Reduction in Oviposition of Poultry Red Mite (Dermanyssus gallinae) in Hens Vaccinated with Recombinant Akirin

The poultry red mite (PRM), Dermanyssus gallinae, is a hematophagous ectoparasite of birds with worldwide distribution that causes economic losses in the egg-production sector of the poultry industry. Traditional control methods, mainly based on acaricides, have been only partially successful, and new vaccine-based interventions are required for the control of PRM. Vaccination with insect Akirin (AKR) and its homolog in ticks, Subolesin (SUB), have shown protective efficacy for the control of ectoparasite infestations and pathogen infection/transmission. The aim of this study was the identification of the akr gene from D. gallinae (Deg-akr), the production of the recombinant Deg-AKR protein, and evaluation of its efficacy as a vaccine candidate for the control of PRM. The anti-Deg-AKR serum IgY antibodies in hen sera and egg yolk were higher in vaccinated than control animals throughout the experiment. The results demonstrated the efficacy of the vaccination with Deg-AKR for the control of PRM by reducing mite oviposition by 42% following feeding on vaccinated hens. A negative correlation between the levels of serum anti-Deg-AKR IgY and mite oviposition was obtained. These results support Deg-AKR as a candidate protective antigen for the control of PRM population growth.


Introduction
The poultry red mite (PRM), Dermanyssus gallinae (De Geer, 1778), is a hematophagous parasitic mite of birds. It has a worldwide distribution and is considered the major pest for the poultry industry in Europe and Asia with severe economic loses for the egg-production sector [1,2]. The PRM hides in furniture in the hen's surroundings and feeds on the hens in darkness [3,4]. The lifecycle of PRM includes five stages: egg, larvae, protonymph, deutonymph, and adult, and it is usually completed in 2 weeks [5]. Severe infestations by PRM increases hen mortality by provoking severe anemia and behavioral disorders [6][7][8]. PRM has also been shown to be a vector for multiple pathogenic viruses and bacteria [2,9,10].
The control of PRM is primarily based on the use of acaricides but there are a number of associated drawbacks such as the selection of resistant mite populations, environmental/food contamination, and limited success once the infestation is established [2,11,12]. Alternative control methods against PRM are under development, and vaccination is a promising intervention as it is environmentally sound, reduces the use of acaricides, and avoids the selection of resistant mites. The use of recombinant proteins for vaccinating hens against PRM has shown favorable results under in-vitro mite-feeding conditions [13][14][15][16].
Akirin (AKR) is a protein encoded by highly-conserved akirin (akr) genes involved in different biological processes, including the innate immune response in all metazoans [17]. The AKR ortholog in ticks, Subolesin (SUB, also known as 4D8), is involved in several biological processes such as tick response to infection, feeding, stress response, development, and reproduction [17]. Most vertebrates have two AKR homologues (AKR1 and AKR2) while insects and ticks have only one SUB/AKR [18]. The SUB/AKR orthologues have shown vaccine efficacy for the reduction of ectoparasite infestations and pathogen infection/transmission [17,19]. Recombinant AKR from Aedes albopictus has been previously evaluated as a vaccine candidate against PRM, resulting in statistically significantly higher mite mortality when fed on blood from vaccinated hens when compared to control birds [15]. However, one limitation of this previous study is the use of mosquito AKR as the PRM homologue sequence was unavailable at that time. The mosquito AKR may therefore have limited protection efficacy against PRM in vaccinated hens when compared to endogenous AKR antigen.
To address this limitation, in this study we identified the akr gene sequence from D. gallinae (Deg-akr) from the newly-available draft genome resource [20]. The recombinant Deg-AKR protein was then produced in Escherichia coli and used to evaluate its efficacy as a vaccine candidate for the control of PRM using a novel on-hen feeding device [21].

Ethics Statement
Animal experiments were conducted in strict accordance with the guidelines of the European Community Directive 2010/63/EU. Animals were housed in the experimental farm of the Institute for Game and Wildlife Research (IREC) with the approval and supervision of the Ethics Committee on Animal Experimentation of the University of Castilla La Mancha (Registry number PR-2018-11-20).

Mites
Dermanyssus gallinae of mixed developmental stages and sexes were obtained from a commercial egg-laying farm in Consuegra (Toledo, Spain). Mites were stored in vented 75 cm 2 tissue culture flasks (Corning, NY, USA) at room temperature (RT) for 10 days. Adult females were selected based on size and morphology, and protonymphs were obtained from larvae hatched from previously-harvested mite eggs.

Cloning of the Gene Coding for Deg-AKR
In order to identify the Deg-akr gene in PRM, the AKR amino acid sequence from Metaseiulus occidentalis (XM_003738959.2) was used as a reference sequence in a local tBLASTn search against the draft genome of the PRM with GenBank accession number QVRM01000000 [20]. Based on sequence similarity to the M. occidentalis AKR gene, exons encoding the Deg-akr ortholog were identified in the D. gallinae genome, and the full-length coding sequence (CDS) was manually assembled. The full-length CDS was PCR-amplified from D. gallinae cDNA generated from mixed-population D. gallinae mites collected from a commercial egg-laying facility (Scotland, UK) as described previously [22]. PCR amplification of the Deg-akr CDS was performed using the oligonucleotide primers Sub_F1: 5 -ATGGCATGTGCGACGCTAAAACGTC-3 and Sub_F2: 5 -TTACGAGCAATAGGACGGGGCGC-3t hat were designed to amplify the full CDS including the putative initiating methionine and stop codon that were conserved between different species of the Acari (Figure 1). PCR amplification was completed using proof-reading DNA polymerase and performed on an Applied Biosystems 2720 thermal cycler with the following conditions: 94 • C for 2 min, followed by 30 cycles at 94 • C for 30 s, 61 • C for 30 s and 72 • C for 1 min. A multiple amino acid sequence alignment was performed using the Clustal Omega algorithm [23] to identify conserved regions. The amplification products were ligated into the ChampionTM pET SUMO vector (Thermo Fisher Scientific, Waltham, MA, USA) and confirmed by DNA sequencing (Eurofin Genomics, Luxemburg). The sequence of Deg-akr was submitted to GenBank under accession number MN310557.

Production of Recombinant Deg-AKR and Vaccine Formulation
The Deg-akr coding sequence was sub-cloned into ChampionTM pET101 expression vector (Thermo Fisher Scientific) and used to transform BL21 StarTM (DE3) E. coli cells. For Deg-AKR protein production, cells were grown at 37 °C with shaking at 200 rpm until OD600 = 0.6, and then cultures were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cultured for a further 4.5 h. Recombinant Deg-AKR was purified from insoluble cell-lysates in the presence of 7 M urea by Ni affinity chromatography (Genscript Corporation, Piscataway, NJ, USA) as previously described [21] using 1 mL HisTrap FF columns mounted on an AKTA-FPLC system (GE Healthcare, Piscataway, NJ, USA). Purified recombinant Deg-AKR was refolded by dialysis against 1000 volumes of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.4 for 12 h at 4 °C. For vaccine formulation, recombinant protein or PBS saline control were adjuvanted in MontanideTM ISA 71 VG (SEPPIC, Paris, France) as previously described [25]. The final concentration for Deg-AKR was 100 µg/mL in a vaccination dose volume of 0.5 mL.

Hen Vaccination and PRM Infestation
Five 25-week-old Lohman Brown hens per group were randomly assigned to two groups (Group 1 and Group 2) and housed together. Hens were vaccinated on days 0 (V1), 14 (V2) and 28 (V3) with a 0.5-mL intramuscular injection in the breast muscle. Group 1 birds were vaccinated with 50-µg Deg-AKR formulated in MontanideTM ISA 71 VG, and Group 2 birds were vaccinated with an equivalent volume of PBS in MontanideTM ISA 71 VG. PRM exposure was carried out using an on-hen feeding device as described previously [22]. Briefly, a feeding device was placed onto each thigh that had been previously plucked, attached to the skin with Leucoplast tape (BSN Medical, Hamburg,

Production of Recombinant Deg-AKR and Vaccine Formulation
The Deg-akr coding sequence was sub-cloned into ChampionTM pET101 expression vector (Thermo Fisher Scientific) and used to transform BL21 StarTM (DE3) E. coli cells. For Deg-AKR protein production, cells were grown at 37 • C with shaking at 200 rpm until OD 600 = 0.6, and then cultures were induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) and cultured for a further 4.5 h. Recombinant Deg-AKR was purified from insoluble cell-lysates in the presence of 7 M urea by Ni affinity chromatography (Genscript Corporation, Piscataway, NJ, USA) as previously described [21] using 1 mL HisTrap FF columns mounted on an AKTA-FPLC system (GE Healthcare, Piscataway, NJ, USA). Purified recombinant Deg-AKR was refolded by dialysis against 1000 volumes of PBS (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4), pH 7.4 for 12 h at 4 • C. For vaccine formulation, recombinant protein or PBS saline control were adjuvanted in MontanideTM ISA 71 VG (SEPPIC, Paris, France) as previously described [25]. The final concentration for Deg-AKR was 100 µg/mL in a vaccination dose volume of 0.5 mL.

Hen Vaccination and PRM Infestation
Five 25-week-old Lohman Brown hens per group were randomly assigned to two groups (Group 1 and Group 2) and housed together. Hens were vaccinated on days 0 (V1), 14 (V2) and 28 (V3) with a 0.5-mL intramuscular injection in the breast muscle. Group 1 birds were vaccinated with 50-µg Deg-AKR formulated in MontanideTM ISA 71 VG, and Group 2 birds were vaccinated with an equivalent volume of PBS in MontanideTM ISA 71 VG. PRM exposure was carried out using an on-hen feeding device as described previously [22]. Briefly, a feeding device was placed onto each thigh that had been previously plucked, attached to the skin with Leucoplast tape (BSN Medical, Hamburg, Germany) and secured with cohesive bandage (Henry Schein Inc., Melville, NY, USA). Each device contained 100 starved female adults and 75 protonymphs. Deuthonymphs were not included in the infestation because they are difficult to differentiate from adult males while preparing the pouches for infestation. Mites fed on hens for 3 h. On-hen feeding experiments were repeated independently four times using fresh mites on each assay on days 42, 44, 46, and 49 from the first vaccination.

Assessment of Vaccine Efficacy
Mites were considered fed when they had taken a fresh blood meal and were fully engorged; and partially fed when they had fresh blood ingested but they were not fully engorged. PRM were considered dead if they did not show any movement and were unresponsive to touch stimulus. The number of fed, unfed and partially fed adults and protonymphs per hen were counted individually in each assay. Both fully engorged and partially fed protonymphs and adult mites were collected from the devices and transferred into individual wells on a 96-well tissue culture plate (Corning Costar, Sigma-Aldrich, St. Louis, MO, USA) and sealed with AeraSeal film (Sigma-Aldrich, A9224-50EA, St. Louis, MO, USA). Plates containing fed mites were placed into an incubator at 25 • C and 85% relative humidity and checked daily for 7 days. Adult mites were monitored for mortality, oviposition and egg fertility, while protonymphs were monitored for mortality and molting ( Figure 2). In order to determine differences in engorgement between vaccinated and control groups, a body size of five to six fully engorged adult mites per hen was measured by scanning electron microscopy (SEM). The mites used for SEM photography were dehydrated in absolute ethanol for 24 h. Specimens were mounted onto standard aluminum SEM stubs using conductive carbon adhesive tabs. Mites were observed and photographed with a field emission scanning electron microscope (Zeiss GeminiSEM 500, Oberkochen, Germany) operating in high vacuum mode at an accelerating voltage of 2 kV in the absence of metallic coating. Mite body length and width were measured, and body area calculated using a Fiji ImageJ Software [26]. This procedure does not affect acari body size if they are not damaged, which are the samples selected for analysis. Germany) and secured with cohesive bandage (Henry Schein Inc., Melville, NY, USA). Each device contained 100 starved female adults and 75 protonymphs. Deuthonymphs were not included in the infestation because they are difficult to differentiate from adult males while preparing the pouches for infestation. Mites fed on hens for 3 h. On-hen feeding experiments were repeated independently four times using fresh mites on each assay on days 42, 44, 46, and 49 from the first vaccination.

Assessment of Vaccine Efficacy
Mites were considered fed when they had taken a fresh blood meal and were fully engorged; and partially fed when they had fresh blood ingested but they were not fully engorged. PRM were considered dead if they did not show any movement and were unresponsive to touch stimulus. The number of fed, unfed and partially fed adults and protonymphs per hen were counted individually in each assay. Both fully engorged and partially fed protonymphs and adult mites were collected from the devices and transferred into individual wells on a 96-well tissue culture plate (Corning Costar, Sigma-Aldrich, St. Louis, MO, USA) and sealed with AeraSeal film (Sigma-Aldrich, A9224-50EA, St. Louis, MO, USA). Plates containing fed mites were placed into an incubator at 25 °C and 85% relative humidity and checked daily for 7 days. Adult mites were monitored for mortality, oviposition and egg fertility, while protonymphs were monitored for mortality and molting ( Figure  2). In order to determine differences in engorgement between vaccinated and control groups, a body size of five to six fully engorged adult mites per hen was measured by scanning electron microscopy (SEM). The mites used for SEM photography were dehydrated in absolute ethanol for 24 h. Specimens were mounted onto standard aluminum SEM stubs using conductive carbon adhesive tabs. Mites were observed and photographed with a field emission scanning electron microscope (Zeiss GeminiSEM 500, Oberkochen, Germany) operating in high vacuum mode at an accelerating voltage of 2 kV in the absence of metallic coating. Mite body length and width were measured, and body area calculated using a Fiji ImageJ Software [26]. This procedure does not affect acari body size if they are not damaged, which are the samples selected for analysis.

Analysis of Immune Response to Deg-AKR in Hens by ELISA
Blood samples from each hen were collected before each vaccination dose on days 0, 14 and 28 and before PRM feeding on day 42. After each blood collection, serum was separated by centrifugation and stored at −20 • C. Hen eggs (8-10 eggs per group) were collected during 2 consecutive days on days 15-16 (T1), 29-30 (T2) and 42-43 (T3). During egg collection, experimental groups were housed separately. Antibodies from egg yolk were extracted as previously described [27] and stored at −20 • C. The IgY antibody levels against Deg-AKR and a mite-soluble protein extract obtained as previously described [28] were determined by ELISA in sera and egg yolks. High absorption-capacity 96-well plates (Immunoplate, SPL Life Sciences, Syeonggi-do, Korea) were coated overnight at 4 • C with 0.25 µg/well of each antigen. ELISA plates were washed four times with 200 µL PBST (PBS containing 0.05% v/v Tween-20) and incubated with 200 µL/well of blocking buffer (5% w/v skimmed milk powder in PBS, Condalab, Torrejón de Ardoz, Spain) for 2 h at RT on a shaker. Then, 50 µL of serum samples diluted in PBS to 1/2000 and 50 µL of yolk samples diluted to 1/1800 were added. After incubating for 1 h at RT, the plates were washed four times and then incubated for 1 h at RT with 50 µL/well of rabbit anti-IgY-peroxidase conjugate (Sigma-Aldrich) diluted 1/1000 in PBST. After four washes, the reaction was visualized by adding 100 µL of 3,3, 5,5-tetramethylbenzidine (TMB) (Promega, Madison, WI, USA) and incubated for 10 min in the dark at RT. After stopping the reaction with 50 µL sulfuric acid, the optical density (OD) was measured at 450 nm using a Multiskan GO microplate reader (Thermo Fisher Scientific, Waltham, MA, USA).

Analysis of Hen Immune Response to Deg-AKR by Western Blot
Western blot analysis was carried out as previously described [29]. Briefly, 10 µg of purified recombinant Deg-AKR was separated by electrophoresis on an SDS-12% polyacrylamide gel (Life Science, Hercules, CA, USA) and then stained with Coomassie Brilliant Blue (Sigma-Aldrich, St. Louis, MO, USA) or transferred to a nitrocellulose membrane. The membrane was blocked with 5% BSA (Sigma-Aldrich) for 2 h at RT and washed four times with TBST (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5% Tween 20). Pooled sera from the day 42 of blood extraction were used as primary antibodies diluted to 1/1000 in TBS. Rabbit anti-IgY-peroxidase conjugate (Sigma-Aldrich, St. Louis, MO, USA) diluted 1/1000 in TBS with 3% BSA was used as the secondary antibody. The membrane was washed five times with TBST and developed with TMB stabilized substrate for HRP (Promega, Madrid, Spain).

Statistical Analysis
Anti-AKR IgY levels between vaccinated and control groups were compared by Mann-Whitney U test (p = 0.05). Partially fed and dead, or missing mites were removed from the analysis of the effect of vaccination on PRM reproduction. Pearson´s correlation coefficient was used to determine if the specific AKR antibody levels at OD450 nm of individual vaccinated and control hens at day 42 (the time when mite infestations were performed) are correlated with the total number of eggs laid per mite fed on individual hens. A generalized linear mixed model (GLMM) was performed to determine the group differences in oviposition based on a Negative Binomial distribution and log as link function. The model included the total number of eggs laid per mite (oviposition) as the dependent variable. The treatment group was included as a fixed effect and hen number and assay replicate as random factors. Data exploration, correlations and GLMM were done using the IBM SPSS Statistics v23 software.

Results and Discussion
Mosquito AKR was previously used as a vaccine candidate against PRM in an experiment that provided support for the role of AKR as a protective antigen [15]. The aim of our study was to identify and determine the protective capacity of the Deg-AKR for the control of PRM infestations in vaccinated hens. The experimental design included the evaluation of vaccination effect on the PRM life cycle including mite (adult and protonymph) mortality, oviposition, and molting (larvae to protonymph and protonymph to deutonymph) ( Figure 2). Alignment of the amino acid sequences obtained from Deg-AKR with AKR/SUB from mites (M. occidentalis and Varroa destructor), tick (Ixodes ricinus) and mosquito (A. albopictus) showed the presence of conserved regions in the coding sequence (Figure 1), further supporting the evolutionary conservation of AKR/SUB between different species [17].
After vaccination with Deg-AKR, hens developed an antigen-specific IgY immune response, which was detected in both hen sera ( Figure 3A) and egg yolk ( Figure 3B). Although IgY antibodies in egg yolk do not affect vaccine efficacy, this approach has been used as a non-invasive method to monitor antibody titers in hens [27]. The anti-Deg-AKR serum IgY antibody levels increased during the entire experiment from day 0 to day 42 and remained higher than in control animals until the end of the experiment ( Figure 3A). In the egg yolk, the anti-Deg-AKR IgY levels were also higher in the vaccinated than in the adjuvant-only control group ( Figure 3B). The IgY response to the soluble mite protein extracts was also higher in the vaccinated than the control group but the difference was only statistically significantly (p < 0.05) higher on days 14 and 28 and days 28 and 42 after vaccination for serum and egg yolk, respectively ( Figure 3C,D). The soluble PRM protein extract is a complex protein mix, which may reflect an AKR dilution effect that can explain the differences in the antibody levels against Deg-AKR and PRM protein extract ( Figure 3A,B vs. Figure 3C,D). The serum IgY recognition of Deg-AKR (25.9 kDa) in vaccinated hens was also corroborated by Western blot ( Figure 3E). Serum antibodies also detected the presence of potential Deg-AKR multimers and some protein degradation products, which commonly occur with AKR and SUB [15,17]. and determine the protective capacity of the Deg-AKR for the control of PRM infestations in vaccinated hens. The experimental design included the evaluation of vaccination effect on the PRM life cycle including mite (adult and protonymph) mortality, oviposition, and molting (larvae to protonymph and protonymph to deutonymph) ( Figure 2). Alignment of the amino acid sequences obtained from Deg-AKR with AKR/SUB from mites (M. occidentalis and Varroa destructor), tick (Ixodes ricinus) and mosquito (A. albopictus) showed the presence of conserved regions in the coding sequence (Figure 1), further supporting the evolutionary conservation of AKR/SUB between different species [17].
After vaccination with Deg-AKR, hens developed an antigen-specific IgY immune response, which was detected in both hen sera ( Figure 3A) and egg yolk ( Figure 3B). Although IgY antibodies in egg yolk do not affect vaccine efficacy, this approach has been used as a non-invasive method to monitor antibody titers in hens [27]. The anti-Deg-AKR serum IgY antibody levels increased during the entire experiment from day 0 to day 42 and remained higher than in control animals until the end of the experiment ( Figure 3A). In the egg yolk, the anti-Deg-AKR IgY levels were also higher in the vaccinated than in the adjuvant-only control group ( Figure 3B). The IgY response to the soluble mite protein extracts was also higher in the vaccinated than the control group but the difference was only statistically significantly (p < 0.05) higher on days 14 and 28 and days 28 and 42 after vaccination for serum and egg yolk, respectively ( Figure 3C, D). The soluble PRM protein extract is a complex protein mix, which may reflect an AKR dilution effect that can explain the differences in the antibody levels against Deg-AKR and PRM protein extract ( Figure 3A, B vs. Figure 3C, D). The serum IgY recognition of Deg-AKR (25.9 kDa) in vaccinated hens was also corroborated by Western blot ( Figure 3E). Serum antibodies also detected the presence of potential Deg-AKR multimers and some protein degradation products, which commonly occur with AKR and SUB [15,17]. Other recognized protein bands correspond to potential multimerization or degradation products of the recombinant protein which can also be observed in the Deg-AKR after purification by Ni affinity chromatography (dots). Abbreviation: MW, molecular weight marker (spectra multicolor broad range protein ladder; Thermo Fisher Scientific).
Vaccination against ectoparasites is not solely focused on the prevention of the infestation but also in the reduction of the parasite population [30]. This reduction can be achieved by affecting parasite feeding, reproduction and/or development by antigen-specific antibodies ingested after feeding on vaccinated animals [31]. Vaccination with Deg-AKR resulted in a 42% reduction in mite oviposition (GLMM, F = 6.060, df1 = 1, df2 = 747, p = 0.014) ( Table 1 and Figure 2). However, in this experiment, partially engorged adult mites did not oviposit and among fed mites only 22.4% (168 out of 749) oviposited. These facts resulted in a lower number of eggs/mite (0.3 to 1.0; Table 1) when compared to previous reports (>3.0) [32,33]. The effect of vaccination with AKR on the oviposition in D. gallinae has not been reported before, but SUB/AKR has demonstrated a reduction in the oviposition of several ectoparasite species after feeding on vaccinated hosts [17,32,34,35].
As seen in previous vaccine efficacy experiments with SUB/AKR in ticks and insects [29,31,[35][36][37], the anti-Deg-AKR IgY levels present in the sera and egg yolk showed a significant negative correlation (r = −0.106 (sera) and r = −0.125 (yolk), p < 0.01) with the amount of eggs laid per fed mite ( Figure 4A,B). In ticks, the effect of the vaccination on the oviposition is generally correlated with a reduction in the body size and weight of engorged females [31,32]. However, herein we did not detect variations in the body size of the fully engorged mites that were collected from vaccinated and control hens ( Figure 4C,D). The average body length in engorged female mites was 1039 µm (from 830 to 1230 µm), the average body width was 563 µm (from 465 to 657 µm), and the average body area was 463 mm 2 (from 317 to 633 mm 2 ). Nevertheless, although selected mites looked like they were fully engorged when sorted with the stereoscope, the variation in body size suggested different blood volumes ingested during the blood meal. These experiments suggested that the use of SEM is a useful tool for measuring blood meal ingestion in individual organisms or developmental stages that cannot be weighed. As seen in previous vaccine efficacy experiments with SUB/AKR in ticks and insects [29,31,[35][36][37], the anti-Deg-AKR IgY levels present in the sera and egg yolk showed a significant negative correlation (r = −0.106 (sera) and r = −0.125 (yolk), p < 0.01) with the amount of eggs laid per fed mite ( Figure 4A, B). In ticks, the effect of the vaccination on the oviposition is generally correlated with a reduction in the body size and weight of engorged females [31,32]. However, herein we did not detect variations in the body size of the fully engorged mites that were collected from vaccinated and control hens ( Figure 4C, D). The average body length in engorged female mites was 1039 µm (from 830 to 1230 µm), the average body width was 563 µm (from 465 to 657 µm), and the average body area was 463 mm 2 (from 317 to 633 mm 2 ). Nevertheless, although selected mites looked like they were fully engorged when sorted with the stereoscope, the variation in body size suggested different blood volumes ingested during the blood meal. These experiments suggested that the use of SEM is a useful tool for measuring blood meal ingestion in individual organisms or developmental stages that cannot be weighed. In this study, vaccination with Deg-AKR did not significantly affect other mite developmental stages. No statistically significant effect was observed after vaccination with the Deg-AKR on feeding rates and mite mortality in adults or in protonymphs or development (i.e., molting rates in fed and partially fed protonymphs and larval hatching) ( Table 2). All eggs laid by females mites hatched by day 7 without differences between groups. During this experiment, lower feeding rates in adult mites   Data shown are a compilation from the four assays. Feeding rates were scored on day 1 of the assay, immediately following removal of the feeding devices from the hens. Oviposition and mortality were scored on day 7. Data were analyzed statistically to compare results between mites fed on vaccinated and control hens by GLMM (F = 6.06, p = 0.014, gL1 = 1, gL2 = 747). In this study, vaccination with Deg-AKR did not significantly affect other mite developmental stages. No statistically significant effect was observed after vaccination with the Deg-AKR on feeding rates and mite mortality in adults or in protonymphs or development (i.e., molting rates in fed and partially fed protonymphs and larval hatching) ( Table 2). All eggs laid by females mites hatched by day 7 without differences between groups. During this experiment, lower feeding rates in adult mites were observed when compared to other studies where the on-hen feeding device was applied [21,25]. In our study, the 19 ± 6 % of the starved adult females fully fed across the four replicates. Of the protonymphs, 9 ± 4% were completely engorged, which is a similar feeding rate to that obtained in previous experiments [21]. Adult mites were starved at RT for 10 days, which is a different conditioning protocol from the suggested 3 weeks of starving at 4 • C [21]. However, we used these mites because they appeared healthier than the ones we starved under the recommended conditions [21]. Of the adult mites recovered, 7 ± 4 % were partially fed, but no differences in the number of partially-fed mites were observed between groups. Vaccination with the mosquito AKR was described to increase mite mortality by 35% after an in-vitro feeding assay [15]. However, in-vitro tests have been shown to present a high background mortality of mites [22]. The in-vitro test employs heparinized blood from vaccinated and control hens and mites feed on it through a natural or artificial membrane in an incubator at 36 • C and 75-85% relative humidity [15]. Therefore, mite mortality effects seen in in-vitro feeding systems may be induced by vaccination alone or exacerbated by the high temperatures required to induce mite feeding in the chamber and the presence of anticoagulants in the blood [22,38]. The use of the on-hen feeding device allows mites to feed directly on the hen which circumvents many of these limitations with the in-vitro feeding device and is more likely to present a true reflection of vaccine effects.

Conclusions
The results obtained in this study identified the D. gallinae AKR homolog and demonstrated the efficacy of the vaccination with the Deg-AKR antigen for the control of PRM in hens. The 42% reduction in mite oviposition generated by the vaccination supports the Deg-AKR as a candidate protective antigen for the control of PRM.