A distinct mitochondrial cytochrome c oxidase subunit I lineage of the poultry red mite Dermanyssus gallinae in Tunisian egg-layer flocks

Abstract

The poultry red mite, Dermanyssus gallinae is a globally significant ectoparasite affecting layer hens, with substantial economic and animal welfare impacts. Despite its importance, the genetic diversity and population structure of Dermanyssus gallinae in North Africa remain completely uncharacterized. We conducted the first molecular phylogenetic characterization of D. gallinae in Tunisia, analyzing mitochondrial cytochrome c oxidase subunit I (COI) gene sequences from 18 specimens collected across 20 commercial layer farms in four distinct regions (Tabarka, Manouba, Nabeul, and Sfax). Phylogenetic analyses, population genetic parameters, and demographic history were assessed using maximum likelihood methods, haplotype networks, and neutrality tests. Six distinct haplotypes were identified, with a single dominant haplotype (H1) present in 72.2% of samples across all regions. Critically, all Tunisian haplotypes formed a well-supported, divergent monophyletic cluster separated from European haplotypes by a minimum of 12 mutational steps, representing a previously uncharacterized mitochondrial lineage. Genetic diversity was moderate (Hd = 0.490) with low nucleotide diversity (π = 0.01188), and demographic analyses revealed signatures of recent population expansion (Tajima's D = -2.223, p < 0.01). Population structure was limited, with 87.5% of variation occurring within populations, though significant differentiation was detected between Tabarka and Sfax (FST = 0.345, p < 0.05). This study reveals a distinct Tunisian mitochondrial lineage of D. gallinae and provides evidence for recent demographic expansion driven by poultry industry intensification and human-mediated dispersal. These findings expand the known genetic diversity of this economically important parasite and underscore the need for region-specific management strategies incorporating improved biosecurity measures to limit mite transmission through contaminated equipment and supply chains.

Introduction

The poultry red mite, Dermanyssus gallinae (Mesostigmata: Dermanyssidae), is a globally significant ectoparasite of birds and an obligatory, temporary hematophagous pest (Sárkány et al., 2025; Sigognault Flochlay et al., 2017). While primarily affecting layer hens, it exhibits a broad host range, including wild birds and mammals, and is a documented cause of gamasoidosis in humans (Cafiero et al., 2019; George et al., 2015). Its infestation constitutes a severe challenge to poultry production systems worldwide, with direct consequences for hen health, welfare, and productivity (Decru et al., 2020; Yevstafieva and Petrunenko, 2024). These effects, which scale with infestation severity, include dermatitis, behavioral stress, immunomodulation, anemia, and in severe cases, increased mortality (Fujisawa, 2022; Temple et al., 2020). Furthermore, D. gallinae acts as a vector for several bacterial and viral pathogens (Schiavone et al., 2022; Valiente Moro et al., 2009), exacerbating disease spread within flocks and raising additional public health concerns (George et al., 2015). The economic impact is substantial, manifesting as reduced egg yield and quality, increased feed conversion ratios, and elevated costs for control measures (Sigognault Flochlay et al., 2017; Sparagano et al., 2011).

In Tunisia, poultry farming, particularly egg production, is a vital agricultural sector contributing to national food security and nutrition. However, intensive production systems face growing threats from Poultry Red Mite (PRM) infestation, which is reported to cause significant economic losses and occupational challenges. Actually, poultry farmers frequently report PRM infestations as a management concern, although quantitative estimates of associated economic losses are currently unavailable.

Despite its clear importance, the genetic identity and population structure of Dermanyssus gallinae in Tunisia—and more broadly across North Africa remain entirely uncharacterized.

Molecular tools, particularly mitochondrial DNA markers, are powerful for elucidating acarine population genetics and phylogeography. The cytochrome c oxidase subunit I (COI) gene is highly informative for such intraspecific studies due to its appropriate evolutionary rate and extensive reference database (Hebert et al., 2003). To our knowledge, no D. gallinae COI sequences from Africa were available in GenBank prior to this study, leaving the contribution of North African populations to the species’ global mitochondrial diversity largely unknown.

This study presents the first molecular phylogenetic characterization of D. gallinae in Tunisia. We analyzed COI gene sequences from mites collected across 20 commercial layer farms in four distinct regions to: (1) determine the haplogroups present, (2) assess the genetic diversity and structure of Tunisian populations, and (3) establish a foundational genetic dataset for future comparative studies and integrated pest management programs in the region.

Materials and methods

Mite sampling and morphological identification

Sampling was conducted between 2021 and 2022 across 20 intensive egg-layer farms in four distinct geographical regions of Tunisia: Tabarka, Manouba, Nabeul, and Sfax. Flocks were aged 38 - 49 weeks and housed 4 000 to 15 000 chickens per unit.

Sample collect was during the summer season in all four regions to minimize seasonal variation in mite abundance and population structure.

Mites were collected using corrugated cardboard traps placed in microhabitats favored by D. gallinae, including beneath feed troughs, inside cages, on walls, and under manure belts (Nordenfors and Chirico, 2001). Additional specimens were collected directly from dust and housing structures using a fine brush.

Although traps occasionally contained other arthropods and mite-like specimens commonly associated with poultry house environments, only individuals morphologically consistent with Dermanyssus gallinae were retained for molecular analyses. Morphological identification was performed using the diagnostic criteria described by Di Palma et al. (2012) (Di Palma et al., 2012), including characters allowing differentiation from other poultry-associated mites such as Ornithonyssus sylviarum. To minimize the risk of misidentification, all retained specimens were subsequently confirmed molecularly through amplification of the mitochondrial 16S rRNA marker using D. gallinae-specific primers. All analyzed specimens were confirmed as D. gallinae. A subset of specimens from each farm was preserved in 95% ethanol or at −20°C for subsequent molecular analysis (Desloire et al., 2006).

DNA extraction

Genomic DNA was extracted from five individual mites per farm using the Wizard® Genomic DNA Purification Kit (Promega, USA), species identity was confirme by sequencing the mitochondrial 16S rRNA marker. One representative specimen per farm was selected for COI sequencing and population genetic analyses, yielding 20 candidate COI sequences. Two sequences were excluded from downstream analyses because of poor chromatogram quality or suspected contamination. The final COI dataset therefore consisted of 18 high-quality sequences.

Briefly, individual mites were washed in distilled water, homogenized in nuclei lysis solution, and digested with proteinase K at 56°C overnight. Subsequent steps followed the manufacturer’s protocol. DNA concentration and purity were assessed spectrophotometrically, and extracts were stored at −20°C.

PCR amplification, sequencing, and sequence confirmation

To confirm the morphological identification and DNA extraction quality, a 377 bp fragment of the mitochondrial 16S rRNA gene was amplified for all samples using the D. gallinae-specific primers F16 and R16 (Desloire et al., 2006).

For phylogenetic analysis, a ∼400 bp fragment of the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using the universal primers COI farward (COIF) and COI reverse (COIR) (Marangi et al., 2009). All PCR reactions were performed in a 25 µL volume containing 12.5 µL of 2x Taq PCR Master Mix (BIOMATIK), 10 pmol of each primer, and 2 µL of DNA template. Bovine Serum Albumin (BSA; 1.25 µL) was added to the COI reactions to inhibit potential PCR inhibitors. A no-template negative control was included in each PCR run to monitor contamination, and no amplification was detected in these controls.

Amplifications were carried out in an Esco Swift Max Pro thermocycler. Products were visualized on 1.2% agarose gels stained with ethidium bromide.

Forward and reverse chromatograms were visually inspected, assembled into consensus sequences, and trimmed to remove low-quality ends. Sequences containing ambiguous base calls, poor-quality chromatograms, or evidence of contamination were excluded from downstream analyses.

PCR amplicons of the COI gene were purified using the Wizard® SV Gel and PCR Clean-Up System (Promega, USA). Purified products were sequenced bidirectionally using the same primers on an ABI 3730xl DNA analyzer (Applied Biosystems, USA) with BigDye Terminator v3.1 chemistry. Sequences were edited, assembled, and trimmed to a standardized length using ChromasPro (v.1.7.4). Derived COI sequences were confirmed as D. gallinae by BLASTn search against the NCBI GenBank database.

Genetic and phylogenetic analyses

Sequence Alignment and Diversity. Edited COI sequences were aligned using the ClustalW algorithm in BioEdit (v7.2.5). The final COI alignment retained for analysis was 410 bp after trimming low-quality ends and standardizing sequence length across all samples.

Haplotypes were identified using DNA Sequence Polymorphism (DnaSP) (v6.12.03). Population genetic parameters, including the number of haplotypes (H), haplotype diversity (Hd), nucleotide diversity (π), and the number of polymorphic sites (S), were calculated in DnaSP.

Population Structure and Demography. Genetic differentiation among the four Tunisian regional populations was estimated by computing pairwise Fixation Index (F_ST) values with 10,000 permutations in Arlequin (v3.5.2.2). Analysis of Molecular Variance (AMOVA) was performed to partition genetic variation within and among populations. Demographic history was investigated using neutrality tests (Tajima’s D and Fu’s Fs) and mismatch distribution analysis in Arlequin. The sum of squared deviations (SSD) and Harpending’s raggedness index (R²) were calculated to test the goodness-of-fit of the observed mismatch distribution to a model of sudden population expansion.

Phylogenetic reconstruction and haplotype network. A phylogenetic tree was constructed using a dataset comprising the 18 unique Tunisian sequences identified in this study and 30 publicly available D. gallinae COI sequences from Eurasia (see Table 1 for references). The best-fit nucleotide substitution model (T92+G) was selected using ModelFinder within IQ-TREE 2 (v2.2.0). A maximum likelihood (ML) tree was inferred with 1000 replicates for both ultrafast bootstrap (UFBoot) and the SH-aLRT branch test. The tree was rooted using a sequence from a congeneric species (Dermanyssus hirundinis). A median-joining haplotype network was constructed in PopART (v1.7) to visualize relationships among haplotypes.

Table 1.

Dermanyssus gallinae COI sequences included in the phylogenetic analysis, with GeneBank accession numbers and geographic origin.

No.	Sequence (GenBank ID)	Country	Reference
1	LR812424.1	Greece	Karp-Thatham et al. (2020)
2	LR812434.1
3	LR812449.1
4	LR812451.1
5	LR812452.1
6	LR812406.1	Slovenia
7	LR812357.1
8	LR812358.1
9	LR812359.1
10	LR812390.1	UK
11	LR812345.1	Romania
12	LR812304.1	Portugal
13	LR812307.1
14	LR812311.1
15	LR812312.1
16	LR812316.1	Czech Republic
17	LR812317.1
18	LR812322.1
19	LR812290.1	Albania
20	LR812285.1
21	LR812352.1	Turkey
22	LR812326.1	Denmark
23	LR812295.1	Croatia
24	LR812296.1
25	AM921853.1	France
26	FN432489.1
27	HQ842406.1		Roy and Buronfosse (2011)
28	FN650451.1	Norway	Øines and Brännström (2011)
29	FN650350.1
30	MN249082.1	Korea	Oh et al. (2019)
31	PV630662.1	Tunisia, Sfax	Present study
32	PV654484.1
33	PV654483.1
34	PV635347.1
35	PV635350.1
36	PV654414.1	Tunisia, Nabeul
37	PV637898.1
38	PV654485.1
39	PV635354.1
40	PV635357.1	Tunisia, Manouba
41	PV654514.1
42	PV635355.1
43	PV636424.1
44	PV654521.1
45	PV631211.1	Tunisia, Tabarka
46	PV631207.1
47	PV635346.1
48	PV631174.1

Results

Genetic diversity and haplotype distribution

Six distinct haplotypes (H1-H6) were identified in the final COI dataset of 18 D. gallinae sequences representing 18 farms across four Tunisian regions. A single, widespread haplotype (H1) was dominant, present in 13 samples (72.2%) and found in all four regions (Figs. 1& 2). The remaining five haplotypes were private, each unique to a single region (Fig. 1).

Fig. 1.

Geographic location of the four populations of Dermanyssus gallinae. Pie charts show the frequencies of the COI haplotypes detected.

Fig. 2.

Median-joining haplotype network of the 6 haplotypes determined in Dermanyssus gallinae specimens from four localities in Tunisia using the COI marker. The size of the haplotypes is proportional to the number of individuals. The small black nodes = median vectors represent internal haplotypes that are absent from the dataset. The length of the connecting lines is mostly proportional to the number of substitutions between the 6 haplotypes. Node sizes are proportional to haplotype frequency.

Overall haplotype diversity (Hd) was moderate (0.490), while nucleotide diversity (π) was low (0.01188) (Table 2). This pattern is indicative of a population that has undergone recent expansion from a common source. Regional values of π were uniformly low, ranging from 0.0061 to 0.0259. The aligned 400 bp COI dataset contained 28 polymorphic sites.

Table 2.

Genetic diversity indices based on COI sequences of Dermanyssus gallinae populations from four Tunisian regions.

Population	N	h	Hd	K	Pi
Tabarka (Tab)	4	3	0.8333 ± 0.2222	8.500	0.02671 ± 0.00855
Manouba (Ma)	5	2	0.4000 ± 0.2370	3.200	0.01042 ± 0.00618
Nabeul (Nab)	5	2	0.5000 ± 0.2650	2.400	0.00914 ± 0.00485
Sfax (Sf)	4	2	0.5000 ± 2650	2.000	0.00541 ± 0.00287
Total	18	6	0.4900 ± 0.1420	3.895	0.01188 ± 0.00420

The combination of moderate haplotype diversity and low nucleotide diversity is consistent with a population that has undergone a recent expansion following a founder event.

Population Genetic Structure and Differentiation. Pairwise genetic distance estimates were calculated between Tunisian COI haplotypes and representative Eurasian reference sequences to quantify the divergence observed in the phylogenetic tree and haplotype network. These distances confirmed that Tunisian haplotypes were differentiated from the available European and Asian reference sequences.

Pairwise genetic differentiation (F_ST) among the four regional populations revealed a complex structure, with values ranging from −0.107 to 0.345 (Table 3). Notably, a moderate and significant level of differentiation was observed between the geographically distant Tabarka and Sfax populations (F_ST = 0.345, p < 0.05). In contrast, most other pairwise comparisons showed negligible to absent genetic structure (F_ST values near or below zero), suggesting high gene flow or a shared recent ancestry among the Manouba, Nabeul, and Sfax populations. Negative F_ST values are interpreted as zero, indicating no measurable genetic differentiation between the corresponding populations, where historical connections have resulted in similar genetic makeups. This can occur through processes such as recent common ancestry or admixture events that blend genetic material from different lineages.

Table 3.

Pairwise genetic differentiation statistics (F_ST) between populations of Dermanyssus gallinae.

Population 1	Population 2	FST
Tabarka	Manouba	0.02500
Tabarka	Sfax	0.34480
Tabarka	Nabeul	0.02128
Manouba	Sfax	0.00000
Manouba	Nabeul	−0.10714
Sfax	Nabeul	−0.05263

Negative FST values were treated as zero for biological interpretation.

Analysis of Molecular Variance (AMOVA) indicated that the majority of genetic variation (87.5%) occurred within populations, with only 12.5% attributed to variation among regions, further supporting limited geographical structuring.

Demographic history. Neutrality tests and mismatch distribution analyses were conducted to infer the demographic history of D. gallinae populations in Tunisia. For the pooled Tunisian dataset, neutrality tests revealed a significantly negative Tajima’s D value (D = −2.223, p < 0.01) whereas Fu’s Fs was positive and non-significant. At the regional level, neutrality statistics were not significant (p < 0,01). Therefore, the neutrality tests did not provide fully concordant evidence for demographic expansion.

This discrepancy suggests that demographic expansion signals are more apparent at the national scale than at the local population level, and highlights the limitations of single-locus demographic inference when sample sizes per population are modest.

The observed mismatch distribution for the pooled sample was unimodal. Goodness-of-fit tests showed no significant departure from the expected distribution under a sudden expansion model (Sum of Squared Deviations, SSD, p > 0.05; Raggedness Index, r = 0.275, p > 0.05).

Together, these genetic patterns—a unimodal mismatch distribution with a non-significant raggedness index, the combination of moderate haplotype diversity (Hd = 0.490) with low nucleotide diversity (π = 0.01188), and significantly negative Tajima’s D and Fu & Li’s statistics—are collectively consistent with a recent demographic expansion of D. gallinae in Tunisia. This expansion likely followed a founder event associated with the rapid colonization of modern poultry farming infrastructure.

In contrast, the mismatch distribution for the Tabarka population alone was polymodal, suggesting this group has experienced a different demographic history, potentially indicative of a longer-established or more stable population (Fig. 3 and Table 4).

Fig. 3.

Mismatch distributions of COI sequences for Dermanyssus gallinae populations from Tunisia. Panels correspond to: (A) Tabarka, (B) Manouba, (C) Nabeul, (D) Sfax, and (E) pooled Tunisian dataset. Observed pairwise differences are shown together with the expected distribution under a sudden expansion model.

Table 4.

Neutrality tests for Dermanyssus gallinae populations with COI marker.

Population	Tajima’s D	Fu’s Fs	SSD (p-value)	Raggedness index R2	Interpretation
Tabarka	−0.852	2.449	<0.05	<0.05	No significant deviation from neutrality, probably a stable population.
Manouba	−1.193	4.234	>0.05	>0.05	Relatively negative values, but not significant → expansion possible but weak evidence.
Cap Bon	−0.817	3.225	>0.05	>0.05	Like Manouba, no strong evidence of selection or expansion.
Sfax	−0.780	2.197	>0.05	>0.05	Very similar results in Cap Bon.
Total	−2.223 (p < 0.01)	1.477	>0.05	0.275	Significant, suggesting recent demographic expansion or positive selection on a global scale.

Phylogenetic Analysis Reveals a deeply divergent mitochondrial COI lineage. The Maximum Likelihood phylogenetic tree, including our sequences and publicly available Eurasian haplotypes, revealed a distinct and well-supported cluster (UFBoot >85%, SH-aLRT >80%) comprising all Tunisian D. gallinae haplotypes (Fig. 4). This Tunisian cluster formed a monophyletic group characterized by notably long branch lengths relative to the compact, geographically defined clusters from Europe (e.g., Balkan, Iberian).

Fig. 4.

Maximum-likelihood phylogenetic tree based on COI sequences of Dermanyssus gallinae. Previously described haplogroups are indicated following Karp-Thatham et al. (2020). Tunisian sequences obtained in the present study are indicated as a distinct mitochondrial COI cluster. The Tunisian cluster may represent a candidate mitochondrial lineage, pending confirmation using multilocus datasets and broader geographic sampling.

The median-joining haplotype network visualized this deep divergence (Fig. 2). The Tunisian haplotypes (H1-H6) were separated from the nearest European haplotypes by a minimum of 12 mutational steps across the 410 pb COI fragment, forming a clearly isolated star-like network centered on the dominant H1 haplotype.

Discussion

This study provides the first molecular characterization of Dermanyssus gallinae populations from Tunisian commercial layer farms and documents a distinct mitochondrial COI cluster together with evidence of limited population structuring and recent demographic expansion at the national scale. These findings broaden the current geographic framework of D. gallinae mitochondrial diversity and provide baseline data for North Africa. Given the recognized economic and veterinary importance of this ectoparasite in intensive laying systems worldwide, expanding regional genetic knowledge remains essential for understanding transmission and improving control strategies (George et al., 2015; Roy et al., 2021; Sigognault Flochlay et al., 2017).

A distinct mitochondrial lineage in Tunisia

The most important finding of this study is the identification of a well-supported Tunisian mitochondrial COI cluster including all detected haplotypes and clearly separated from currently available Eurasian reference sequences. This cluster was characterized by long branch lengths in the phylogenetic tree and by a minimum of 12 mutational steps from the nearest European haplotypes in the median-joining network. This marked separation was further supported by pairwise sequence divergence analysis in DnaSP. Across the 176 ungapped COI positions retained for comparison, Tunisian and Eurasian datasets differed by 75 fixed nucleotide substitutions and showed no shared polymorphisms. Average nucleotide divergence between populations was high (Dxy = 0.44545; Da = 0.43974), reinforcing the phylogenetic and haplotype network evidence that Tunisian COI sequences are strongly differentiated from the currently available Eurasian references. Comparable studies based on mitochondrial COI have demonstrated the utility of this marker for detecting phylogeographic structuring and mitochondrial divergence in D. gallinae and other arthropod taxa (Marangi et al., 2014; Roy et al., 2021). However, because these estimates were calculated on the subset of comparable ungapped positions and rely on a single mitochondrial marker, they should be interpreted cautiously and considered evidence of marked mitochondrial differentiation within the present dataset rather than definitive taxonomic separation or independent evolutionary status (Roy and Buronfosse, 2011; Roy et al., 2021). The significance of this result is strengthened by the limited molecular characterization previously available for North African D. gallinae populations. While European and Asian studies have described several mitochondrial haplogroups and regional substructure (Chu et al., 2015; Karp-Thatham et al., 2020; Marangi et al., 2014), the Tunisian sequences did not cluster closely with those previously reported lineages. This finding highlights the importance of expanding geographic sampling beyond the regions currently represented in public databases (Roy et al., 2011, 2021). At the same time, comparisons with published datasets require caution because studies differ considerably in geographic scale, sample size, production systems, and sampling design. Accordingly, the present dataset should be interpreted as an initial regional baseline for Tunisia and North Africa rather than as directly equivalent to larger continental surveys (Karp-Thatham et al., 2020; Koziatek-Sadłowska and Sokół, 2022). The origin of this mitochondrial lineage remains unresolved. Although trade with Europe may represent a plausible route for mite movement, the Tunisian sequences formed a differentiated mitochondrial cluster relative to the available European sequences. Possible explanations include long-term regional persistence, introduction from currently unsampled Mediterranean or North African populations, or historical introduction followed by local diversification. However, because the inference is based on a single mitochondrial marker, this cluster should be interpreted conservatively as a distinct COI lineage pending confirmation using broader geographic sampling across North Africa and the Mediterranean basin and nuclear markers (Hebert et al., 2003; Roy et al., 2021).

Genetic diversity patterns and population structure

Despite this mitochondrial differentiation, genetic diversity within Tunisia remained limited. Six haplotypes were identified among 18 sequences, with H1 dominating and occurring in all four regions. The combination of moderate haplotype diversity and low nucleotide diversity is compatible with expansion from a limited founder pool and has been described in arthropod populations following recent establishment or range expansion (Excoffier et al., 2009; Oh et al., 2019). This pattern is broadly consistent with previous regional studies in Poland and Korea, where dominant haplotypes and relatively limited within-country diversity were also reported, whereas broader European surveys documented substantially higher diversity (Chu et al., 2015; Karp-Thatham et al., 2020; Koziatek-Sadłowska and Sokół, 2022). Together, these comparisons suggest that continental-scale diversity in D. gallinae may coexist with relatively restricted diversity at national or regional scales. Population structure analyses indicated generally weak differentiation, with most variation occurring within populations. The only significant pairwise differentiation was observed between Tabarka and Sfax, this differentiation may reflect their locations in contrasting Tunisian bioclimatic zones—humid and arid, respectively. Climatic conditions, particularly humidity and temperature, can influence the biology, reproductive dynamics, and survival of D. gallinae (De Wit, 2021; Nordenfors et al., 1999). This pattern suggests that geographic distance alone is unlikely to explain population structure and that additional bioclimatic and anthropogenic factors may contribute to local differentiation (Roy et al., 2021; Roy and Buronfosse, 2011). Tabarka, which showed the highest haplotype and nucleotide diversity, differs markedly from the other study regions: it lies within a humid Mediterranean bioclimate in a cork oak forest zone, hosts a more diverse avifauna, and is close to the Algerian border. These features raise the hypothesis that Tabarka’s higher mite genetic diversity reflects greater historical connectivity or multiple introduction events, driven by cross‑border exchanges and contacts with resident and migratory bird species, and/or by more favorable local bioclimatic conditions. Testing this hypothesis will require broader local and transboundary sampling combined with systematic recording of bioclimatic indicators.

Evidence for recent demographic expansion

At the pooled national scale, demographic analyses were broadly consistent with recent expansion. The significantly negative Tajima’s D and unimodal mismatch distribution support an excess of low-frequency variants and a demographic scenario compatible with recent population growth (Excoffier et al., 2009; Tajima, 1989). However, this interpretation should remain cautious because Fu’s Fs was positive and non-significant, and demographic inference based on a single mitochondrial locus has limited resolution (Ramírez-Soriano et al., 2008; Ramos-Onsins and Rozas, 2002). The discordance between neutrality statistics may reflect sample size, population structure, or marker-specific limitations.

The Tabarka population showed a polymodal mismatch distribution, suggesting a demographic history distinct from the other sampled regions. This may indicate a more stable local population or admixture among divergent mitochondrial variants which is consistent with Tabarka’s elevated genetic diversity, although these scenarios cannot be distinguished with the present dataset (Excoffier et al., 2009).

At the epidemiological level, recent expansion is biologically plausible in the context of poultry intensification, as large-scale laying systems provide stable host availability and favorable conditions for rapid establishment of D. gallinae populations (Mul et al., 2009; Sparagano et al., 2014).

Human-mediated dispersal and biosecurity implications

The widespread distribution of the dominant haplotype H1 across all four sampled regions supports some level of connectivity among farms. This pattern is consistent with human-mediated dispersal through poultry production networks rather than strict geographic isolation (Roy and Buronfosse, 2011; Sparagano et al., 2014). Similar observations have been reported elsewhere, with identical or closely related haplotypes detected across distant regions in Europe and Asia (Chu et al., 2015; Koziatek-Sadłowska and Sokół, 2022; Sioutas et al., 2024). In Tunisia, shared equipment, transport materials, egg trays, and routine movement within commercial production systems may facilitate mite transfer between farms, as previously reported in poultry production environments (Mul et al., 2009; Sparagano et al., 2014). The differentiation observed between Tabarka and Sfax nevertheless indicates that regional heterogeneity may still occur despite overall connectivity, highlighting the importance of integrating farm-level biosecurity with attention to regional movement networks. Although this study did not evaluate acaricide resistance, the observed genetic connectivity among multiple populations suggests that supply-chain networks and trade could facilitate the spread of resistant red poultry mite (RPM) populations across Tunisian farms. However, genetic data alone cannot determine whether resistant mites are circulating; confirming resistance would require targeted phenotypic assays and molecular resistance screening (Decru et al., 2020; Roy et al., 2021).

Methodological considerations

Several limitations should be acknowledged in our study. The COI fragment analyzed was shorter than the standard barcode region, which may reduce phylogenetic resolution and comparability with some published datasets (Hebert et al., 2003). In addition, mitochondrial DNA alone cannot fully resolve population history or distinguish among processes such as introgression or male-mediated gene flow (Roy and Buronfosse, 2011). The modest number of sequences also limits regional inference. Consequently, the present results likely capture the dominant genetic signal in the sampled farms but may underestimate broader diversity, particularly in underrepresented ecological contexts. Future work should therefore incorporate multilocus approaches and broader geographic coverage, including additional Tunisian regions and neighboring North African populations. The addition of nuclear markers such as ITS, microsatellites, or SNPs would substantially improve phylogeographic and demographic resolution (Roy et al., 2021; Roy and Buronfosse, 2011).

Comparative context and future directions

In a broader phylogeographic context, the Tunisian population shares with other regional studies the presence of dominant haplotypes and relatively limited within-country diversity (Chu et al., 2015; Koziatek-Sadłowska and Sokół, 2022). However, the presence of a differentiated Tunisian mitochondrial cluster distinguishes the present dataset from currently available Eurasian references and expands the known diversity of the species. Further studies should prioritize broader Mediterranean and North African sampling, incorporation of nuclear markers, and integration of genetic data with bioclimatic features and farm management and movement networks. Combining population genetics with epidemiological, environmental and zootechnical data would improve identification of transmission pathways and help refine integrated pest management strategies (Decru et al., 2020; George et al., 2015). Linking genetic structure with acaricide susceptibility would also strengthen interpretation of dispersal and control dynamics and could clarify whether connected poultry networks contribute to the dissemination of particular adaptive traits (Roy et al., 2021).

In summary, this study provides the first COI-based molecular dataset for Dermanyssus gallinae from Tunisia and identifies a distinct Tunisian mitochondrial cluster within the currently available reference framework. Genetic diversity was limited overall, with one dominant haplotype distributed across all regions and only moderate regional differentiation. Demographic analyses were broadly compatible with recent expansion at the national scale, although this remains provisional given the use of a single mitochondrial marker. Taken together, these findings provide a baseline for future phylogeographic and epidemiological studies of D. gallinae in North Africa and support the importance of regionally adapted biosecurity and integrated management strategies for poultry production systems (Roy et al., 2021; Sparagano et al., 2014).

Statement regarding ethical approval

I hereby confirm that the research presented in our manuscript did not involve the use of vertebrate animals as experimental subjects. Mite specimens were collected from commercial layer farm environments using non-invasive trapping methods. No handling, manipulation or experimental procedures were performed on live hens or any other vertebrate animals. Although, we obtained the approval from the Ethics Committee for Animal Experimentation under Tunisian National Guidelines and institutional policies at the National School of veterinary Medicine of Sidi Thabet.

CRediT authorship contribution statement

Maroua Bettaieb: Writing – review & editing, Writing – original draft, Visualization, Validation, Software, Resources, Methodology, Investigation, Data curation, Conceptualization. Safa Amairia: Formal analysis, Data curation. Mohammed Ridha Rjeibi: Visualization, Software. Mohammed Ghorbel: Data curation. Tarek Hajji: Visualization, Validation, Supervision, Project administration, Methodology, Conceptualization. Mohammed Aziz Darghouth: Visualization, Validation, Supervision, Project administration, Methodology, Conceptualization.

Disclosures

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.