengthen species-level assignment.

6. Functional Evidence for Thermal Adaptation – Unsupported Claims

The manuscript discusses thermotolerance but does not provide mechanistic or functional validation.

If heat tolerance is proposed as a biological trait, it should be supported by:

• Growth curves at multiple temperatures.

• Statistical comparison of proliferation rates.

• Gene expression assays (e.g., RT-qPCR) evaluating HSP70, HSP90, desaturases, and flagellar proteins.

• Normalization against housekeeping genes such as GAPDH.

The mention of multiple copies of HSP-related genes in the genome is insufficient to infer functional adaptation without expression data.

Currently, conclusions regarding thermal adaptation are speculative.

REVIEWER #3

The authors have adequately addressed all the concerns raised during the first round of review. The revisions have improved the clarity and quality of the manuscript. I therefore support the acceptance of the manuscript.

AUTHORS' RESPONSE TO THE REVIEWER

Manuscript ID: MIOC-2025-0286

Blechomonas campbelli from Ctenocephalides felis: in vitro development, thermotolerance, and phylogenetic insights

Dear Dr. Dias-Lopes,

We thank you and the reviewers for the careful evaluation of our revised manuscript.

Following the second round of review, two reviewers (Reviewers 1 and 3) recommended acceptance without further comments, while Reviewer 2 provided several minor suggestions. All comments have been carefully addressed point by point, with revisions made to improve clarity, methodological transparency, and interpretation. Where suggestions extended beyond the scope of the study, we clarified the limitations and adjusted the text accordingly.

Below, we provide a detailed response to all comments, with references to the tracked-changes version of the manuscript.

We hope that the revised version meets the Journal’s standards and can be accepted for publication.

Sincerely,

Felipe Dutra Rêgo

On behalf of all authors

Instituto René Rachou - Oswaldo Cruz Foundation

Reviewer 2

Reviewer comments to the authors

Reviewer #2:

a) Abstract - The abstract would benefit from:

• Clearer specification of the methodological approaches used for host identification and parasite confirmation.

• More precise wording regarding the strength of the phylogenetic support.

• Avoiding overstatement of novelty without robust comparative evidence.

Authors’ comment: The abstract has been revised to improve precision and to better reflect the methodological and interpretative scope of the study. Specifically, the following adjustments were made: (1) a brief clarification on host identification was added (line 23). Given the word limit constraints, detailed diagnostic criteria were not included in the abstract but are provided in the Methods section; (2) the wording describing phylogenetic inference was adjusted to avoid overstatement, replacing “identified as” with the more conservative “placed within” which more accurately reflects the level of support provided by 18S rRNA and gGAPDH sequence data (lines 30-31), and (3) minor modifications were introduced to moderate the tone of the conclusions and avoid overstating novelty, while preserving the main findings and their relevance (lines 33-37).

b) Originality and Importance of the Contribution - The originality claim is weakened by:

• Insufficient comparison with previously described isolates.

• Lack of detailed morphometric comparison with the original description by Votypka & Suková (2013).

• Limited discussion of prior regional reports of monoxenic trypanosomatids in fleas (e.g., reports from Minas Gerais).

Authors’ comment: The manuscript has been revised to strengthen the contextualization of the findings and to highlight the contribution of the study to the current knowledge on trypanosomatid flagellates in fleas. Specifically, the following adjustments were made: (1) We performed a morphological comparison of our strain with the type strain described by of B. campbelli by Votýpka et al. (2013), but noted that the extremely concise nature of the original description renders such a comparison unreliable, whereas available molecular sequences clearly indicate that both belong to the same species (see Results and Discussion, lines 273-282). (2) Prior reports of trypanosomatids in fleas from Brazil, including studies conducted in Minas Gerais, have been mentioned (lines 333-338). We highlight that morphometric data present in one of these studies overlap with those estimated by us, suggesting similarity of the isolated parasites, although molecular evidence for this is lacking. (3) The overall wording of the manuscript has been adjusted to more accurately reflect the nature of the contribution.

c) Methodology, Results, and Discussion:

1. Host Identification:

The identification of Ctenocephalides felis is not sufficiently documented.

• No detailed morphological description is provided.

• No indication of chemical clearing procedures for proper visualization of cephalic and comb structures.

• No molecular confirmation (e.g., COI barcoding) is reported.

• No explicit differentiation from Ctenocephalides canis or other related taxa.

Authors’ comment: The manuscript has been revised to improve the documentation of host identification: (1) a representative figure has been added (Figure 1) showing diagnostic morphological characters used for flea identification, now also listed in the text; and (2) it has been clarified in Material and Methods that identification was performed on fresh specimens under stereomicroscopy using standard taxonomic keys (lines 99-102). Although chemical clearing and slide mounting can facilitate detailed morphological examination in arthropods, these procedures are not required for species-level identification in Ctenocephalides, where key diagnostic characters are readily visible in untreated specimens.

Molecular confirmation (COI barcoding) was not performed, as morphological identification remains the standard approach in studies involving Ctenocephalides spp., particularly in regions where closely related species can be reliably distinguished using well-established morphological traits. Moreover, specimen processing for parasite isolation (maceration and cultivation) was not compatible with clearing or molecular barcoding.

Species identification was conducted by Dr. Daniel Avelar, co-author of this study and curator of the Siphonaptera collection originally established by Prof. Pedro Marcos Linardi, ensuring taxonomic accuracy and consistency with established expertise in the group.

2. Isolation Procedures and Parasite Detection - The manuscript does not provide adequate methodological details regarding:

• Time and conditions between flea capture and processing.

• Dissection procedures.

• Culture medium composition.

• Temperature conditions.

• Subculturing frequency.

• Contamination control measures.

Authors’ comment: The manuscript has been revised to provide additional methodological details regarding parasite isolation and cultivation. Specifically, information has been added on (1) conditions between flea collection and processing, including transport under controlled temperature and humidity and same-day processing (lines 94-98); (2) clarification of the culture medium composition, explicitly defining the biphasic NNN/LIT medium (lines 107-109); (3) subculturing frequency (lines 110-111).

Contamination control measures, including antibiotic and antifungal supplementation, incubation conditions, and pre-incubation steps to reduce microbial load were already described in the previous version of the manuscript.

3. Anatomical Localization – The anatomical localization of the parasite within the flea is not clearly described. Given that Blechomonas species are commonly associated with the hindgut surface, and the authors hypothesize the presence of a “ uromonad ” stage involved in attachment, the study should:

• Specify where parasites were observed.

• Provide microscopy images supporting localization.

• Compare findings with previous descriptions.

Authors’ comment: The anatomical localization of the parasite within the flea was not assessed in the present study, as fleas were processed by whole-body maceration for culture-based isolation, and subsequent characterization was performed exclusively in culture. We acknowledge that information on parasite localization within the host would be valuable, especially given that it not available in the original description. However, addressing this question was beyond the scope of the present work.

The identification of “uromonads” as an attachment stage is based on observations of their behaviour in culture and comparisons with known attachment stages (e.g., haptomonads) described in other trypanosomatids, as well as previously reported hindgut-associated forms of Blechomonas spp. This interpretation has been revised in the manuscript to avoid overstatement.

We have also explicitly acknowledged the lack of anatomical localization as a limitation and clarified that future studies using dissected specimens and in situ approaches will be necessary to confirm the localization and attachment mechanisms of this species within the flea host (lines 258-263).

4. Morphological Characterization and Taxonomic Context – Although morphometric measurements were performed, there is no structured comparison with the original description of Blechomonas campbelli.

This is particularly important because:

• Species in this genus exhibit overlapping morphological traits.

• Morphology alone is insufficient for reliable identification.

• Previous cases (e.g., reclassification of Leptomonas pulexsimulantis to Blechomonas ) demonstrate the limitations of morphology-based taxonomy.

Authors’ comment:

The aim of this study is not taxonomic revision or species delimitation, but the characterization of a cultured isolate and its biology. We agree that morphology alone (particularly at the light microscopy level) is insufficient for reliable species identification in trypanosomatids and that morphological traits may overlap among species. Species identification was therefore performed based on sequence data, while the morphological analysis was intended to expand the currently limited information available for Blechomonas campbelli, rather than to serve as a basis for taxonomic discrimination. The original description of this species contains only minimal morphological data, which limits the scope for a structured comparative analysis (see also response to comment 1b). In our study, the morphological analysis primarily aims to provide insights into the parasite’s life cycle, with appropriate acknowledgment of its limitations (see response to comment 3).

The manuscript has been revised to include a comparison of our isolate with the original description of B. campbelli and an earlier paper providing morphometric data on trypanosomatids from cat fleas (lines 283-294).

5. Phylogenetic Analyses - The phylogenetic analysis would benefit from:

• Broader inclusion of representative isolates from multiple trypanosomatid genera.

Restricted sampling can artificially inflate bootstrap or posterior probability values due to reduced topological alternatives. Strong support values under limited sampling do not necessarily reflect true phylogenetic stability. Broader sampling would improve clade stability and strengthen species-level assignment.

Authors’ comment: Our phylogenetic analysis was specifically designed to resolve relationships within a single genus (Blechomonas) rather than across multiple trypanosomatid genera, which has been already efficiently achieved by a phylogenomic analysis (Kostygov et al., 2024). To this end, we included all available sequences representing multiple species and strains within that genus, ensuring dense and representative ingroup sampling. Because this genus does not have a well-established close sister lineage at the genus or subfamily level and instead branches as sister to a large clade comprising most other trypanosomatid genera, we selected a limited set of representatives from this clade, along with a more distant taxon (Trypanosoma), for outgroup rooting. Expanding the sampling across this diverse clade would not increase resolution within the focal genus but would substantially increase dataset complexity and overall tree size without clear benefit for the question addressed here.

In this context, support values are driven primarily by ingroup sampling, which is comprehensive in our dataset, rather than by the breadth of representation of more distantly related taxa.

6. Functional Evidence for Thermal Adaptation – The manuscript discusses thermotolerance but does not provide mechanistic or functional validation.

If heat tolerance is proposed as a biological trait, it should be supported by:

• Growth curves at multiple temperatures.

• Statistical comparison of proliferation rates.

• Gene expression assays (e.g., RT-qPCR) evaluating HSP70, HSP90, desaturases, and flagellar proteins.

• Normalization against housekeeping genes such as GAPDH.

Authors’ comment: Our study does not aim to investigate the mechanisms of thermotolerance, but rather to assess temperature-dependent growth as one of the traits characterizing the strain. In this context, our data demonstrate a lack of thermotolerance, as evidenced by the absence of proliferation at 35 °C and reduced growth at 30 °C (Figure 2 and Supplementary Data - Sheet1). The additional experiments proposed (e.g., gene expression analyses of heat shock proteins) would address the molecular basis of thermotolerance and represent a distinct line of investigation beyond the scope of this work. We have clarified this point in the revised manuscript (lines 200-202).

The raw statistical data are provided in Supplementary Material - Sheet1.

• peer review recommendation: accept

History

• Received
  14 Oct 2025
• Accepted
  11 May 2026

REVIEWERS COMMENTS

About the reviewer

REVIEWER #1

No comments.

REVIEWER #2

No comments.

REVIEWER #3

No comments.

• peer review recommendation: accept

History

• Received
  14 Oct 2025
• Accepted
  11 May 2026

Publication Dates

• Publication in this collection
  24 Aug 2026
• Date of issue
  2026

History

• Received
  14 Oct 2025
• Accepted
  11 May 2026