The microbiome of the sucking louse Linognathus stenopsis (Phthiraptera, Anoplura, Linognathiidae)

930 © The Author(s) 2025. Published by Oxford University Press on behalf of Entomological Society of America. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (https://creativecommons. org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact [email protected] for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact [email protected]. Vector/Pathogen/Host Interaction, Transmission The microbiome of the sucking louse Linognathus stenopsis (Phthiraptera, Anoplura, Linognathiidae) Francisco J. Márquez1,, José E. Granados2,, Luz M. Canet3,, Manuel de Rojas4,, Antonio J. Caruz5,, and Jesús M. Pérez1,*, 1Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus Las Lagunillas, s.n., E-23071 Jaén, Spain 2Delegación Territorial de Sostenibilidad y Medio Ambiente en Granada, Joaquina Eguaras 2 (Edificio Almanjáyar), E-18013, Granada, Spain 3Unidad de Genómica, Instituto de Parasitología y Biomedicina “Lopez-Neyra”, Parque Tecnológico Ciencias de la Salud, Avda. Conocimiento 17, Armilla E-18016 Granada, Spain 4Departamento de Microbiología y Parasitología, Facultad de Farmacia, Universidad de Sevilla, Profesor García González 2, E-41012 Sevilla, Spain 5Departamento de Biología Experimental, Universidad de Jaén, Campus Las Lagunillas, s.n., E-23071 Jaén, Spain *Corresponding author. Departamento de Biología Animal, Biología Vegetal y Ecología, Universidad de Jaén, Campus Las Lagunillas, s.n., E-23071 Jaén, Spain (Email: [email protected]). Subject Editor: Stephen Rich Received on 15 October 2024; revised on 10 April 2025; accepted on 17 April 202527June2025 Graphical Abstract Keywords: Anoplura, Linognathus stenopsis, microbiome Journal of Medical Entomology, 62(4), 2025, 930–937 https://doi.org/10.1093/jme/tjaf060 Advance Access Publication Date: 27 June 2025 Research Downloaded from https://academic.oup.com/jme/article/62/4/930/8175087 by guest on 31 October 2025 931Journal of Medical Entomology, 2025, Vol. 62, No. 4 Epidemiological studies aimed at understanding the vector role of lice as transmitters of bacteria are notably scarce. The objective of our study was to expand our knowledge about the vector role of Linognathus stenopsis, a sucking louse found on the Iberian ibex (Capra pyrenaica), and determine the diversity of bacteria it harbors. To achieve the above-mentioned goal, we used next-generation DNA sequencing. This study allowed the detection of an endosymbiont closely related to genus Sodalis, together with several genera that include human and animal pathogens, including Ricketssia sp., Coxiella sp. Francisella sp, and Acinetobacter sp. Sodalis sp. are important endosymbiont of L. stenopsis. The presence of potentially pathogenic bacteria can be ascribed to the bacterial community that may be shared/transferred by the ectoparasite community feeding on a same host individual (infracommunity). Sucking lice (Phthiraptera: Anoplura) are hemimetabolous, obligate blood-sucking insects that exclusively parasitize eutherian mammals (Light et al. 2010). These host-specific ectoparasites exhibit dorsoventral flattening, lack wings, and possess tibio-tarsal claws for clinging to host hair. Their piercing mouthparts are adapted for feeding on blood (Durden 2019). Linognathus stenopsis (Burmeister, 1838) (Anoplura: Linognathidae) (Fig. 1) is a sucking louse that parasitizes goats and chamois (Capra spp. and Rupicapra spp.) worldwide (Durden and Musser 1994). It is a common ectoparasite of the Iberian ibex, Capra pyrenaica (Fernández-Muñoz et al. 2023). A vicariant species, Linognathus africanus, which parasitizes the previously mentioned hosts and deer (Odocoileus spp.) in Africa, the Middle East, and North America, and has also been frequently cited in several Mediterranean countries, including Spain (Durden 2019, Nizamov and Prelezov 2021). Heavy infestations of L. africanus may cause severe alopecia, depression, poor growth and increase the probability of pathogen transmission in mule and white-tailed deer (Nelson et al. 1977, Foreyt et al. 1986). While data on pathogen detection in L. stenopsis is sparse, in several louse species sampled in Hungary, including L. stenopsis, the presence of Anaplasma spp. has been detected in both bovine and ovine hosts (Hornok et al. 2010). This louse has also been found to harbor A. ovis in Algeria (Ouarti et al. 2021), as well as Anaplasma, Bartonella, Ehrlichia, Mycoplasma, and Rickettsia in Mexico (Balados-González et al. 2023). While these findings suggest a potential association between the louse and these potentially or known pathogenic agents, additional studies are needed. Sucking lice have specialized organs, namely bacteriomes, harboring ensosymbiotic bacteria (belonging to Enterobacteriaceae) which can be vertically transmitted from mother to offspring (Fukatsu et al. 2009, Boyd et al. 2016). Recent technological developments in DNA sequencing, like next-generation sequencing or (NGS) (Malla et al. 2019, Dubey et al. 2022) have enabled “the microbiome revolution”. This paradigm shift has allowed the reformulation of scientific issues to include the microbiota as a fundamental element in the study of certain biological phenomena, from the animal to the disease, that allow us to extend our knowledge to include “non-culturable” species of bacteria. The post-genomic era holds great potential for identifying and classifying the γ-proteobacterial primary endosymbionts of true lice. Unlike free-living and pathogenic bacteria, louse primary endosymbionts cannot be cultured and classified using traditional microbiological methods. Sequencing of the 16S rRNA gene by PCR has provided valuable insights into the diversity of primary endosymbionts of all insects (Boyd and Reed 2012). Within this context, we have developed a strategy aimed at revealing the internal microbiome of L. stenopsis. Arthropod-bacteria endosymbiosis is a relatively common phenomenon. The presence of obligate endosymbiotic bacteria complements the nutritional supply of insects that depend on nutritionally incomplete diets, providing their hosts with the essential nutrients they lack (Dillon and Dillon 2004). Acquiring an endosymbiont can provide significant selective advantages to the host, enabling them to exploit highly specialized niches with limited dietary resources (Moran et al. 2003). Endosymbiotic bacteria, residing either within the intestine or as primary, intracellular symbionts housed in bacteriomes, play crucial roles in host physiology. They contribute to the host’s health and significantly supplement its nutritional requirements (Moran et al. 2019). For instance, endosymbionts provide essential vitamins that are absent or deficient in the diets of lice (Boyd et al. 2016). Molecular phylogenetic analyses of bacteria inhabiting bacteriomes suggest that louse-bacterium endosymbiotic associations have evolved independently multiple times, or alternatively, that endosymbionts have been repeatedly replaced within louse lineages (Boyd et al. 2016). Using NGS, this study aimed to determine the composition of the bacterial community associated with lice, with an emphasis on identifying bacteria that have established a symbiotic relationship Fig. 1. Linognathus stenopsis. Adult gravid female. Habitus. Downloaded from https://academic.oup.com/jme/article/62/4/930/8175087 by guest on 31 October 2025 932 Márquez et al. with their host and those that belong to genera known to include pathogens of humans or animals. Materials and Methods Study Area, Sample Collection, and Processing Specimens of Linognathus stenopsis were collected from two subadult male Iberian ibexes (Capra pyrenaica) inhabiting the Sierra Nevada Natural Space (36°00′–37°10′N, 2°34′–3°40′W) in southern Spain. The ibexes were chemically immobilized using a combination of xylazine (3 mg/kg) and ketamine (3 mg/kg) (Casas-Díaz et al. 2011). During the immobilization period, animals were also physically immobilized and provided with a mask for preventing ocular lesions and reducing stress. Once sampling was completed, the animals remained under monitoring until revovery. A manual removal of lice was performed using forceps, following a meticulous inspection of the hosts. Specimens were fixed in 70% ethanol for subsequent analysis in the laboratory. The lice underwent thorough surface cleaning and sterilization, involving a 30-s wash in 1% commercial bleach, followed by three subsequent 1-minute rinses in sterile water (Binetruy et al. 2019). Subsequently, each louse was stored individually in a sterile vial containing 70% ethanol as a fixative. Morphological identification and DNA extraction were conducted under sterile conditions within a laboratory setting. The generation of the bacterial profile of the L. stenopsis microbiome began with the preparation of three batches of 12 adult lice. DNA extraction was made under appropriate sterile conditions using the DNeasy Blood and Tissue kit (Macherey-Nagel, Düren, Germany), following the manufacturer’s instructions. Each sample was examined twice in independent experiments. Library preparation and Illumina sequencing targeting the V3-V4 hypervariable region of prokaryotic 16S rDNA were carried out at the Institute of Parasitology and Biomedicine “López-Neyra” (IPBLN) Genomics Facility, part of the Spanish National Research Council (CSIC, Granada, Spain). Amplicon libraries were generated by a two-step PCR strategy and Fig. 2. Comparison of sucking louse microbiota alpha diversity between each replicated sample, including species richness (represented by observed species) and evenness (represented by Shannon and Simpson index). Downloaded from https://academic.oup.com/jme/article/62/4/930/8175087 by guest on 31 October 2025 933Journal of Medical Entomology, 2025, Vol. 62, No. 4 three replicates were made for each of the samples. The V3-V4 region of 16S rDNA was amplified using Pro341F/Pro805R primer set for Bacteria (Takahashi et al. 2014). The PCR reactions were conducted with PCR clamps block the amplification of contaminating sequences from a eukaryotic host, in order to enrich microbial sequences (Lundberg et al. 2013). Conditions for amplification of DNA were with an annealing at 55 °C and with a concentration of 0.1 µM of each primer and 0.25 µM of clamps. Sequencing was conducted using a paired-end, 2 × 275-bp cycle run on an Illumina MiSeq sequencing system, according with Illumina MiSeq library preparation guide. As a part of a bioinformatic study, Fastq files were pre-processed and analyzed using specific analysis of microbiome packages DADA2 v.1.14.0 (Callahan et al. 2016) and Phyloseq (McMurdie and Holmes 2013), complemented with other R packages (microbiome, microbiomeMarker, etc.) in the R 4.2.3 environment (R Core Team 2020). To generate a table of amplicon sequence variants (ASVs) containing higher-resolution analogs of the traditional operative taxonomical units (OTUs) primers and adapters were removed, the quality profiles of the reads were inspected. After that, the forward and reverse reads were merged to obtain the full-denoised sequences. Chimeras were excluded, and the ASV table was finally constructed. Taxonomy was assigned to the ASVs with the function assign taxonomy of DADA2, which uses as input the set of sequences to be classified and the reference sequences of SILVA database version 138 (Quast et al. 2013). Results The resulting metabarcoding data set comprised 449,893 reads from the three samples. The average number of sequences per sample was 74,982.2 ranging from 51,320 to 83,856. The alpha diversity analyses showed statistically significant differences between the six NGS (two per batch of lice) used in this study (Fig. 2). Microorganism taxonomic composition of each replica is represented in Fig. 3A and B. Our results show a high predominance of a sequence related with the order Enterobacterales, which we notated as “Linognathus stenopsis endosymbiont” (LSE), initially identified by the system as belonging to the genus Sodalis (Fig. 4). To obtain more information about the possible identity of these taxa and the phylogenetic relationships of the LSE sequences, we performed a phylogenetic analysis on the V3-V4 region of the 16S rRNA sequences, using Blast to search for the highest sequence identity in GenBank. Blast revealed that the two selected 429 bp sequences, positions 200762 to 201188 in the accession LN854557, Sodalis glossinidius str. ‘morsitans’ isolate B4 genome assembly, have a maximum percentage of identity (id) ranging from 94.61% to 93.01%. The sequences with the greatest homology correspond to Enterobacterales, mainly Pectobacteriacea of the genus Sodalis (Adeolu et al. 2016), Yersiniaceae of the genus Serratia, or several uncultured endosymbiont as “candidatus Steffania” (Havelka et al. 2021), among others. While studying the bacteriome of sucking louse or other hematophagous arthropods, it is important to note that not all bacterial species belonging to genera known for their medical or veterinary significance are necessarily pathogenic. Many of these bacteria are, in fact, endosymbionts that often play crucial roles in the arthropod’s biology, such as providing essential nutrients, aiding in digestion, or modulating the host’s immune response. Therefore, the presence of potential pathogens does not equate to this sucking louse being Fig. 3. (A) Taxonomical composition of Linognathus stenopsis microbiota by family and genus. (B) Taxonomical composition of L. stenopsis microbiota by family and genus, excluding LSE cf Sodalis genus. The order of the colors used consistently corresponds to the order presented in the explanatory key, from top to bottom. Downloaded from https://academic.oup.com/jme/article/62/4/930/8175087 by guest on 31 October 2025 934 Márquez et al. a potential or proven vector due to the intricate host-microbe dynamics. A comprehensive understanding of the complex interactions between hosts and their microbial communities is necessary to accurately assess the risk of disease transmission. In this context, the presence of species of genera probed as relevant veterinary and medical importance should be considered. Such is the cases of Rickettsia sp. (100% id with a large range of species), Coxiella sp. (100% to 96% id with Coxiella-like endosymbiont of the tick Haemaphysalis punctata MT313147 (Chisu et al. 2021 [27]), Francisella sp. (100% id with several endosymbions of ticks of genus Dermacentor, Hyalomma and Haemaphysalis) and Acinetobacter sp. (100% id with Acinetobacter johnsonii MN826149 and several others, and 97.2 with Acinetobacter lwoffii MT192311). An Euler diagram (Fig. 5), shows the relationships between the main bacteria found in each sample at the genus level. Seven genera constitute the core group of bacteria shared between the samples. Sample R1 presents all the significant genes (22), compared to the 12 that appeared in sample R2, and the 8 in R3. Discussion From a veterinary point of view, sucking lice can cause weight loss, skin damage, moderate to severe anemia, hypoproteinaemia, insufficient absorption of food, and reduced vitality in infected goats (Otter et al. 2003, Paul et al. 2012, Nizamov and Iliev 2023). In addition, they can transmit viruses, bacteria, protozoa, and fungi like Rickettsia spp. and Anaplasma ovis (Hornok et al. 2010). The results gathered from our study of the internal microbiome showed that the bacterial composition of L. stenopsis has a high biodiversity. The full microbiome includes several hundreds of taxa, but in our case, we detected at least four genera that include species of medical or veterinary importance, including Rickettsia, Anaplasma, Francisella, and Coxiellasp. Our results are very different from those obtained when the technical approach involves the isolation of bacterial strains (Nizamov 2022), but they seem to endorse those obtained by various authors who have developed the specific molecular detection of certain genera of bacteria (Hornok et al. 2010, Kumsa et al. 2012). Insects can establish a variety of symbiotic associations with bacteria that can have a significant impact on their evolutionary ecology. In the context of the parasitism-mutualism continuum, interactions between insects and symbiont bacteria can be extremely diverse and evolve rapidly (Drew et al. 2021). Organisms linked to obligate hematophagy during their life cycle, in order to compensate for nutritional deficiencies and metabolic integration (Boyd and Reed 2012), have evolutionarily converged in the development of analogous functional microbiomes with nutritional symbionts that provide them with group B vitamins (Duron and Gottlieb 2020). Primary endosymbionts (P-endosymbionts) are obligate and mutualistic bacterial endosymbionts vertically inherited, implicated in strict co-speciation processes (Grossi et al. 2024). In the case of lice, the primary symbionts present in the bacteriocytes, located in the stomach disk, are transmitted from the ovary to the oocytes through transovarian transmission (Duron and Gottlieb 2020). Although molecular phylogenetic studies reveal the presence of both alpha and gamma-Proteobacteria in different lice genera, it seems clear, however, that all currently known primary endosymbionts (obligate intracellular endosymbionts) in this group of insects belong to the γ-Proteobacterial, included in the families Enterobacteriales and Legionellales (McCutcheon et al. 2019). Fig. 4. Heat map of replicas for the top genera. Downloaded from https://academic.oup.com/jme/article/62/4/930/8175087 by guest on 31 October 2025 935Journal of Medical Entomology, 2025, Vol. 62, No. 4 Species of the genus Sodalis are particularly ubiquitous as symbiotic partners. They have established independent symbioses, linked to maternal transmission, in a large range of insect taxa (Renoz et al. 2023). Sodalis is prototrophic for many cofactors and amino acids (Toh et al. 2006), and nutritional provisioning is suspected in other Sodalis-like endosymbionts (Boyd 2014). We consider it highly probable that LSE may correspond to a P-endosymbiont of L. stenopsis, taking into account that it is a key factor in the evolutionary success of approximately 15% of insect species (Buchner, 1965) and attending to the persistence of Sodalis and Sodalis-like taxa among diverse insect groups (McCutcheon et al. 2019, Boyd et al. 2016). Endosymbiont seclusion within host specialized cells (bacteriome), is crucial in protecting them from host immune defenses while avoiding chronic host immune activation (Ferrarini et al. 2022). These types of events generally determine a modification of the abiotic niche of the insect (Lemoine et al. 2020), and an extensive reduction in the size of the genome of the bacteria involved in it, conditioned by a long coevolutionary history with the hosts (Moran and Bennett 2014). Reduction in endosymbiont genomes can operate as horizontal gene transfer from endosymbionts to host (McCutcheon and Moran 2012), or as changes in the partners’ individual gene regulatory networks (Alarcón et al. 2022). Sucking lice have limited dispersal opportunities, meaning that they typically must rely on direct contact between hosts (Hellenthal and Price 2009). This circumstance could determine that endosymbionts have experienced a coevolutionary history similar to that of their host, allowing indirect phylogenetic analysis of the host clade from its endosymbiotic bacteria (Hammoud et al. 2022). Anoplura frequently move between hosts and puncture the skin in several places during each blood meal (Durden 2019). Co-feeding processes (Buysse et al. 2022) could facilitate the horizontal transference of bacteria found in other hematophagous ectoparasites (ticks, horse flies, louse flies, fleas, etc.). Insect-borne pathogens follow the most common horizontal transmission cycle of vector-borne pathogens as has been demonstrated for the flea Ctenocephalides felis and Rickettsia felis (Brown et al. 2015, Fongsaran et al. 2022). In the case of L. stenopsis, its co-occurrence with ticks during more or less prolonged periods on the same host, potentially leading to cofeeding, which can allow horizontal transmission of bacteria between arthropods during feeding without systemic or local infection. This can occur through the ingestion of infected blood or through mechanical contamination via the feces of other arthropods (Fernández-Muñoz et al. 2023 [4]). It is known that communities of microorganisms present positive or negative correlations with other elements that could be part of the same community (Krawczyk et al. 2022). Although the weight of these associations seems to have a limited effect on the composition of the microbiome at the scale of a local population of ectoparasites, it may be relevant to the dynamics of pathogens transmitted by certain individuals (ie ticks). In some of them, microorganisms that are transmitted horizontally converge with those that are transmitted vertically, and that have been acquired in relation to vertebrate species on which they have fed, and the presence of other hematophagous arthropods that can enrich these communities. Fig. 5. Euler diagram showing the relationships between the main bacteria find in each sample at the genus level. Downloaded from https://academic.oup.com/jme/article/62/4/930/8175087 by guest on 31 October 2025 936 Márquez et al. Conclusions This study identified a Pectobacteriaceae endosymbiont, likely associated with the bacteriome of L. stenopsis, closely related to the genus Sodalis. Furthermore, the bacterial community associated with this louse species included several genera that encompass pathogenic species including Rickettsia sp., Coxiella sp., Francisella sp., and Acinetobacter sp. The presence of these pathogens within the louse community suggests potential for pathogen sharing or transmission between individuals within the same host (infracommunity). However, while suggestive, this study does not definitively establish L. stenopsis as a disease vector. Further investigation is crucial to fully understand the complex host–microbe interactions and determine the precise role of this sucking louse in disease etiology. Acknowledgments The technical and human support provided by Unidad de Genómica, Instituto de Parasitologia y Biomedicina “Lopez-Neyra”(CSIC, Granada, Spain) is gratefully acknowledged. Author contributions Francisco Márquez (Conceptualization [Equal], Funding acquisition [Equal], Investigation [Equal], Software [Equal], Validation [Equal], Writing - original draft [Equal], Writing - review & editing [Equal]), José Granados (Investigation [Equal], Writing - review & editing [Equal]), Luz Canet (Investigation [Equal], Writing - review & editing [Equal]), Manuel de Rojas (Resources [Equal], Writing - review & editing [Equal]), Antonio J. Caruz (Funding acquisition [Equal], Resources [Equal], Writing - review & editing [Equal]), and Jesús Pérez (Conceptualization [Equal], Data curation [Equal], Investigation [Equal], Writing - review & editing [Equal]) Funding Research activities of authors are partially funded by the Universidad de Jaén [FEDER-UJA 2020 “Caracterización del microbioma y del viroma de distintas garrapatas (Ixodidae) del Sur de Europa” (code 1380336) and Action 1b] and by the PAIDI, Junta de Andalucía (BIO 294 and RNM 118 groups). Conflicts of interest. 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