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Article

Differentiation of Synanthropic Fleas from Andalusia (Spain) through Geometric Morphometrics Analysis

by
Angela M. García-Sánchez
,
Ignacio Trujillo
,
Antonio Zurita
* and
Cristina Cutillas
Department of Microbiology and Parasitology, Faculty of Pharmacy, University of Seville, Profesor García González 2, 41012 Sevilla, Spain
*
Author to whom correspondence should be addressed.
Animals 2024, 14(11), 1582; https://doi.org/10.3390/ani14111582
Submission received: 3 May 2024 / Revised: 20 May 2024 / Accepted: 24 May 2024 / Published: 27 May 2024
(This article belongs to the Special Issue Parasites and Parasitic Diseases in Small Animals)
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Abstract

:

Simple Summary

Fleas are blood-sucking insects that are not only a nuisance but can also act as vectors for various diseases in animals and humans, including dangerous ailments such as the bubonic plague. Identifying and classifying these insects accurately is crucial for understanding how they spread and how to control them. Geometric morphometrics, a cutting-edge technique, is proving to be an invaluable tool in this regard, alongside traditional methods and molecular biology. In the present study conducted in Andalusia, Spain, this technique successfully differentiated between three populations of fleas, providing insights into their distribution, size, and characteristics. Image processing software was employed to obtain measurements, such as perimeters and areas, of the fleas under study. These findings underscore the importance of geometric morphometrics in studying and managing arthropod populations, particularly in cases where other methods fall short or are not available.

Abstract

Fleas (Siphonaptera) are ectoparasitic hematophagous insects responsible for causing bites and itchy skin conditions in both humans and animals. Furthermore, they can act as vectors of different pathogens of a wide variety of diseases worldwide, including bartonellosis, rickettsiosis, and bubonic plague. Accurate identification of fleas is necessary for the study of their epidemiology, prevention, and control. In addition to traditional morphological classification approaches and molecular biology techniques, geometric morphometrics is increasingly proving to be a useful complementary tool for discriminating between Siphonaptera taxa. With the objective of determining the capacity of this technique to identify and differentiate synanthropic fleas, a principal component analysis was carried out on populations of Ctenocephalides felis, Pulex irritans, and Archaeopsylla erinacei collected in distinct regions of Andalusia (Spain). The analysis carried out on 81 male and female specimens revealed factorial maps that allowed the differentiation of the populations under study, with only partial overlaps that did not prevent their correct identification. Global size differences were also detected, with a slightly larger size in P. irritans males and a bigger size in A. erinacei females. Therefore, the present study emphasizes the role of geometric morphometrics as a useful complementary technique in taxonomic studies of arthropods, especially in the case of flea specimens lacking representative morphological features.

1. Introduction

Fleas (Siphonaptera) comprise a highly specialized order of holometabolous ectoparasitic insects with a cosmopolitan distribution and about 2700 species described so far [1]. In addition to being able to provoke bites and pruritic welts on the skin, these arthropods are also known to be vectors of different pathogens, responsible for causing a wide variety of diseases worldwide, including bartonellosis, rickettsiosis, and bubonic plague [2,3,4,5,6,7,8,9]. This is due to the nature of some flea species, which present a low host specificity that facilitates the exchanging of microorganisms, posing a potential threat to the health of both humans and animals [10,11,12].
The prevention and control of fleas require a large investment of money per year, which represents a significant economic burden [13]. It is crucial to enhance our knowledge about the taxonomy of fleas to develop effective strategies to reduce flea infestations and their negative impact on our environment.
Recently, in addition to the traditional morphological identification and molecular biology approaches, geometric morphometrics has proven to be a useful complementary technique for discriminating taxa across different groups [14,15,16]. One of the main features of geometric morphometrics is that it is especially helpful in cases of taxa that present morphological ambiguity [15], a situation relatively common in fleas [17,18,19]. This scenario invited the exploration of the affordable criterion offered by geometric morphometrics in systematic studies on flea genera, with promising results in Ctenocephalides Stiles & Collins, 1930 [20,21], Ctenophthalmus Kolenati, 1856 [22], Pulex Linnaeus, 1758 [23], and Stenoponia Jordan & Rothschild, 1911 [24].
In Europe, there is evidence of an escalating frequency of vector-borne diseases and heightened pathogen circulation, primarily influenced by human-related factors [25]. One region remarkably affected by arthropod-borne diseases is Andalusia, situated in the southern of Spain, where the West Nile Virus circulation is more widespread than initially considered [26], and outbreaks have been reported in recent years [27].
Furthermore, the incidence of murine typhus, a zoonosis caused by Rickettsia typhi da Rocha Lima, 1916 transmitted to humans by fleas, seems to be increasing slowly in Andalusia [28], an aspect that reveals the ability of some flea species present in the region to transmit pathogenic bacteria. Hence, it is essential to resort to techniques that allow us to safely discern between taxa.
The cat flea, Ctenocephalides felis Bouché, 1835, succeeded in its expansion as a global parasite, and it is one of the most common flea species identified in domestic dogs and cats worldwide [11,19]. In Southwestern Europe, C. felis is the dominant species, although, in Eastern Europe, the infestation by Ctenocephalides canis Curtis, 1826 and Pulex irritans Linnaeus, 1758 also occurs [19,29,30,31].
On the other hand, hedgehogs inhabit rural, urban, and suburban environments, and they are frequently parasitized by blood-sucking arthropods, such as hard ticks and fleas, including the hedgehog flea, Archaeopsylla erinacei Bouché, 1835, and other flea species such as C. felis, C. canis, and Nosopsyllus fasciatus Bosc, 1800 [32,33,34,35]. Since they usually cohabit with pets and humans, they can potentially act as reservoirs of pathogen microorganisms responsible for zoonoses [12].
The main objective of the present study was to determine the capacity of geometric morphometric analysis to identify and discriminate fleas from populations of C. felis, P. irritans, and A. erinacei collected in Andalusia, in order to strengthen its role as a useful complementary technique in arthropod taxonomical studies.

2. Materials and Methods

2.1. Collection of Samples

Over a period of 19 months, we collected flea samples from dogs (Canis lupus familiaris Linnaeus, 1758) and one hedgehog (Erinaceus europaeus Linnaeus, 1758) that coexisted with other dogs.
To gather flea samples from the hosts, we reached out to some veterinary clinics, veterinary hospitals, pet shelters, and some pet owners. In total, we contacted 145 veterinary clinics and 30 pet shelters and kennels. Among these, 18 centers agreed to participate in the sample collection (see Acknowledgements). All participants volunteered for this sampling process. Only animals parasitized by fleas were sampled. Veterinary practitioners performed an initial inspection of pets brought to their facilities. Each pet was checked for fleas and examined by a veterinarian who recorded clinical signs related to flea infestation. Adult flea counts were conducted according to the World Association for the Advancement of Veterinary Parasitology guidelines [36]. In brief, the animals were combed over their entire bodies with a fine-toothed comb for 5–10 min.
All captured fleas from each infested host were transferred to a plastic 1.5 mL tube containing 96% ethanol for subsequent identification and morphometrics analyses.

2.2. Morphological Identification and Metric Data Processing

For morphological analysis, all specimens were initially examined under an optical microscope for specific classification. Following this, the specimens were cleared using 10% KOH, prepared, and mounted on glass slides following conventional procedures with the EUKITT mounting medium (O. Kindler GmbH & Co., Freiburg, Germany) [37]. The cleared and mounted specimens were examined again for a more detailed morphological analysis using a BX61 microscope (Olympus, Tokyo, Japan) and submitted to image capture processes with the imaging software cellSens Standard version 4.2 (Olympus, Tokyo, Japan). The diagnostic morphological characters of all samples were analyzed by comparison with figures, keys, and descriptions reported previously [38,39,40,41,42]. The measurement images of each flea were made using the image analysis software Image-PRO v11 (Media Cybernetics, Rockville, MD, USA). A total of 28 different parameters were measured for males and 36 for females (Table 1 and Table 2).
Descriptive univariate statistics based on arithmetic mean, standard deviation, and coefficient of variation for all parameters were determined for male and female populations. The data were subjected to one-way ANOVA (analysis of variance) for statistical analysis of the parameters. The results were statistically significant when p < 0.05. Statistical analysis was performed using Microsoft Excel for Microsoft 365 MSO (v2402). In addition, biometric characters of fleas were compared between different species and the most significant parameters were assayed for a morphometrics study.
Morphological variation was quantified using geometric morphometrics [43], a technique that provides an estimate of size integrating different growth axes into a single variable known as “centroid size” [44]. The estimate of size was represented by a single variable that reflected variation in multiple directions, as many as there were landmarks under study, and shape was defined as their relative positions after correction for size, position, and orientation. With these informative data, and the corresponding software freely available to conduct complex analyses, significant biological and epidemiological features can be quantified more accurately [45].
Multivariate analyses were applied to assess phenotypic variations among the samples, using size-free canonical discriminant analysis on the covariance of log-transformed measurements. These analyses are applied to exclude the effect of within-group ontogenetic variations by reducing the effect of each character on the first pooled within-group principal component (a multivariate size estimator) [46]. principal component analysis (PCA) was used to summarize most of the variations in a multivariate dataset in a few dimensions [47]. Morphometric data were explored using multivariate analysis in three parameters in males (TW, HW, and AW) (Table 1) and females (Global Circularity, BULGAP, and HL) in females (Table 2) using BAC v.2 software [21,48].

3. Results

A total of 81 fleas (34 males and 47 females) were collected from different regions of Andalusia and classified as follows: 39 as C. felis (18 males and 21 females), 19 as P. irritans (6 males and 13 females), and 23 as A. erinacei (10 males and 13 females) (Table 3). All C. felis specimens and three P. irritans (the only male from Huelva and two female fleas from Seville) were collected from dogs (Canis lupus familiaris), whereas A. erinacei were collected from a hedgehog host. The rest of the P. irritans fleas were collected off-host from a neglected horse stable (Table 3).
To carry out the classification of the samples, we considered descriptions used traditionally to discern between these species and, additionally, remarkable morphological features based on the measurements performed. Statistical tests showed several significant measurements for subsequent morphometric analyses. Therefore, the following parameters were used: total width (TW), total width of the head (HW), and apex width (AW) in males (Table 1) and total length of the head (HL), perimeter of the bulga (BULGAP), and Global Circularity in females (Table 2). This perimeter is the length of the bulga’s boundary, whereas the circularity is calculated as the ratio of the area of an object against a circle whose diameter is equal to the object’s maximum feret. The influence of size was analyzed using PCA in C. felis, P. irritans, and A. erinacei, involving the regression of each character separately on the within-group first principal component (PC1). The resulting factor maps for male and female populations are shown in Figure 1 and Figure 2, respectively.
Male variables significantly correlated with PC1, contributing 71% to the overall variation. The male factor maps showed global size differences in the flea populations, with a slightly larger size in P. irritans males (Figure 1). The three male communities are well grouped in the factor map, with a lack of noteworthy overlapping areas between them. Only C. felis and P. irritans showed a partial overlap but with no inconvenience in their identification.
On the other hand, female variables significantly correlated with PC1, contributing 90% to the overall variation. The resulting factor maps (Figure 2) clearly illustrate global size differences in the populations analyzed, including a bigger size in A. erinacei. As in the previous factor maps, there is a lack of notable overlapping areas between the female populations. As in the male factor maps, C. felis and P. irritans showed a partial overlap that did not prevent their identification.

4. Discussion

The accurate classification of fleas requires careful morphological examination or molecular confirmation, and therefore, the possibility that prior studies may have inadvertently misidentified fleas cannot be discarded [19,49]. In fact, authors like Ménier and Beaucournu reported numerous misidentifications in specimens of the genus Ctenocephalides [50]. The study of flea epidemiology, control, and prevention requires the accurate identification of species and subspecies.
In general, the classification of genera and species of fleas is based on external morphological characters. The presence or absence of combs and eyes, along with the length of the head, are typically significant features in morphological identification [51]. However, the size itself could never represent a way to reliably recognize the sex or the species of a flea specimen [52], and some flea species do not have easily identifiable morphological characters. For instance, A. erinacei and P. irritans do not possess pronotal or genal combs, so their classification process can be more complex in case the required specialized skills in flea identification are lacking. It is necessary to pay great attention to detail to recognize developmental stages and adequate sex identification [52]. Additionally, C. felis is a good example of a flea species known for its morphological ambiguity and the underlying issues in the study of their global populations [19]. In terms of molecular biology techniques, the notable lack of large-scale phylogenetic data for flea taxa causes some genera like Ctenocephalides to not have a defined genetic identity [17,19,40], especially if we consider subspecies [19].
Furthermore, it is still surprising that, despite the considerable veterinary and public health significance of dog fleas, studies investigating the diversity of these ectoparasite species on pets and the occurrence of flea-borne pathogens are scarce in certain regions [20].
Given the difficulties associated with flea morphological identification, the limited genetic information available, and the insufficient knowledge of the common pathogens of each flea species, the need to resort to complementary diagnosis techniques arises.
Geometric morphometrics analysis is one of these novel approaches applied to parasitological diagnosis, usually employed in arthropod identification [43]. The technique is based on the utilization of computer software for data processing and interpretation, with the advantage being that costly reagents and equipment are not required. Its affordability and the simplicity of data collection make it especially useful in low-resource settings [52,53].
To the best of our knowledge, the present work is the first in which flea measurements have been obtained using the imaging software Image-PRO. This program has been used before to study arthropods and other parasites [54,55,56,57]. It allowed us to incorporate into the analysis accurate measures such as areas, perimeters and circularities of the flea specimens for the first time. Global Circularity and BULGAP were revealed as useful features that contribute to the identification of flea species via geometric morphometrics.
The distribution of the three analyzed flea species showed a comparable pattern in both factor maps. A. erinacei appeared distant from C. felis and P. irritans, showing an appreciably larger size in the case of females. C. felis and P. irritans presented a small overlapping area, which did not prevent their individual identification. In both cases, P. irritans has always appeared larger than C. felis.
Although A. erinacei and C. felis are part of the Pulicinae family, the factor maps illustrated that fleas at the same taxonomic level are not necessarily closer at the morphological level since A. erinacei appears further away from both C. felis and P. irritans.
The selection of representative measurements for the morphometrics analyses was in accordance with previously published works. Total width (TW), head width (HW), and apex width (AW) are consolidated as useful parameters that define the morphological identity in males [21,50,58], as well as the total length of the head (HL) and the perimeter of the bulga (BULGAP) for females [21]. Due to the lack of genal ctenidium in P. irritans and A. erinacei, the difference in length between first and second spines (DEG parameter [21]) could not be considered in the present analysis, whereas the inclusion of the degree of elongation of the apical part (hilla) in females offered similar results but with a bit more overlap between P. irritans and C. felis. This is why the parameter APEHILL [21] was substituted by BULGAP, which permits the best differentiation between species.
The three analyzed flea species are among the most frequent in our environment, and, as a result, there is a notable risk of encountering them, with consequently associated parasitism suffered by humans and animals. After applying geometric morphometrics to differentiate flea communities of the same genera [21,22,23,24], the present work represents a step further, since this technique allowed us to identify different flea genera.

5. Conclusions

Accurate identification of fleas is necessary for studying the epidemiology, prevention, and control of these arthropods. In situations of uncertainty, alternative approaches are required to ensure correct classification. Geometric morphometrics is increasingly recognized as a reliable complementary technique for identifying flea species, particularly valuable in environments with limited resources.
In the present work, we were able to discern between the flea species A. erinacei, P. irritans, and C. felis using principal component analysis of males and females. Differences in overall size were also detected: A. erinacei presented the largest size in females, whereas P. irritans was slightly larger in males. Therefore, morphometrics is a relevant technique with great potential for application in the field of fleas, considering the existence of species that have traditionally posed challenges in their identification.

Author Contributions

Conceptualization, C.C. and A.Z.; methodology, A.M.G.-S.; software, A.M.G.-S. and I.T.; validation, A.Z. and C.C.; formal analysis, C.C.; investigation, A.M.G.-S. and I.T.; resources, C.C.; data curation, A.M.G.-S.; writing—original draft preparation, A.M.G.-S.; writing—review and editing, A.Z. and C.C.; visualization, C.C.; supervision, A.Z. and C.C.; project administration, C.C.; funding acquisition, C.C. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by a project from the Junta de Andalucía, European Regional Development Fund (FEDER) (Ref. P20_00544).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The data presented in this study are available in the article.

Acknowledgments

We would like to express our gratitude to these participating veterinary clinics, pet shelters, and kennels for their continued support and timely submission of samples, which greatly contributed to the success of our study. Veterinary clinics: El acuario, Ayamonte, Huelva; Animal-Vetx El Saladillo, Huelva; Cuenca minera, Minas de Río Tinto, Huelva; Consultorio veterinario Galaroza, Galaroza, Huelva; Roncal, Pilas, Sevilla; Mascotasalud, Tomares, Sevilla; Ronda de capuchinos, Sevilla; Vilovet, Cazalla de la Sierra, Sevilla; Taxana, Los Rosales, Sevilla; Sobrino E Illescas, Ecija, Sevilla; SyR, Estepa, Sevilla; Alfavet, Fuente Palmeras, Córdoba; Mascovet, Posadas, Córdoba; El Pinar, Puerto de Santa María, Cádiz; El Parque, San Fernando, Cádiz. Pet shelters and kennels: Mascotomares, Tomares, Sevilla; Arca, Sevilla; Sociedad protectora de animales y plantas, Sevilla.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Hastriter, M.W.; Bossard, R.L.; Lewis, R.E. Flea (Siphonaptera) World Species List (Spreadsheet). 2018. Available online: https://esanetworks.org/group/fleanews/page/flea-species-of-theworld-spreadsheetupdated-3-february-2018 (accessed on 3 December 2023).
  2. Fleta Zaragozano, J. Rickettsiosis transmitidas por piojos, pulgas y ácaros. Med. Integr. 2002, 39, 147–152. [Google Scholar]
  3. Parola, P.; Davoust, B.; Raoult, D. Tick- and flea-borne rickettsial emerging zoonoses. Vet. Res. 2005, 36, 469–492. [Google Scholar] [CrossRef] [PubMed]
  4. De Sousa, R.; Fournier, P.E.; Santos-Silva, M.; Amaro, F.; Bacellar, F.; Raoult, D. Molecular detection of Rickettsia felis, Rickettsia typhi and two genotypes closely related to Bartonella elizabethae. Am. J. Trop. Med. Hyg. 2006, 75, 727–731. [Google Scholar] [CrossRef] [PubMed]
  5. Beliavskaia, A.; Tan, K.K.; Sinha, A.; Husin, N.A.; Lim, F.S.; Loong, S.K.; Bell-Sakyi, L.; Carlow, C.K.S.; Abubakar, S.; Darby, A.C.; et al. Metagenomics of culture isolates and insect tissue illuminate the evolution of Wolbachia, Rickettsia and Bartonella symbionts in Ctenocephalides spp. Fleas. Microb. Genom. 2023, 9, 001045. [Google Scholar] [CrossRef] [PubMed]
  6. Boyer, S.; Gillespie, T.R.; Miarinjara, A. Xenopsylla cheopis (rat flea). Trends Parasitol. 2022, 38, 607–608. [Google Scholar] [CrossRef] [PubMed]
  7. Millán, J.; Sepúlveda-García, P.; Di Cataldo, S.; Canales, N.; Sallaberry-Pincheira, N.; Painean, J.; Cevidanes, A.; Müller, A. Molecular identification of Bartonella spp. and Rickettsia felis in fox fleas; Chile. Comp. Immunol. Microb. 2023, 96, 101983. [Google Scholar] [CrossRef] [PubMed]
  8. Sepúlveda-García, P.; Alabi, A.; Álvarez, K.; Rojas, L.; Mella, A.; Gonçalves, L.R.; André, M.R.; Machado, R.; Müller, A.; Monti, G. Bartonella spp. in households with cats: Risk factors for infection in cats and human exposure. One Health 2023, 16, 100545. [Google Scholar] [CrossRef]
  9. Yomogida, K.; Kjemtrup, A.; Martínez-López, B.; Ibrahim, M.; Contreras, Z.; Ngo, V.; Halai, U.A.; Balter, S.; Feaster, M.; Zahn, M.; et al. Surveillance of Flea-Borne Typhus in California; 2011–2019. Am. J. Trop. Med. Hyg. 2024, 110, 142–149. [Google Scholar] [CrossRef] [PubMed]
  10. Kernif, T.; Socolovschi, C.; Wells, K.; Lakim, M.B.; Inthalad, S.; Slesak, G.; Boudebouch, N.; Beaucournu, J.C.; Newton, P.N.; Raoult, D.; et al. Bartonella and Rickettsia in arthropods from the Lao PDR and from Borneo; Malaysia. Comp. Immunol. Microbiol. Inf. Dis. 2012, 35, 51–57. [Google Scholar] [CrossRef]
  11. Clark, N.J.; Seddon, J.M.; Šlapeta, J.; Wells, K. Parasite spread at the domestic animal-wildlife interface: Anthropogenic habitat use, phylogeny and body mass drive risk of cat and dog flea (Ctenocephalides spp.) infestation in wild mammals. Parasit. Vectors. 2018, 11, 8. [Google Scholar] [CrossRef]
  12. Benkacimi, L.; Diarra, A.Z.; Bompar, J.M.; Bérenger, J.M.; Parola, P. Microorganisms associated with hedgehog arthropods. Parasit. Vectors 2023, 16, 211. [Google Scholar] [CrossRef]
  13. Blagburn, B.L.; Dryden, M.W. Biology; treatment; and control of flea and tick infestations. Vet. Clin. N. Am. Small Anim. 2009, 39, 1173–1200. [Google Scholar] [CrossRef] [PubMed]
  14. Villemant, C.; Simbolotti, G.; Kenis, M. Discrimination of Eubazus (Hymenoptera; Braconidae) sibling species using geometric morphometrics analysis of wing venation. Syst. Biol. 2007, 32, 625–634. [Google Scholar] [CrossRef]
  15. Ruane, S. Using geometric morphometrics for integrative taxonomy: An examination of head shapes of milksnakes (genus Lampropeltis). Zool. J. Linn. Soc. 2015, 174, 394–413. [Google Scholar] [CrossRef]
  16. Li, S.; Ricchiardi, E.; Bai, M.; Yang, X. A taxonomy review of Oreoderus Burmeister, 1842 from China with a geometric morphometric evaluation (Coleoptera; Scarabaeidae; Valgini). ZooKeys 2016, 552, 67–89. [Google Scholar]
  17. Beaucournu, J.C.; Ménier, K. Le genre Ctenocephalides Stiles et Collins; 1930 (Siphonaptera; Pulicidae). Parasite 1998, 5, 3–16. [Google Scholar] [CrossRef]
  18. Zurita, A.; Callejón, R.; de Rojas, M.; Cutillas, C. Morphological and molecular study of the genus Nosopsyllus (Siphonaptera: Ceratophyllidae). Nosopsyllus barbarus (Jordan & Rothschild 1912) as a junior synonym of Nosopsyllus fasciatus (Bosc d’Antic; 1800). Insect Syst. Evol. 2018, 49, 81–101. [Google Scholar]
  19. Lawrence, A.L.; Webb, C.E.; Clark, N.J.; Halajian, A.; Mihalca, A.D.; Miret, J.; D’Amico, G.; Brown, G.; Kumsa, B.; Modrý, D.; et al. Out-of-Africa, human-mediated dispersal of the common cat flea, Ctenocephalides felis: The hitchhiker’s guide to world domination. Int. J. Parasitol. 2019, 49, 321–336. [Google Scholar] [CrossRef]
  20. Deak, G.; Safarov, A.; Xie, X.C.; Wang, R.; Mihalca, A.D.; Šlapeta, J. Fleas from the Silk Road in Central Asia: Identifcation of Ctenocephalides canis and Ctenocephalides orientis on owned dogs in Uzbekistan using molecular identification and geometric morphometrics. Parasite. Vectors 2022, 15, 345. [Google Scholar] [CrossRef]
  21. García-Sánchez, A.M.; 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, 707. [Google Scholar] [CrossRef]
  22. Zurita, A.; García-Sánchez, Á.M.; Cutillas, C. Ctenophthalmus baeticus boisseauorum (Beaucournu; 1968) and Ctenophthalmus apertus allani (Smit; 1955) (Siphonaptera: Ctenophthalmidae) as synonymous taxa: Morphometric; phylogenetic; and molecular characterization. Bull. Entomol. Res. 2020, 110, 663–676. [Google Scholar] [CrossRef]
  23. Zurita, A.; Callejón, R.; García-Sánchez, Á.M.; Urdapilleta, M.; Lareschi, M.; Cutillas, C. Origin, evolution, phylogeny and taxonomy of Pulex irritans. Med. Vet. Entomol. 2019, 33, 296–311. [Google Scholar] [CrossRef] [PubMed]
  24. Zurita, A.; García-Sánchez, Á.M.; Cutillas, C. Comparative molecular and morphological study of Stenoponia tripectinata tripectinata (Siphonaptera: Stenoponiidae) from the Canary Islands and Corsica. Bull. Entomol. Res. 2022, 112, 681–690. [Google Scholar] [CrossRef] [PubMed]
  25. Beugnet, F.; Marié, J.L. Emerging arthropod-borne diseases of companion animals in Europe. Vet. Parasitol. 2009, 163, 298–305. [Google Scholar] [CrossRef]
  26. García-Bocanegra, I.; Arenas-Montes, A.; Napp, S.; Jaén-Téllez, J.A.; Fernández-Morente, M.; Fernández-Molera, V.; Arenas, A. Seroprevalence and risk factors associated to West Nile virus in horses from Andalusia; Southern Spain. Vet. Microbiol. 2012, 160, 341–346. [Google Scholar] [CrossRef]
  27. Hiraldo, J.D.G.; Fuerte-Hortigón, A.; Domínguez-Mayoral, A.; De la Rosa Riestra, S.; Palacios-Baena, Z.R.; Fernández, F.S.; Ruiz, R.L.; Pascual-Vaca, D.; de León, C.M.; Hurtado, R.J.; et al. Uncovering the neurological effects of West Nile virus during a record-breaking southern Spain outbreak in 2020–2021. J. Neuroimmunol. 2023, 383, 578179. [Google Scholar] [CrossRef]
  28. Rodríguez-Alonso, B.; Almeida, H.; Alonso-Sardón, M.; Velasco-Tirado, V.; Robaina Bordón, J.M.; Carranza Rodríguez, C.; Pérez Arellano, J.L.; Belhassen-García, M. Murine typhus. How does it affect us in the 21st century? The epidemiology of inpatients in Spain (1997–2015). Int. J. Infect. Dis. 2020, 96, 165–171. [Google Scholar] [CrossRef]
  29. Beugnet, F.; Labuschagne, M.; Fourie, J.; Jacques, G.; Farkas, R.; Cozma, V.; Halos, L.; Hellmann, K.; Knaus, M.; Rehbein, S. Occurrence of Dipylidium caninum in fleas from client-owned cats and dogs in Europe using a new PCR detection assay. Vet. Parasitol. 2014, 205, 300–306. [Google Scholar] [CrossRef] [PubMed]
  30. Zurita, A.; Benkacimi, L.; El Karkouri, K.; Cutillas, C.; Parola, P.; Laroche, M. New records of bacteria in different species of fleas from France and Spain. Comp. Immunol. Microbiol. Inf. Dis. 2021, 76, 101648. [Google Scholar] [CrossRef]
  31. Zurita, A.; Trujillo, I.; García-Sánchez, Á.M.; Cutillas, C. Survey of flea infestation in cats and dogs in Western Andalusia; Spain: Seasonality and other risk factors for flea infestation. Med. Vet. Entomol. 2024, 38, 244–251. [Google Scholar] [CrossRef]
  32. Beck, W.; Clark, H.H. Differentialdiagnose medizinisch relevanter Flohspezies und ihre Bedeutung in der Dermatologie. Der Hautarzt 1997, 48, 714–719. [Google Scholar] [CrossRef] [PubMed]
  33. Beck, W. Animal parasites and dermatophytes as a cause of epizoonoses in humans. Der Prakt. Tierarzt 2005, 86, 426–434. [Google Scholar]
  34. Gaglio, G.; Allen, S.; Bowden, L.; Bryant, M.; Morgan, E.R. Parasites of European hedgehogs (Erinaceus europaeus) in Britain: Epidemiological study and coprological test evaluation. Eur. J. Wildl. Res. 2010, 56, 839–844. [Google Scholar] [CrossRef]
  35. Dudek, K.; Földvári, G.; Majláthová, V.; Majláth, I.; Rigó, K.; Molnár, V.; Tóth, M.; Jankowiak, L.; Tryjanowski, P. Patterns in the distribution and directional asymmetry of fleas living on the northern white-breasted hedgehog Erinaceus roumanicus. Folia Parasit. 2017, 64, 026. [Google Scholar] [CrossRef] [PubMed]
  36. Marchiondo, A.A.; Holdsworth, P.A.; Fourie, L.J.; Rugg, D.; Hellmann, K.; Snyder, D.E.; Dryden, M.W. World Association for the Advancement of Veterinary Parasitology (W.A.A.V.P.) second edition: Guidelines for evaluating the efficacy of parasiticides for the treatment; prevention and control of flea and tick infestations on dogs and cats. Vet. Parasitol. 2013, 194, 84–97. [Google Scholar] [PubMed]
  37. Lewis, R.E. Notes on the geographical distribution and host preferences in the order Siphonaptera. Part 8. New taxa described between 1984 and 1990, with a current classification of the order. J. Med. Entomol. 1993, 30, 239–256. [Google Scholar] [CrossRef] [PubMed]
  38. Hopkins, G.H.E.; Rothschild, M. An Illustrated Catalogue of the Rothschild Collection of Fleas in the British Museum (Nat. Hist.), Vol. III. Hystrichopsyllidae; Cambridge University Press: Cambridge, UK, 1962. [Google Scholar]
  39. Beaucournu, J.C.; Launay, H. Les Puces (Siphonaptera) de France et du Bassin Méditerranéen Occidental, Faune de France; Fedération Française des Sociétés des Sciences Naturelles: Paris, France, 1990; Volume 76. [Google Scholar]
  40. Lawrence, A.L.; Brown, G.K.; Peters, B.; Spielman, D.S.; Morin-Adeline, V.; Slapeta, J. High phylogenetic diversity of the cat flea (Ctenocephalides felis) at two mitochondrial DNA markers. Med. Vet. Entomol. 2014, 28, 330–336. [Google Scholar] [CrossRef] [PubMed]
  41. Zurita, A.; Callejón, R.; de Rojas, M.; Cutillas, C. Morphological, biometrical and molecular characterization of Archaeopsylla erinacei (Bouché; 1835). Bull. Entomol. Res. 2018, 108, 726–738. [Google Scholar] [CrossRef]
  42. van der Mescht, L.; Matthee, S.; Matthee, C.A. New taxonomic and evolutionary insights relevant to the cat flea, Ctenocephalides felis: A geographic perspective. Mol. Phylogenet. Evol. 2021, 155, 106990. [Google Scholar] [CrossRef]
  43. Rohlf, F.J.; Marcus, L.F. A revolution morphometrics. Trends Ecol. Evol. 1993, 8, 129–132. [Google Scholar] [CrossRef]
  44. Bookstein, F.L. Size and shape: A comment on semantics. Syst. Zool. 1989, 38, 173–180. [Google Scholar] [CrossRef]
  45. Dujardin, J.P. Morphometrics applied to medical entomology. Infect. Genet. Evol. 2008, 8, 875–890. [Google Scholar] [CrossRef] [PubMed]
  46. Dos Reis, S.P.; Pessoa, L.M.; Strauss, R.E. Application of size-free canonical discriminant analysis to studies of geographic differentiation. Braz. J. Genet. 1990, 13, 509–520. [Google Scholar]
  47. Dujardin, J.P.; Le Pont, F. Geographical variation of metric properties within the neotropical sandflies. Infect. Genet. Evol. 2004, 4, 353–359. [Google Scholar] [CrossRef] [PubMed]
  48. Dujardin, J.P. BAC Software; Institut de Recherches pour le Développement (IRD): Paris, France, 2002; Available online: http://www.fsf.org/copyleft/gpl.html (accessed on 10 April 2024).
  49. Hii, S.F.; Lawrence, A.L.; Cuttell, L.; Tynas, R.; Abd Rani, P.A.M.; Šlapeta, J.; Traub, R.J. Evidence for a specifc host-endosymbiont relationship between ‘Rickettsia sp. genotype RF2125’ and Ctenocephalides felis orientis infesting dogs in India. Parasit. Vectors 2015, 8, 169. [Google Scholar] [CrossRef] [PubMed]
  50. Ménier, K.; Beaucournu, J.C. Taxonomic study of the genus Ctenocephalides Stiles & Collins; 1930 (Insecta: Siphonaptera: Pulicidae) by using aedeagus characters. J. Med. Entomol. 1998, 35, 883–890. [Google Scholar] [PubMed]
  51. Güvendi, M.; Can, H.; Köseoğlu, A.F.; Alak, S.E.; Kandemir, C.; Taskın, T.; Sürgeç, E.; Demir, S.; Değirmenci Döşkaya, A.; Karakavuk, M.; et al. Investigation of the genetic diversity and flea-borne pathogens in Ctenocephalides felis samples collected from goats in İzmir and Şanlıurfa provinces of Turkey. Comp. Immunol. Microbiol. Infect. Dis. 2022, 90–91, 101896. [Google Scholar]
  52. Chotelersak, K.; Puttikamonkul, S.; Samung, Y.; Chaiphongpachara, T.; Dujardin, J.P.; Sumruayphol, S. Ctenocephalides orientis and Ctenocephalides felis in Thailand: Head geometry by species, sex and geography. Med. Vet. Entomol. 2024, 38, 179–188. [Google Scholar] [CrossRef]
  53. Ruenchit, P. State-of-the-Art Techniques for Diagnosis of Medical Parasites and Arthropods. Diagnostics 2021, 11, 1545. [Google Scholar] [CrossRef]
  54. Ghosh, N.; Silva, J.; Vazquez, A.; Das, A.B.; Smith, D.W. Use of fluorescence and scanning electron microscopy as tools in teaching biology. In Scanning Microscopies 2011: Advanced Microscopy Technologies for Defense, Homeland Security, Forensic, Life, Environmental, and Industrial Sciences; Postek, M.T., Newbury, D.E., Platek, S.F., Joy, D.C., Maugel, T.K., Eds.; SPIE: Bellingham, WA, USA, 2011; Volume 8036, pp. 217–227. [Google Scholar]
  55. García-Sánchez, A.M.; Rivero, J.; Callejón, R.; Zurita, A.; Reguera-Gomez, M.; Valero, M.A.; Cutillas, C. Differentiation of Trichuris species using a morphometric approach. Int. J. Parasitol. Parasites Wildl. 2019, 31, 218–223. [Google Scholar] [CrossRef]
  56. Escobar-Chavarría, O.; Cossío-Bayúgar, R.; Ramírez-Noguera, P.; Prado-Ochoa, M.G.; Velázquez-Sánchez, A.M.; Muñoz-Guzmán, M.A.; Angeles, E.; Alba-Hurtado, F. In vivo and in vitro apoptosis induced by new acaricidal ethyl-carbamates in Rhipicephalus microplus. Ticks Tick-Borne Dis. 2021, 12, 101603. [Google Scholar] [CrossRef] [PubMed]
  57. Kassa, B.; Lee, M.H.; Kumar, R.; Mickael, C.; Sanders, L.; Tuder, R.M.; Mentink-Kane, M.; Graham, B.B. Experimental Schistosoma japonicum-induced pulmonary hypertension. PLoS Neglect. Trop. Dis. 2022, 16, e0010343. [Google Scholar] [CrossRef] [PubMed]
  58. Marrugal, A.; Callejón, R.; de Rojas, M.; Halajian, A.; Cutillas, C. Morphological, biometrical, and molecular characterization of Ctenocephalides felis and Ctenocephalides canis isolated from dogs from different geographical regions. Parasitol. Res. 2013, 112, 2289–2298. [Google Scholar] [CrossRef]
Animals 14 01582 g001
Figure 1. Factor map corresponding to C. felis, P. irritans, and A. erinacei male adults. Samples are projected onto the first and second principal components: PC1 (71%) and PC2 (26%). Each group is represented by its perimeter.
Figure 1. Factor map corresponding to C. felis, P. irritans, and A. erinacei male adults. Samples are projected onto the first and second principal components: PC1 (71%) and PC2 (26%). Each group is represented by its perimeter.
Animals 14 01582 g001
Animals 14 01582 g002
Figure 2. Factor map corresponding to C. felis, P. irritans, and A. erinacei female adults. Samples are projected onto the first and second principal components: PC1 (90%) and PC2 (8%). Each group is represented by its perimeter.
Figure 2. Factor map corresponding to C. felis, P. irritans, and A. erinacei female adults. Samples are projected onto the first and second principal components: PC1 (90%) and PC2 (8%). Each group is represented by its perimeter.
Animals 14 01582 g002
Table 1. Biometrical data of males of C. felis, P. irritans, and A. erinacei collected from Canis lupus familiaris and Erinaceus europaeus from Andalucia (Spain).
Table 1. Biometrical data of males of C. felis, P. irritans, and A. erinacei collected from Canis lupus familiaris and Erinaceus europaeus from Andalucia (Spain).
C. felisP. irritansA. erinacei
MAXMINMeanSDVCMAXMINMeanSDVCMAXMINMeanSDVC
Global measures
Area (mm2) †1058.4659.3847.3123.4151379.8688.61009.6276271436.7990.31225.5153.313
Roundness †2.412.022.200.1152.061.881.950.0842.482.092.320.146
Circularity †0.360.300.330.0250.50.420.460.0360.430.350.380.026
Perimeter (µm) †5642.44213.94828.5418.395676.44114.84925.4649.5136685.85424.45958410.77
TL (µm)20841563.01777.7138.382027.71312.21650.0285.8172094.51667.31877.9158.58
TW (µm) †815.1627.5722.152.671064.1772.3926.0120.913989.4810.1890.056.46
Head measures
Area (µm2) †78,28753,98268,19463189100,67374,31883,921952011145,712114,111127,33411,3279
Roundness †1.391.291.350.0221.311.231.270.0321.241.171.190.022
Circularity †0.460.420.440.0130.500.450.480.0240.630.590.610.023
Perimeter (µm) †1153953.81072.649.851261.01071.61153.968.661502.91302.71379.864.05
HL (μm) †391.1306.836022.96357.0293.4317.625.08453.2378.3412.623.26
HW (μm) †251.2210.0228.412.15263.0228.3248.215.16378.1323.4352.216.25
Prothorax measures
Area (µm2) †24,84814,28719,19233951820,140963513,08137122833,26818,71128,802403814
Roundness †1.451.231.370.0642.081.681.900.1471.601.251.430.118
Circularity †0.580.460.510.0480.360.260.320.04110.610.380.460.0716
Perimeter (µm) †665.5478.9570.250.39651.1466.6550.160.411778.0594.9715.253.07
PROTW (μm) †120.482.0100.811.01183.546.160.112.821151.088.6120.716.614
Mesothorax measures
Area (µm2) †31,14214,04322,35845182028,396858118,18673474033,77223,28928,643371613
Roundness †1.641.331.460.0862.051.651.810.19101.921.401.720.1911
Circularity †0.570.420.480.0480.430.290.370.05150.550.370.420.0513
Perimeter (µm) †723.9512.8634.163.310764.6467.7624.0107.417858.8670.8783.064.78
MESOW (μm) †137.488.7109.013.813103.547.776.221.829137.099.3111.313.612
Metathorax measures
Area (µm2) †37,15220,30528,52850611853,52220,28035,56912,2103448,48633,65938,673420911
Roundness †1.571.281.460.0751.641.381.530.1071.811.471.600.107
Circularity †0.570.390.440.04100.470.420.440.0250.420.340.390.026
Perimeter (µm) †877.8625.5758.671.691024.4667.7857.8123.5141023.2883.7922.741.95
METW (μm) †142.8102.3120.412.610177.197.5131.430.423157.1109.7134.213.910
AW (μm) †34.912.920.76.02951.319.237.012.23331.311.323.16.227
TL = total length, TW = total width, HL = total length of the head, HW = total width of the head, PROTW = total width of the prothorax, MESOW = total width of the mesothorax, METW = total width of the metathorax, AW: apex width, MAX = maximum, MIN = minimum, SD = standard deviation, Mean = arithmetic mean, VC = coefficient of variation (percentage converted), † = significant differences between groups (p < 0.005).
Table 2. Biometrical data of females of C. felis, P. irritans, and A. erinacei collected from Canis lupus familiaris and Erinaceus europaeus from Andalucia (Spain).
Table 2. Biometrical data of females of C. felis, P. irritans, and A. erinacei collected from Canis lupus familiaris and Erinaceus europaeus from Andalucia (Spain).
C. felisP. irritansA. erinacei
MAXMINMeanSDVCMAXMINMeanSDVCMAXMINMeanSDVC
Global measures
Area (mm2)2290.4827.81724.7438.2252876.61180.31596.4567.1362874.61368.82023.6434.121
Roundness †2.571.822.100.20102.111.801.900.0842.341.762.020.178
Circularity †0.400.330.360.0250.500.400.460.0250.490.360.410.038
Perimeter (µm) †7967.94583.86661.7837.0138379.85310.46092.9993.7168733.56038.37127.7902.613
TL (mm) †2859.51616.22439.8337.8142861.01757.82041.3372.8182938.31885.72425.1332.814
TW (mm) †1208.6767.41038.7136.2131495.91003.51150.2144.0131487.51058.31220.5112.79
Head measures
Area (µm2) †100,16967,74983,173926411135,02387,601111,13712,66911163,067107,663135,80018,24313
Roundness †1.551.351.450.0531.401.221.300.0641.261.161.200.022
Circularity †0.460.380.420.0250.500.430.460.0250.580.510.550.024
Perimeter (µm) †1354.01102.81229.373.261455.01238.61342.465.351564.91273.11423.189.76
HL (μm) †449.5357.9406.627.37428.5344.0371.524.47463.8380.5421.326.46
HW (μm) †296.8243.2265.415.16330.1243.7287.622.28383.9274.2324.730.59
Prothorax measures
Area (µm2) †36,97215,84425,77453472129,11816,76422,84236021648,28725,72937,136754220
Roundness †1.921.401.650.1482.221.531.780.18101.501.261.420.085
Circularity †0.530.360.450.05100.430.260.340.04130.590.410.480.0511
Perimeter (µm) †876.7576.8723.874.310812.5615.0709.351.47879.0691.0805.671.89
PROTW (μm) †139.067.7105.018.31798.656.984.812.214168.1106.3138.220.215
Mesothorax measures
Area (µm2)48,41920,03235,00283982449,24327,11237,91457341550,80029,39740,732654316
Roundness †1.691.241.510.1171.861.521.640.1061.861.501.600.127
Circularity †0.600.330.460.06130.460.350.420.0370.510.400.470.036
Perimeter (µm) †967.0638.1807.6101.4131031.0732.8879.578.291012.9783.6899.375.28
MESOW (μm) †161.488.1135.219.014145.5105.9124.510.08168.1113.8140.815.611
Metathorax measures
Area (µm2) †63,42126,20746,24010,4482373,62148,60363,11778541266,05444,55554,053742514
Roundness †2.091.411.680.17101.731.371.510.1071.591.311.480.075
Circularity †0.480.340.410.0390.480.370.440.0380.500.400.440.037
Perimeter (µm) †1185.4772.5979.9128.9131199.2943.21090.170.361116.9897.5997.973.27
METW (μm) †175.8109.0147.518.513206.9145.3178.415.89188.3144.9170.216.19
Spermatheca measures
Area (µm2) †308821622619259103489243930623091011,76838757474212028
Roundness †1.211.051.140.0441.061.011.030.0111.251.051.120.066
Circularity †0.760.590.650.0570.890.760.850.0450.810.530.660.0812
BULGAP (µm) †212.1167.8192.710.96210.8176.3198.010.35396.7227.0320.445.614
BULGAL (μm) †71.552.661.25.0870.654.363.94.77127.080.399.715.015
BULGAW (μm) †58.246.450.93.0666.755.659.63.15133.757.890.822.625
APEHILL (μm) †52.822.935.69.12564.226.946.912.226118.938.483.421.025
APEHILW (μm) †31.717.024.74.41864.523.431.710.73457.920.938.19.425
DBMV (μm) †34913125157.12348529037856.715503.8150.3293.4116.540
TL = total length, TW = total width, HL = total length of the head, HW = total width of the head, PROTW = total width of the prothorax, MESOW = total width of the mesothorax, METW = total width of the metathorax, BULGAP: perimeter of the bulga, BULGAL: total length of the bulga, BULGAW: total width of the bulga, APEHILL: total length of the apex of the hilla, APEHILW: total width of the apex of the hilla, DBMV = distance from bulga to the ventral margin of the body, MAX = maximum, MIN = minimum, SD = standard deviation, Mean = arithmetic mean, VC = coefficient of variation (percentage converted), † = significant differences between groups (p < 0.005).
Table 3. Distribution of fleas collected from dogs from different geographical origins.
Table 3. Distribution of fleas collected from dogs from different geographical origins.
Geographical OriginC. felis
(Number of Fleas)		P. irritans
(Number of Fleas)		A. erinaceid
(Number of Fleas)
MaleFemaleMaleFemaleMaleFemale
Sanlúcar de Barrameda (Cadiz, Spain)1821----
Seville (Seville, Spain)--513--
Huelva (Huelva, Spain)--1---
Dos Hermanas (Seville, Spain)----1013
Total18216131013
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García-Sánchez, A.M.; Trujillo, I.; Zurita, A.; Cutillas, C. Differentiation of Synanthropic Fleas from Andalusia (Spain) through Geometric Morphometrics Analysis. Animals 2024, 14, 1582. https://doi.org/10.3390/ani14111582

AMA Style

García-Sánchez AM, Trujillo I, Zurita A, Cutillas C. Differentiation of Synanthropic Fleas from Andalusia (Spain) through Geometric Morphometrics Analysis. Animals. 2024; 14(11):1582. https://doi.org/10.3390/ani14111582

Chicago/Turabian Style

García-Sánchez, Angela M., Ignacio Trujillo, Antonio Zurita, and Cristina Cutillas. 2024. "Differentiation of Synanthropic Fleas from Andalusia (Spain) through Geometric Morphometrics Analysis" Animals 14, no. 11: 1582. https://doi.org/10.3390/ani14111582

APA Style

García-Sánchez, A. M., Trujillo, I., Zurita, A., & Cutillas, C. (2024). Differentiation of Synanthropic Fleas from Andalusia (Spain) through Geometric Morphometrics Analysis. Animals, 14(11), 1582. https://doi.org/10.3390/ani14111582

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