ISSN2423-9143

福岡県農林業総合試験場特別報告

第 12 号

Studies on a novel control system ofDermanyssus gallinae

令和２年３月

福岡県農林業総合試験場（福岡県筑紫野市大字吉木）

SPECIAL BULLETIN
OF
THE FUKUOKA AGRICULTURE AND FORESTRYRESEARCH CENTER
NO.12

Studies on a novel control system ofDermanyssus gallinae
byMakiko ODAKA

THE FUKUOKA AGRICULTURE AND FORESTRYRESEARCH CENTER
Chikushino, Fukuoka 818-8549, Japan

March

2020

CONTENTS

CHAPTER 1 General introduction of Dermanyssus gallinae

・・・・・・・・・・・・・ 1

CHAPTER 2 Development of a trap for Dermanyssus gallinae

・・・・・・・・・・・・ 6

CHAPTER 3 Correlation between the proportion of stained eggs and the number of mites(Dermanyssus gallinae) monitored using a “non-parallel board trap” ・・・ 12

CHAPTER 4 Efficacy of a novel mixture of substances derived from food and food additives
for controlling Dermanyssus gallinae (Mesostigmata: Dermanyssidae) ・・・ 21

CHAPTER 5 General conclusion ・・・・・・・・・・・・・・・・・・・・・・・・ 30

ACKNOWLEDGEMENTS

REFERENCES

・・・・・・・・・・・・・・・・・・・・・・・・・・ 32

・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 33

和文摘要 ・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・・ 42

CHAPTER 1
General introduction of Dermanyssus gallinae
1.1. Taxonomy and common nameDermanyssus gallinae

D. gallinae is a blood-sucking

ectoparacite belongs to the parasitiformes order Mesostigmata (Gamasida), in the suborder
Monogynaspida, cohort Gamasina, superfamily Dermanyssoidea, family Dermanyssidae (Lindquist etal., 2009). The common name is poultry red mite or chicken mite.

1.2. Life cycle
Dermanyssus gallinae has five development stages: egg, larva, protonymph, deutonymph, andadult (Maurer and Baumgärtner, 1992; Axtell, 1999). Eggs of D. gallinae are laid in cracks, crevices,or litter in the poultry house and hatch into six-legged larvae in 2 to 3 days. The larvae molt into
protonymphs within 1 to 2 days without feeding. From protonymph stage, this mite has eight legs
(Gray and Barry, 2009). The protonymphs feed and molt into deutonymphs within 1 to 2 days. Thenthe deutonymphs take another blood meal in order to molt into adults within 2 to 3 days. Adults matesoon after molting. After mating, females lay 1-9 eggs every 12 to 24 h following a blood meal.
Females will lay about 30 eggs in their lifetime (Sparagano et al., 2014). It takes 7 days to complete
development stages (egg-to-egg) under optimal conditions (Chauve, 1998).

1.3. Morphology
The egg of D. gallinae is small (0.4mm x 0.3mm), oval, smooth and pearly white (Chauve, 1998).
The larva is white and same size with egg. From protonymph stages, the mite varies in length and incolor from gray to brown/red depending on feeding status (Sparagano et al., 2014). Adult is about 1.0- 1.5 mm in length (Sikes and Chamberlain, 1954; Sparagano et al., 2014). Adult has a prominent
dorsal shield that is oval and tapers toward the posterior part (Murano, 2006; Di Palma et al., 2012).
The posterior margin of the dorsal shi
pairs of setae. This mite has a roundish sub-triangular anal plate at posterior ventral surface. The anusis open on the posterior part of this plate. Adult female has long stylet-like chelicerae, and adult malehas scissor-like chelicerae (Murano, 2006).

1.4. Ecology
-1-

- 1 -

Dermanyssus gallinae usually feeds for 30 to 90 minutes at night every 2 to 4 days (Maurer et al.,1988). This mite parasitizes on its host only during blood-feeding (Chauve, 1998). After blood-feeding,it spends away from host and hides in the cracks, crevices and litter in the poultry farm. Temperatures
below -20°C and above 45°C are lethal for D. gallinae (Mul et al., 2009). The optimum conditions for
growth of D. gallinae are at 25°C to 30°C with a humidity of 70 to 85% (Tucci et al., 2008). This mitecan survive for 9 months without feeding (Nordenfors et al., 1999).

1.5. Hosts
Dermanyssus gallinae feeds blood of more than 30 species of domestic and wild birds (Roy and
Chauve, 2007) and attacks laying hens in particular (Chauve, 1998; Sparagano et al., 2014). It
occasionally bites mammals such as dogs (Ramsay et al., 1975; De Clerq and Nachtegaele, 1993), cats(Grant, 1989), horses (Mignon and Losson, 2008), rodents (Lucky et al., 2001) and even humans(Rosen et al., 2002; Abdigoudarzi et al., 2014; Sparagano et al., 2014) in the absence of birds.

1.6. Prevalence
Dermanyssus gallinae is distributed worldwide (Sparagano et al., 2014). The massive infestation
causes serious problems for poultry production in Europe such as Demark, France, The Netherlands,
Germany, Belgium (Mul, 2013), Norway (Øines and Brännström, 2011), Sweden (Höglund et al.,1995), Poland (Tomasz, 2003), Italy (Marangi et al., 2014), UK (Guy et al., 2004; Fiddes et al., 2005),
and in United States (Ruff, 1999; Gray and Barry, 2009) and in Africa such as Kenya (Mungube et al.,2008) and Tunisia (Gharbi et al., 2013). Also in Asia, prevalence rate of D. gallinae in commercial
egg layer farms is 64% and 85% in China (Wang et al., 2010) and Japan (Sparagano et al., 2009),
respectively.

1.7. Consequences of infestation
Dermanyssus gallinae affect animal and public health in particular relating to humans closely
associated with poultry as well as the productivity of the egg industry (Chauve, 1998; Sparagano etal., 2014; George et al., 2015).

1.7.1. Clinical sign in hens
In hens, Dermanyssus gallinae causes higher levels of preening, head-scratching, feather-pecking,cannibalistic behavior, decreasing of growth rates and feed conversion, anaemia and even death
-2-

- 2 -

(Chauve, 1998; Mul et al., 2009; Flochlay et al., 2017). This mite can lead to decrease in egg quality
due to blood spots on the shells and in egg production (Chauve, 1998). In addition,

infested chickens fall into immunosuppression, such as the suppression of antibody production(; Kaoud and El-Dahshan, 2010) and downregulation of Th1 and pro-inflammatory cytokines/chemokines (Harrington et al., 2010).

1.7.2. Clinical sign in humans
Dermanyssus gallinae can cause irritation, eczema, and allergic skin reactions (Potenza et al.,2008; Sahibi et al., 2008; Cafiero et al., 2011). Therefore, D. gallinae is being regarded as an
occupational hazard for poultry farm workers in Europe (Cafiero et al., 2011). In Japan, infestation of
D. gallinae have resulted in turnover of poultry farm employees (Murano, 2007; Murano et al., 2015).

1.7.3. Transmission diseases as a vector
Dermanyssus gallinae may serve as a disease vector for numerous pathogens including Salmonella
gallinarum and Salmonella enterica, Erysipelothrix rhusiopathiae, Listeria monocytogenes, Coxiellaburnetii, Nocardia brasiliensis, Mycoplasma synoviae, Newcastle disease, Fowlpox virus, Saint-Louis
encephalitis, the Eastern, Western, and Venezuelan equine encephalomyelitis viruses and tick-borne
encephalitis virus (Valiente Moro et al., 2005, 2009; Chu et al., 2014; George et al., 2015). Its vectorial
role in maintaining these pathogens on poultry farms is enhanced by its strong viability enduring tolong starvation (Sylejmani et al., 2016).
When D. gallinae attacks humans, there is a possiblility of transmission of spirochetes including
Borrelia burgdorferi, rickettsiae, Salmonellae, Bartonellae, Pasteurellae, Sporozoa, hemogregarines,
flagellates filariae and avian influenza A virus (Litwin, 1961; Sommer et al., 2016).

1.7.4. Economic impact
The annual economic cost for control of this mite and productivity losses caused by D. gallinae

Japan, respectively (van Emous, 2005; Sparagano et al., 2009). However, recently this estimated cost

population and infestation rate of D. gallinae.

1.8. Control management
1.8.1. Conventional methods
Cleaning of poultry farms in particular during the unoccupied period and maintaining good
-3-

- 3 -

hygiene practices help to reduce mite populations of D. gallinae, while only these methods are notsufficient for control the mite in most cases. The control of D. gallinae has relied on the applicationof synthetic acaricides, over 35 compounds (e.g., organochlorines, organophosphates, pyrethrin,
pyrethroids, carbamates, amitraz, and endectocides) (Chauve, 1998; Thind and Ford, 2007; Marangi
et al., 2012). However, many countries now restrict acaricides which can be used against D. gallinae
due to regulations on active ingredients. In addition, resistance of D. gallinae to acaricides has beenreported in many countries, including Japan (Beugnet et al., 1997; Nordenfors et al., 2001; Fiddes etal., 2005; Thind and Ford, 2007; Marangi et al., 2009; Murano et al., 2015). For example, more than
60% of poultry farms had experienced infestations of acaricide-resistant mites in British (Guy et al.,2004). Moreover, the misuse and abuse of acaricides often result in residual acaricides in the organs
and tissues of the laying hens (Marangi et al., 2012).

1.8.2. Emerging and future control strategies
1.8.2.1. Vaccines
A vaccine under development reduced number of D. gallinae in mite-infested birds (Harrington etal., 2009; Bartley et al., 2015). There is potential to develop a vaccine against D. gallinae based on
somatic or recombinant proteins, but their effects are still insufficient.

1.8.2.2. Biological methods
Biological control methods, such as natural enemies and entomopathogenic fungi, have been
examined in D. gallinae. As candidates of the natural enemies, several predatory mites consuming D.
gallinae

Lesna et al., 2009; Ali et al., 2012; Lesna et al., 2012). However, field

effect was strongly dependent on various factors such as temperature, alternative prey, contact
frequency between natural enemies and D. gallinae (Ali et al., 2012; Lesna et al., 2012). Someentomopathogenic fungus such as Metarhizium anisopliae were fetal to D. gallinae under laboratory
conditions (Kaoud, 2010; Tavassoli et al., 2011). However, their efficacy was insufficient even in semifield condition, because fungi require very high humidity or free waters to act.

1.8.2.3. Physical methods
D. gallinae in the
Netherlands and Norway (Mul et al., 2009). The disadvantage of heat-treatment is its high cost anddamage to the equipment in poultry houses (Mul et al., 2009). Therefore, this method is not suitable
to poultry houses which are large or have plastic equipment components (Mul et al., 2009).
Inert substances include diatomaceous earth, kaolin, and silicas. Many products of inert substances
are widely used (e.g., InsectoSec, BIOFA, Germany, and Decimite+, BASF, Germany). These products
-4-

- 4 -

absorb cuticular lipids from the surface of mites, resulting in death by dehydration (Vincent et al.,2003). However, these substances are difficult to apply in practical situations. For example, D. gallinae
becomes active during hot and humid periods in the poultry house (Nordenfors and Höglund, 2000;
Othman et al.
and Steenberg, 2009; Maurer et al., 2009). Their effect also was greatly affected by the quality of the
raw materials (Mul et al., 2009; Schulz et al., 2014). In addition, silica products were banned in the
Netherlands recently, because they pose serious risk for humans and animal due to irritation of
respiratory tract caused by inhaled silica particles (Flochlay et al., 2017).
The acaricidal effects of oils against D. gallinae have been reported in vitro, but not in the poultry
farm (Maurer et al., 2009). In addition, mineral oils, such as diesel, which was more toxic to phytoseiid
mites than plant oils (Momen et al., 2006), are not recommended for use in poultry houses because of
their unpleasant odor and the risk of egg contamination (Hoop, 2008).

1.9. The aim of our study
Dermanyssus gallinae can not be easily controlled due to an unclear optional timing for control ofthis mites in poultry farms. It is difficult for farmers to discover D. gallinae in early stages ofinfestation, because this mite is too small and hides during the daytime. After infestation of D. gallinae
prevailed in a poultry house, it is practically impossible to control the mites because of its high viability(Nordenfors et al., 1999) with reproductive potential (Maurer and Baumgärtner, 1992). Therefore, toclarify optimal timing of treatment with synthetic acaricidal substances against this mite is important.
Moreover, only applications of acaricides for controlling this mite has led a reduction of theireffectiveness (Beugnet et al., 1997; Nordenfors et al., 2001) as well as residual acaricides in poultry
products resulting in a threat for human health (Marangi et al., 2012). Therefore, alternatives control
methods against D. gallinae have been required.
of D. gallinae to monitor mite population in poultry farms. In order to clarify the indicator of optimal
timing for treatment with acaricidal substances, we analyzed the relationship between the damage
caused by mites and the mite population monitored using this trap. Furthermore, as an alternative
control measure, a mixture of substances derived from food and food additives (SFF), including apolysaccharide extract from laver seaweed, was examined for acaricidal effects on D. gallinae in vitro
and in vivo. Moreover, the mechanism of its acaricidal effect was examined.

-5-

- 5 -

CHAPTER 2
Development of a trap for Dermanyssus gallinae
2.1. Abstract
We have constructed a novel trap based on the behavior of the Dermanyssus gallinae andcompared it with the other traps. First, we prepared two types of trap made of two overlapping boards.
One type had the boards not touching each other (non-parallel board trap) and the other consisted of
two boards kept apart along the entire board (parallel board trap). Then we compared the rate ofcollected mites. As the result, the non-parallel board trap collected more mites than the parallel-board
trap. Next, to determine which materials would be most effective, we tested non-parallel board traps
were made of cedar, Japanese cypress, bamboo and polyvinyl chloride. The cedar and Japanese cypress
traps collected significantly more mites than the other traps. Furthermore, our non-parallel board trap
made of cedar collected significantly more mites than the corrugated cardboard and thick card traps.
In conclusion, the novel non-parallel board trap could capture the mites efficiently. This trap could bea useful tool for monitoring and exterminating mites and studying on the ecology of the poultry red
mite in poultry farms.

2.2. Introduction
Dermanyssus gallinae sucks blood only at night and hides in cracks, such as the connection
between cages, egg channels and conveyor belts for eggs during the daytime (Harrison, 1963; Oshio,1979). Recently, it has been confirmed that D. gallinae parasitizes daytime and night without leaving
poultry that is heavily infested by northern foul mite (Nakamae et al., 1997). It is difficult for farmers
to discover D. gallinae in the early stages of infestation, because it is only about 1.0mm in length andhides during the daytime (Sikes and Chamberlain, 1954). After infestation of D. gallinae prevailed ina poultry house, it is practically impossible to control the mites because they increase in the poultry
house rapidly due to high reproductive potential (Chuave, 1998). Therefore, Zenner et al. (2009) havesuggested to determine the threshold for starting extermination based on population of D. gallinae
monitored in poultry houses. Furthermore, we should take appropriate and effective measures at theearly stages of infestation following to the threshold. In order to determine the optimal timing forcontrol, it is necessary to use traps which have high ability for capture of D. gallinae as well aspracticality for monitoring mite population. The trap made of corrugated cardboard (cardboard trap)(Nordenfors and Chirico, 2001) has been evaluated to be effective. However, it is impossible tovisually count the number of mites in the cardboard trap without disassembling the trap. In addition,

-6-

- 6 -

the cardboard trap is not suitable for monitoring since it is vulnerable to moisture. None of the othereffective traps have been reported, and traps for D. gallinae are not generally used in Japan.
In this study, a novel trap with a higher capture rate than previously reported trap was developed.

2.3. Materials and methods
2.3.1. Dermanyssus gallinae
Dermanyssus gallinae used in this study was collected from one layer farm. Then we identified
them as D. gallinae with a stereomicroscope morphologically (Baker et al., 1956; Moss, 1968; Oshio,1979). Next, in order to breed D. gallinae, this mite was kept in a mouse rearing container (46.0cm x
46.0cm x 60cm) along with a young chicken for blood sucking (Boris Brown or Julia between 7 and
30 days old, male) and cedar traps for proliferation of D. gallinae (proliferation traps). We confirmedthat D. gallinae had naturally proliferated in the proliferation traps after regular observation.
Furthermore, in order to prevent escape of the mites, the container was sealed with adhesive tape, its
inner sides were lubricated with vaseline, and it was soaked in a limonene solution.

2.3.2. General test methods
A rearing container with over 50 mites as described above was placed in an incubator set at 28°C
and 75% relative humidity (RH). The traps for the test were placed on both sides of the proliferation
trap overnight (Fig. 2. 1). Then the number of mites caught in the traps was counted using a magnifying
glass. Since the test was conducted simultaneously with proliferation of the mites, the number of mites
in the container fluctuated. Therefore, we evaluated the ratio of the number of mites caught in the test
traps to the number of mites in the proliferation trap as the capture rate. For animal welfare, several
chickens were prepared and rotated for blood sucking. The time for a single chicken in the rearing
container was less than 15 hours.

2.3.3. Development of the trap
2.3.3.1. Gap shape
Two traps were made from 2 wood pieces each (4.5cm x 8.5cm x 1.0cm), and a gap was madeusing a hinge (length 25mm) to the short sides of the wooden pieces and connecting the two pieces.
One type was a non-parallel board trap, as the opposite side from the hinge was allowed to touch (Fig.
2. 2 A-a). The other type of trap was inspired by Hagimori et al. (2004) and small wooden dowel wasplaced in the gap on the opposite end from the hinge, resulting in the wooden pieces being parallel(Fig. 2. 2 A-b). Both traps were made of cedar wood with equal distances between the wood at thehinged end. We then compared the capture rate of D. gallinae. Experiments were performed five times.
-7-

- 7 -

2.3.3.2. Gap width
In order to determine the gap width of the hinged end, the gap width of 1.0mm, 2.0mm and 3.0mm
were examined. The trap with a 3.0mm gap width was used as a standard and traps with the other gap
widths were simultaneously examined. Experiments were performed three to five times.

2.3.3.3. Trap materials
Finally, in order to select the best material with high capture rate, non-parallel board traps with agap width of 3.0mm were made of cedar, Japanese cypress, bamboo and polyvinyl chloride. The cedar
trap was used as a standard and the other traps were simultaneously examined. Experiments performed
four to five times.

2.3.4. Comparison between the reported traps and our developed trap
The other traps were made according to the previous report (Nordenfors and Chirico, 2001) as abase. Corrugated cardboard (3.0 mm thick) was cut into 10cm x 7cm. For another type, we used thickcardboard (210 g/m2, size 20cm x 7cm) folded in half based on the previous report by Levot (1991).
Specifically, we put 100 adult mites (male and female) saturated with blood, and the two traps into arearing container. After a night, we then recorded the number of mites collected in each trap.
Experiments were replicated six times.

2.3.5. Statistical analysis
The data was analyzed using statistical software R-3.0.1. An F-test for equality of variance was
performed after checking the normal distribution using the Kolmogorov-Smirnov verification method.
According to these results, a Student's t-test or Welch's t-test were carried out. The significance level
was set to p <0.05. The capture rate of the mite was arcsine-transformed and statistically processed.

2.4. Results and discussion
Gap shape of the traps did not significantly affect their capture of D. gallinae. However, at all gap
widths, there was a tendency that the non-parallel board trap had a higher capture rate than the parallelboard trap (Table 2. 1). In the parallel board trap, D. gallinae developed clusters against the small
wooden dowel on the unhinged end of the trap (Fig. 2. 2 B-b). In contrast, the mites congregated along
the unhinged side of the trap in the non-parallel board trap (Fig. 2. 2 B-a). This difference may beaffected by darkness of the gap in the non-parallel board trap that had less light than the parallel board
trap, because D. gallinae avoid light. In addition, the non-parallel board trap with a variation in the

-8-

- 8 -

gap may have easily captured different stage of the mites who prefer a tight fit. Moreover, the nonparallel board trap is more practical than the parallel board trap, because it is simple and stable.
Although the gap width test showed no statistically significant differences, there was a tendency
that traps with a 3.0mm gap width captured more mites than the others (data not shown).
As for comparison of trap material, the cedar trap had significantly (p <0.01) higher capture rateof D. gallinae than those made of bamboo or polyvinyl chloride (Table 2. 2). The Japanese cypress
trap had the same capture rate as well as the cedar trap. Among the test materials in this study,
polyvinyl chloride was better than the other materials in reusability. Dermanyssus gallinae sometimes
develop clusters on the polyvinyl chloride feeding troughs. However, the polyvinyl chloride wasinferior to cedar in its capture rate, even when wood derived ingredients and scents were added or theinside of the trap was made to be rough (data not shown). In the case of the poultry red mite,
protonymphs have the highest survival rate at 70 to 90% RH (Nordenfors et al., 1999). Since cedar
and Japanese cypress absorb and retain much more moisture than polyvinyl chloride, mites must prefer
the cedar and Japanese cypress traps.
As the result, we chose the non-parallel boards made of cedar, which costs less than Japanesecypress, with a gap width of 3.0mm as our developed trap.
Our developed trap captured significantly (p <0.01) more mites than the cardboard trap or the thickcard trap (Table 2. 3). This indicated that our novel developed trap is superior to the previous traps in
capturing D. gallinae. The thick card trap is similar in design to our developed trap. Therefore, we
assumed that the difference in capture rates might be related to properties of the trap surface andingredients contained in the material.
Mul et al. (2009) described four types of traps for the poultry red mite: ADAS traps, cardboard
traps, perch traps and tube traps. Several studies on the dynamics of populations of D. gallinae using
traps have already been reported (Harrison, 1963; Levot, 1991; Nordenfors and Chirico, 2001; Zeneret al., 2009). Of these four traps, the cardboard trap was effective to some extent. Even though thistrap is useful for extermination, it is difficult to count number of mites in the trap. For monitoring
purposes, we have to take apart its cardboard before counting the mites. In addition, this trap could not
be reused, if this trap was wet (Zenner et al., 2009). In contrast, our developed trap makes it easy tocount the number of mites, and its capture ability is not easily affected by moisture. As the result, ourdeveloped trap is superior in usefulness and capture ability. Therefore, our trap could be an ideal tool
to monitor an accurate mite population in the farm.
In application of mite trap, an exterminating method for D. gallinae using cardboard traps
containing neem oil or acaricides has been reported (Chirico and Tauson, 2002; Lundh et al., 2005).
In addition, using cardboard traps only as a capturing tool by discarding the trap after mites are

-9-

- 9 -

captured can also be considered. If our developed trap is available, our trap would be comparatively
effective due to its higher capture ability.
The ecophysiology of the poultry red mite is unclear. The effective accumulated temperature forD. gallinae has been still unknown although appearance of many agricultural pests can be predictedusing effective accumulated temperature. By using our developed trap as observation tool of mites,
the effective accumulated temperature for D. gallinae must be clarified.

Table 2. 1 Comparison of capture rate of Dermanysus gallinae
between traps with different gap widths
Capture rate of D. gallinae 1) (%Gap width1

Non-parallel board trap

parallel board trap

1.0mm

32.3±36.1

7.9±9.8

2.0mm

10.1±4.2

7.2±9.8

3.0mm

16.5±13.2

10.1±7.0

Capture rate of D. gallinae = (number of mites in test trap/number of mites in proliferation trap) x100

Table 2. 2 Comparison of capture rate of Dermanysus gallinae
between traps made of different materials
Capture rate of D. gallinae1) (%Materials
Cedar:

Japanese cypress

22.1±11.6:

18.5±11.0

Cedar

:

Bamboo11.6±5.8A:

0.1±0.2B

Cedar:

Polyvinyl chloride

55.6±9.1 A:

0.8±1.1B

1

Capture rate of D. gallinae = (number of mites in test trap/number of mites in proliferation trap) x100

AB

Different alphabet letters indicate significant difference (p <0.01)

Table 2. 3 Comparison of number of Dermanysus gallinae captured byour developed trap and previous traps
Type of trap

1

Number of mites per trap

Developed trap1):

Cardboard trap

78±18.6A:

6±3.8B

Developed trap:

Thick card trap

87±9.7A:

0±0.4BThe developed trap is non-parallel with a 3.0mm gap, cedar woodAB

Different alphabet letters indicate significant difference (p <0.01)- 10 -- 10 -

Fig. 2. 1 Schematic diagram of trap development test

Fig. 2. 2 Overview of two types of traps
A Outer view of traps
B Opened traps (many mites collected inside trap)a : non-parallel board trapb : parallel board trap

- 11 -

CHAPTER 3
Correlation between the proportion of stained eggs and the number of mites(-

3.1. Abstract
The poultry red mite (Dermanyssus gallinae) is a serious problem for the poultry industry
worldwide. However, the relationship between the mite population and the damage that they cause isstill unclear. In this study, the mite population in poultry houses was examined using an establishedtrap method, and the risk of blood-stained eggs caused by the mites was assessed. Traps were placed
once a week outside the egg channels and/or on the floor in two poultry farms in Fukuoka Prefecture,
Japan, from April 2012 to July 2014. The numbers of blood-stained eggs and total eggs were counted
at weekly intervals. The results showed that the number of mites increased from April to May, andreached a peak around the beginning of June when the average temperature and humidity were >24°C
and 70-90%, respectively. In the segmented model, the correlation between the proportion of bloodstained eggs and the number of mites or temperature was positive over a threshold. In conclusion, ourestablished trap method is useful for monitoring mites and can be used to predict when poultry farms
should be treated to prevent appearance of blood-stained eggs.

3.2. Introduction
Poultry red mite is not easily controlled and once a poultry house is infested it is very difficult toeradicate them (Chauve, 1998) because they can survive long periods of starvation (Nordenfors et al.,1999). Furthermore, it is almost impossible to avoid damage caused by poultry red mite after
infestation has spread in a poultry house because D. gallinae has a high reproduction potential (Maurer
and Baumgärtner, 1992). D. gallinae is small and hides during the daytime which makes it difficult
for farmers to observe them. In addition, resistance of poultry red mite to various synthetic products
has been reported in many countries (Beugenet et al., 1997; Nordenfors et al., 2001; Fiddes et al.,2005; Thind and Ford, 2007; Marangi et al., 2009). In Japan, resistance against all commercial
acaricides has increased recently (Murano et al., 2015). Problems of resistance to synthetic products
have likely worsened due to their misuse. Also acaricide residues in laying hens, eggs and muscle havebeen reported (Aulakh et al., 2006; Marangi et al., 2012).
A sensitive method is therefore required for monitoring populations of D. gallinae on farm tounderstand the problem and to prevent the damage caused by the mite. Also, analysis of the correlation
between the damage caused by mites and the mite population can identify indicators which can beused to control infestations by treatment for poultry red mite. Several studies on the dynamics of- 12 -- 12 -

populations of D. gallinae have already been reported (Nordenfors and Höglund, 2000; Nordenfors
and Chirico, 2001; Zenner et al., 2009). However, the relationship between the mite population andthe proportion of blood-stained eggs is still unclear.
In this study, the population of poultry red mite in poultry houses was examined using ourestablished trap which is more sensitive than the previously reported traps (Odaka et al., 2014) andthe risk of blood-stained eggs caused by mites was assessed. In addition, the relationship between
environmental factors in the poultry house and the mite population or the risk of blood-stained eggs
was revealed.

3.3. Materials and methods
3.3.1. Experimental sites and animals
This study was performed from April 2012 to July 2014 in two poultry farms (A and B) in Fukuoka
Prefecture, Japan. Farm A is located at the southwestern part of Fukuoka Prefecture and the other farm
B is located 44km apart from Farm A. These farms have cage-system poultry houses which arecommon in Japan. Farm A is a low floor poultry house with approximately 320 hens housed in two
rows of two level metal cages (Table 3. 1). Farm B has a high-rise floor poultry house where excretionfrom hens accumulates on the lower floor with approximately 250 hens housed in two rows of twolevel metal cages (Table 3. 1). The hens were kept at their farms for approximately 10 or 18 weeks(Table 3. 1) and D. gallinae had already been observed at both farms.
All examination in this study were carried out under permission by the farm owners and any
special treatment to chicken were not performed.

3.3.2. Measurement of the population of mites using traps-

et al., 2014) was used for monitoring

the population of mites. This trap consisted of two cedar boards (4.5 cm×8.5 cm×1.0 cm each)connected at one end by a hinge.
The traps were placed outside the egg channel using a hook and rubber band (egg channel traps),
and on the floor (floor traps) at intervals of ten cages once a week. Traps were collected after 24 h andthe mites in the traps were immediately put into sealed bags. The bags were cooled to 4 °C and images
were captured with a digital camera before the mites in the bags were returned to each sampling place.
Then, the number of mites in each picture (4,000 × 3,000 pixels) was counted.
The number of traps placed at each farm is shown in Table 3. 1. Traps were placed in only half ofthe space in the poultry house at farm A in 2013 and only egg channel traps were used for the survey
at farm B.
- 13 -- 13 -

3.3.3. Environmental data
Temperature and humidity were recorded hourly by more than one automatic logger (OndotoriTM,
T and D corporation, Nagano, Japan) at both farms during the survey except during the middle of April
2012 and March 2014. Average temperature and humidity were calculated daily.

3.3.4. The number of blood-stained eggs
To elucidate the effect of D. gallinae on egg production, the numbers of blood-stained eggs andtotal eggs were counted once a week. The proportion of stained eggs was calculated by dividing thenumber of stained eggs by the total number of eggs.

3.3.5. Statistical analysis
The number of mites collected by egg channel traps or floor traps at farm A plus 0.5 per trap was
logarithmically transformed. Means were compared between egg channel traps and floor traps
separately for each collection using analysis of variance.
The average number of mites collected by egg channel traps or floor traps per collection at farm
A plus half of the discrete unit, 0.5/m (m: number of traps), was logarithmically transformed(Yamamura, 1999, 2016). The relationship between the number of mites collected using the two traps
was examined by linear regression analysis.
We applied a segmented regression model to examine the relationship between the proportion ofstained eggs and the number of mites, assuming that egg staining is negligible when mite abundanceis below a certain level. In this analysis, the proportion of stained eggs was arcsine-square-root
transformed and the average number of mites per collection at both farms was logarithmicallytransformed after processing as described above to perform a linear regression analysis.
This model can be formulated as follows.
y=0

if x

b (breakpoint)y=a(x-b)

if xb (breakpoint),

where y is arcsine-square-root transformed proportion of stained eggs, x is the log-transformed (N(average number of mites per collection) + 0.5/m (number of traps)), and a and b are slope andbreakpoint to be estimated.
The segmented regression model was also applied to examine the relationship between the
proportion of stained eggs and the average daily temperature when the survey for stained eggs was
performed, assuming that egg staining is negligible when the temperature is below a certain level. In
this analysis, x was the average temperature as an independent variable instead of the number of mites.
Four missing data points of the average temperature at farm A and two at farm B were complemented
with predictive values according to the observatory data by the Meteorological Agency. A 95%- 14 -- 14 -

confidence interval of the breakpoint (b) and the slope (a) was estimated using bootstrapping repeated
10,000 times. The significance level was set to p <0.05. These analyses were performed using the Ri386 3.0.1 and Microsoft Excel 2013.

3.4. Results
3.4.1. Relationship between the number of mites and environmental factors
The fluctuations in mite population are shown in Figs. 3. 1 and 3. 2. The number of mites wereshown as logarithmically transformed (N+ 0.5) to avoid problems caused by 0. The number of mites
started to increase from April to May. Data from Farm A in 2013 (Fig. 3. 1 b-2013) and Farm B in2014 (Fig. 3. 2 b-2014) indicate that the number of mites reached a maximum near the beginning ofJune, after the average temperature was over 24°C and humidity was 70-90%.

3.4.2. Comparison between egg channel and floor traps
In 2012, the floor traps collected more mites than the egg channel traps (Fig.3. 1 b-2012). The
average number of mites in the floor traps was between 1.3 to 20 times greater than the numbers in
the egg channel traps. Significant differences (p <0.01 or p <0.05) in the number of mites wereobserved between the egg channel traps and the floor traps except on 24th April. In 2013, the egg
channel traps collected more mites than the other traps in most sampling periods between February
and late May (Fig. 3. 1 b-2013). In June, more mites were collected in floor traps than in egg channel
traps. Significant differences (p <0.01 or p <0.05) in the number of mites were observed between the
two traps on 19th March, 12th June and 23rd June.
On the other hand, the results of linear regression analysis showed that the number of mites trappedin the egg channel was positively correlated with the number trapped on the floor (p<0.01) (Fig. 3. 3).

3.4.3. Relationship between blood-stained eggs and mite numbers
At farm A, the stained eggs were first observed on 2nd May 2012 (Fig. 3. 1 b-2012) and 19th
March 2013 (Fig. 3. 1 b-2013). The corresponding average number of mites per egg channel trap onthese dates was 68.0 (2012) and 57.8 (2013) and for floor traps was 770.2 (2012) and 14.0 (2013). At
farm B, the stained eggs were first observed on 22nd May 2014 and the average number of mites peregg channel trap was 131.9 (Fig.3. 2 b-2014). The proportion of stained eggs then increased as thenumber of mites increased at both farms.
The segmented model for the relationship between the proportion of stained eggs and the numberof mites in the egg channel traps fits the observed data very well with estimated parameters, a=13.18(95% CI: 10.02-16.72) and b=1.52 (95% CI: 1.32-1.67; R²=0.84, p <0.00001; Fig. 3. 4A). The model- 15 -- 15 -

indicated that stained eggs occur when mite density in the egg channel trap exceeds a threshold of
101.52, 33.11-0.5/m (m; number of traps), and beyond the threshold the arcsine-square-root
transformed proportion of the stained eggs increased by a slope of 13.18 per 10 times increase in mitedensity. Similarly, the segmented model fits well with the observed relationship between the
proportion of stained eggs and the mite density in the floor trap with estimated parameters, a=13.61(95% CI: 10.86-18.37) and b=1.82 (95% CI: 1.61-2.08; R²=0.87, p <0.00001; Fig. 3. 4B). The model
indicated that stained eggs occur when mite density in the floor trap exceeds a threshold of 101.82,
66.07-0.5/m (m; number of traps).

3.4.4. Relationship between stained eggs and average temperature
The segmented model also fitted well with the observed relationship between the proportion ofstained eggs and the average daily temperature with estimated parameters. The model indicated thatthe stained eggs occur when the temperature is > 16.95°C (95% CI: 13.01-18.82°C, R²=0.81, p<0.00001; Fig. 3. 5), and beyond the threshold the arcsine-square-root transformed proportion ofstained eggs increased by a slope of 2.58 (95% CI: 2.35-3.90) per 1°C increase in temperature.

3.5. Discussion
We found an increase in mite numbers during hot and humid periods (between May and June) in
accordance with previous reports (Nordenfors and Höglund, 2000; Othman et al., 2012; Murano et al.,2013). In addition, the optimum conditions for growth of D. gallinae were reported to be attemperatures between 25°C and 30°C with a humidity of 70-85% (Tucci et al., 2008). Our observationof the mite populations is almost consistent with this previous report. The number of mites decreased
gradually because D. gallinae is very sensitive to humidity (Chauve, 1998) and the high humidity
during the rainy season (end of June/beginning of July) in Japan would have had an effect on thegrowth of the mites. A similar phenomenon has been reported in Japan previously (Murano et al.,2013).
A difference in the number of mites collected by the egg channel traps and the floor traps wasobserved between 2012 and 2013 at farm A. Maurer (1993) reported that poultry red mite drops onto
the floor after feeding to seek shelter, manure and litter, where they are able to hide. Mites which drop
onto the floor may have preferred traps to shelter in because manure was scraped out every day at farm
A. Moreover, the temperature in April 2012 was higher than in April 2013. The number of mites in thefloor traps would be influenced by temperature because increasing temperature is effective in
activating D. gallinae (Kilpinen, 2001). However, the number of mites in both traps was positively
correlated, indicating that both could be good methods for monitoring the mite population. On the- 16 -- 16 -

other hand, the above results suggested that the number of mites in the floor traps may be different
depending on the poultry house structure. Therefore, we recommend that the egg channel traps might
be more useful for monitoring mites in cage-system poultry houses.
We have focused on blood-stained eggs as a consequence of poultry red mite. In Japan, poultry
farm workers sometimes leave their jobs (Murano et al., 2008; Murano et al., 2015) because they havebeen suffering from transient skin irritation, more serious eczema and even allergic reactions (Potenza
et al., 2008; Sahibi et al., 2008; Cafiero et al., 2011). In our preliminary study, workers started tocomplain about the mites when the stained eggs were first observed at both farms (data not shown).
van Emous et al. (2005) reported that substantial infestations by the mites may increase blood-stained
eggs. However, the relationship between the stained eggs and the number of poultry red mite in poultry
houses was unknown. In this study, we applied the segmented regression model which provided aguideline for mite density and temperatures which can be used for mite control measures. In this model,
the correlation between the proportion of stained eggs and the number of mites was positive over a
threshold. In particular, the number of mites using the egg channel trap indicated a clear minimum
mite density when stained eggs occur. The number of mites using floor traps and temperature did notpresent clear thresholds, but below these thresholds the proportion of stained eggs was small anyway.
The proportion of stained eggs in Farm A were higher than in Farm B. Furthermore, the averagetemperature around farm A was about 0.4°C higher than those around Farm B from April to July.
Therefore, the rapid increase in the proportion of stained eggs in Farm A might have been influenced
by average temperatures. Further analysis is required to clarify the relationship between the averagetemperature in a poultry house and the proportion of stained eggs.
In conclusion, we confirmed that monitoring mites with our established traps is a useful method
for identifying the time periods where application of mite control measures may be implemented toprevent the appearance of stained eggs. By monitoring poultry red mite, the economic damage causedby them and the resistance to synthetic acaricides may be reduced and the risk of poultry productresidues could be minimized. Control of poultry red mite under the average number of mites (N),33.11-0.5/m (m; number of traps) using egg channel traps, is likely to be effective.

- 17 -- 17 -

Table 3. 1 Hens and mite traps used in this study
Number of hens
Age of hens (weeks old)
Commercial layers
Trap durance
Number of traps

Farm A
320 (2012)
160 (2013)55-64 (2012)53-70 (2013)
Hy-Line Brown, Lohman LSL
24 hr
32 egg channel traps (2012)16 floor traps (2012)16 egg channel traps (2013)
8 floor traps (2013)

Farm B
250 (2014)
48-65 (2014)
Hy-Line Brown
24 hr
24 egg channel traps (2014)

Figure3. 1 Environmental data (a) and dynamics of mites and the proportion of blood-stained eggs(b) at farm A. Horizontal dotted line indicates the average temperature of 24°C.
Asterisks (* and **) indicate significant differences (p <0.05 and p <0.01, respectively)
between egg channel traps and floor traps. N: the average number of mites collectedby egg channel traps or floor traps.

- 18 -- 18 -

a-2014100

Relative humidity
Average temperatureb-2014
5

45
40

80

Egg channel traps

4

30
25

35

20

30

60

3

25
40

15

20

2
10

15

20

1

10
0

5

5

0

0

Figure 3. 2 Environmental data (a) and dynamics of mites and the proportion of blood-stained
eggs (b) at farm B. Horizontal dotted line indicates the average temperature of 24°C.
N: the average number of mites collected by egg channel traps.

Figure 3. 3 Comparison of the number of mites in egg channel traps and floor traps. Values of R2
and p were obtained from linear regression analysis.
N: the average number of mites collected by egg channel traps or floor traps, m:number of traps- 19 -- 19 -

A

B

0.3

0.3

0.2

0.2

0.1

0.1

0

0
0

0

1
2
3
4
Number of mites in egg channel traps x(log10(N 0.5/m))

1
2
3
Number of mites in floor traps x(log10(N 0.5/m))

4

Figure 3. 4 Relationship between the proportion of stained eggs and the average number of mites
collected by the egg channel traps (A) or the floor traps (B). Segmented linear models
were plotted after re-transformation from arcsine-square-root values. Values of R2 andp were obtained from linear regression model in which the proportion of stained eggs
was arcsine-square-root transformed. N: the average number of mites collected by egg
channel traps or floor traps, m: number of traps.

Figure 3. 5 Relationship between the proportion of stained eggs and average temperature.
Segmented linear model was plotted after re-transformation from arcsine-square-rootvalues. Values of R2 and p were obtained from linear regression model in which theproportion of stained eggs was arcsine-square-root transformed.

- 20 -- 20 -

CHAPTER 4
Efficacy of a novel mixture of substances derived from food and food additives forcontrolling Dermanyssus gallinae (Mesostigmata: Dermanyssidae)
4.1. Abstract
Dermanyssus gallinae De Geer (Mesostigmata: Dermanyssidae), also known as poultry red mite,causes serious economic losses to the poultry industry worldwide. In this study, a mixture of
substances derived from food and food additives (SFF), including a polysaccharide extract from laver
seaweed, Pyropia yezoensis (Bangiaceae), was examined for acaricidal effects. Treatment with SFF
was more effective in killing mites by blocking their respiratory organs than SFF without thepolysaccharide extract. In a field trial, treatment with SFF reduced the density of mites in a poultry
farm as well as the proportion of blood-stained eggs. This SFF appears useful for controlling the
population of D. gallinae in poultry farms.

4.2. Introduction
The control of D. gallinae has relied on the application of acaricides (Marangi et al., 2012; Thindand Ford, 2007). However, the extensive and repeated usage of acaricides has resulted in a reductionof their effectiveness due to the acquisition of resistance by poultry red mite (Beugnet et al., 1997;
Nordenfors et al., 2001). Such resistance to acaricides has been reported in many countries, including
Japan (Fiddes et al., 2005; Thind and Ford, 2007; Marangi et al., 2009; Murano et al., 2015). In
addition, the misuse and abuse of acaricides often result in residual acaricides in the organs and tissuesof the laying hens (Marangi et al., 2012). Therefore, we focused on physical control as an alternative
control measure against D. gallinae. Some inert substances and oils have been reported to havephysical control activity against D. gallinae under laboratory conditions (Maurer et al., 2009).
However, previous studies have not identified substances that could exert stable acaricidal effects
under field conditions (Kilpinen and Steenberg, 2009; Maurer et al., 2009).
In this study, we focused on a mixture of substances derived from food and food additives (SFF).
The SFF consisted of vegetable oil, organic acid, lecithin, and polysaccharide. Among these
constituents, oil has been reported to have a blocking effect on the spiracles of several plant pest insects
and mites (de Ong et al., 1927; Davidson et al., 1991; Stadler and Buteler, 2009). In addition,
Katsumoto et al. (2012) reported that lecithin may enhance the acaricidal effect of vegetable oil on
aphids. In particular, we focused on a polysaccharide extract from Pyropia yezoensis (Ueda) M. S.
mainly porphyran withapproximately 0.83 mg/mg of glucose. Porphyran is a sulfated polysaccharide from the hot water-- 21 -- 21 -

soluble portion of the algal cell wall (Morrice et al., 1983), and it has a relatively high viscosity(Sugano et al., 2009; Bhatia et al., 2010). In addition, Bhatia et al. (2010) showed that porphyran had
amphiphilic properties and might be useful as a new polysaccharide surfactant. Therefore, thispolysaccharide was expected to function as an emulsifier in the SFF, similar to other surfactants incommon insecticides.
In this study, the effects of SFF on the mortality of D. gallinae was examined in vitro and in vivo.
Moreover, the mechanism of its acaricidal effect was examined.

4.3. Materials and methods
4.3.1. Dermanyssus gallinae
Adult and immature D. gallinae were collected from one commercial layer farm in Fukuoka
Prefecture, Japan. The collected mites were kept in a plastic cage (46 cm × 46 cm × 60 cm) at 28°C,
70% to 90% relative humidity (RH), and 12-h light:12-h dark photoperiod conditions without bloodsucking. Some non-parallel board traps (Odaka et al., 2014) were placed in the plastic cage to pick up
active mites. Trapped mites were used for the laboratory experiments within 3 days of collection.

4.3.2. Test substances
The SFF (Porufui; MMO Co., Ltd., Fukuoka, Japan) contained vegetable oil, organic acid, lecithin,
and 0.25% of polysaccharide extracted from P. yezoensis as described previously (Sumi et al., 2005).
Briefly, dried powder of P. yezoensi was mixed with water and heated at 100°C and 0.1 MPa for 180
min. The soluble fraction was separated by centrifugation, and debris was removed byConcentrating System 7M4-2P; Noritake Co., Ltd., Aichi, Japan) equipped with ceramic membranes(Membralox® P19-40; nominal filtering accuracy of 50 nm; Pall Corporation, New York, NY, USA).
After filtration with a synthetic adsorbent, the concentrated solution was dried and crushed. SFF
without the polysaccharide extract (SFFWP) was also obtained from MMO Co., Ltd. As controls, two
insecticides, carbamate (Barizon®; Meiji Seika Pharma Co., Ltd., Tokyo, Japan) and organophosphate(Premium Sumithion® 10% emulsifiable concentrate for animal use; SC Environmental Science Co.,
Ltd., Osaka, Japan) were diluted to the median values according to the prescription by the
manufactures before use.

4.3.3. Acaricidal efficacy test
The acaricidal efficacy of SFF was examined as described previously (Busvine and Barnes, 1947;
Hemingway et al., 1993; Koehler et al., 1993) with a minor modification. Although the previous- 22 -- 22 -

studies used filter papers or vinyl tiles to expose insects to pesticides, we used basswood plywood
boards that would maintain the humidity of the test area. Diluted solutions of the test substances, i.e.,
SFF, SFFWP, carbamate, and organophosphate, were sprayed onto the basswood plywood boards (6.0cm × 8.0 cm × 0.4 cm) with a sprayer (Canyon® HI-SPRAYER 500 ml; Asaka Industrial Co., Ltd.,
Osaka, Japan) at a constant pressure 5 cm from the boards. The boards were tilted gently several times
2. Then, 159 to 598 D. gallinae

adults and nymphs were transferred onto the plywood boards with a paintbrush without drying process
as described previously in the acaricidal efficacy test of oils (Maurer et al., 2009). Each plywood board
was covered with a petri dish (5.2 cm in diameter × 1.4 cm in height) and the space between the dishand the board was sealed with adhesive bonding tape so that the mites inside could not escape. Thisapparatus was set in the dark at 28°C and 70% to 90% RH. After 24 h the numbers of surviving mites
and dead mites were recorded under a stereomicroscope. Mites were counted as dead if they did not
respond to a gentle touch with a paintbrush. The mortality rate of D. gallinae was calculated by
dividing the number of dead mites by the total number of mites. Five replicates were performed foreach treatment.

4.3.4. Scanning electron microscopic observation
To observe the effect of the SFF, nine to 15 mites that died after SFF exposure and 9 control mitesthat were exposed to distilled water were treated with osmium tetroxide vapor at room temperatureand then observed with a scanning electron microscope (SEM; TM3000; Hitachi, Tokyo, Japan).

4.3.5. Poultry farm and experimental design of the field experiment
The field experiment was performed from April to July, 2016, in one poultry farm in Fukuoka
Prefecture, Japan. This farm had a cage-system poultry house, which had metal cages arranged in tworows, and 426 hens were bred. This poultry house was divided into three sections: a test section fortreatment with SFF; an untreated control section without any treatment; and a buffer section containing
20 cages that was located between the two other sections for preventing mite contamination (Fig. 4.
1a). The hens used in the experiment are described in Fig. 4. 1b. Before this experiment, the farm wasalready infested with D. gallinae.
To examine the effect of SFF on D. gallinae in the poultry farm, we started spraying SFF when
the average number of mites per trap exceeded 33.11-(0.5/m) (m; the number of traps placed in thepoultry house) as proposed by Odaka et al. (2017). In dilution of the SFF solution, dilution of 100fold or less caused adverse effects on the driving parts of the equipment in the poultry house in our
preliminary experiment. Therefore, the dilution rate of the SFF was decided to be 200-fold or more.
The SFF was sprayed once a week in June and once every 2 weeks in July. Cationic surfactant- 23 -- 23 -

(Cleakil®-100; Tamura-Seiyaku Corp., Tokyo, Japan) was sprayed to clean the equipment on the 20thof June, 2016. The SFF solution was mixed with a Wire Mixer ADL-3LR (Asaba Manufacturing, Inc.,
Nagano, Japan). The spray solution was applied directly onto the surfaces of the cages, cage wires,
feed troughs, and accompanying equipment using a power sprayer with a tank and a nozzle (RoundNozzle® 25 N-KA-055SB; Yamaho Industry Co., Ltd., Wakayama, Japan). The amount of spraysolution used was 0.5 L/m2.

4.3.6. Density of D. gallinae
To monitor the population of D. gallinae, non-parallel board traps were attached to the egg channel
at every 10 cages (Fig. 4. 1a, b) and collected after 24 h. The trapped mites were immediately put intosealed bags, and then stored at 4°C. Images were captured with a digital camera to record the numberof mites in each sealed bag.

4.3.7. Proportion of blood-stained eggs
The numbers of blood-stained eggs and total eggs were recorded every week. The proportions ofblood-stained eggs among the total number of eggs were calculated.

4.3.8. Statistical analysis
All analyses were carried out using R 3.3.2 (R Development Core Team, 2016). The effects of SFF
and SFFWP on the mortality rate of D. gallinae was analyzed by a generalized linear model (GLM).
To examine the effects of SFF, which was diluted to different concentrations, and the two insecticides,
Tukey's test was conducted using the multcomp package (version 3.2.2). Analyses by a generalized
linear mixed model with a Poisson error distribution were carried out using the lme4 package (version
3.2.2) to compare the numbers of mites between the test and control sections. The explanatory
variables were ‘Treatment’, ‘Preapply’, and ‘Day’ as the fixed effects, and ‘Subject’, where the cagetraps were placed, as a random effect. ‘Preapply’, the number of mites on each cage before the firsttreatment, was added as a parameter that accounted for the correlational structure of the repeatedmeasurements. Analyses by GLM were also conducted to evaluate the differences in the proportion ofblood-stained eggs between the test and control sections. The best-fitting models were selected basedon the Akaike information criterion.

4.4. Results
4.4.1. Effect of the polysaccharide extract on the mortality rate of D. gallinae
With the application of 200-fold diluted SFF and SFFWP, the mortality rates of D. gallinae were

- 24 -

85.7% ± 4.5% and 65.0% ± 13.1%, respectively (Fig. 4. 2a). SFF was significantly more effective inkilling mites than SFFWP (p < 0.01) (Fig. 4. 2a).

4.4.2. Concentration-dependent effect of SFF on the mortality of D. gallinae
The mortality rates of D. gallinae after the application of 100-fold, 200-fold, 400-fold, and 800fold diluted SFF were 97.2% ± 1.6%, 85.6% ± 6.3%, 45.4% ± 14.1%, and 22.2% ± 10.9%, respectively(Fig. 4. 2b). The lower diluted SFF was significantly more effective at killing mites than the higher
diluted SFF (p < 0.01). The mortality rates with the application of 750-fold diluted carbamate and 150fold diluted organophosphate were 6.5% ± 4.2% and 34.2% ± 22.7%, respectively.

4.4.3. SEM observation of SFF-treated mites
SEM observation showed that the respiratory organs of D. gallinae, i.e., the peritreme and spiracle,
were blocked after SFF treatment. The states of blocking of the peritreme and spiracle differed among
the individual mites. We classified the states of blocking into three categories: blockage of both theperitreme and spiracle (Fig. 4. 3a); blockage of only the peritreme (Fig. 4. 3b); and no blockage of theperitreme and spiracle (Fig. 4. 3c). The ratios of mites with blockage of both the peritreme and spiracle
after the application of 100-fold, 200-fold, 400-fold, and 800-fold diluted SFF were 100%, 88.9%,
80.0%, and 50.0%, respectively (Fig. 4. 3d). There was a trend that the ratio of mites with blockage ofthe peritreme and spiracle increased in a SFF concentration-dependent manner.

4.4.4. Effect of SFF application on the number of D. gallinae
Application of 200-fold diluted SFF to the poultry farm started on the 3rd of June, when the
average number of mites per trap was 84.6 in the test section. Significant differences (p < 0.01) in thenumbers of mites were observed between the test and control sections (Fig. 4. 4a).

4.4.5. Effect of SFF application on the proportion of blood-stained eggs
The proportion of blood-stained eggs in the test section remained below 1.0%, while that in thecontrol section increased from 4.3% (June 10) to 12.9% (June 29) (Fig. 4. 4b) during the observation
period. This demonstrated that SFF application had a statistically significant effect on the proportionof blood-stained eggs (p < 0.01).

4.5. Discussion
In this study, it was evident that SFF was more effective in controlling D. gallinae in farms with
reduction of effect of conventional insecticides, i.e., carbamate and organophosphate. We- 25 -- 25 -

demonstrated that the application of SFF in a poultry farm could decrease the number of mites as wellas the damage caused by the mites. In the poultry farms around the world, reduction of effect of thecommercial insecticides against D. gallinae has been reported (George et al., 2015). In British, morethan 60% of poultry farms had experienced infestations of acaricide-resistant mites (Guy et al., 2004).
Murano et al. (2015) reported that 18.7% of poultry farms in 29 prefectures had infested by D. gallinae
which acquired resistance against all conventional insecticides (three carbamates, one pyrethroid andthree organophosphates) in Japan.
Both the acaricidal effect of SFF against D. gallinae and the ratio of mites with blockage of theperitreme and spiracle depended on the concentration of SFF (Figs. 4. 2 and 4. 3). These results
indicated that the mites died of suffocation as a result of the blockage of their respiratory organs. The
vegetable oil in SFF was rich in glycerides of fatty acids, which are also known as spiracle-blocking
pesticide for agricultural mites and aphids (Miyata and Masuda, 2006a, 2006b).
SFF was significantly more effective in killing mites than SFFWP, confirming the acaricidal effectcarbohydrates (Kayama et al., 1983; Mumford and Miura, 1988), and approximately 80% of the
carbohydrate component is porphyran (Sugano et al., 2009; Kitano et al., 2012). In the extraction

performed to prevent the extraction of proteins and to remove the low molecular weight portion fromthe extracted substances. Therefore, the polysaccharide extract in SFF consists mainly of highmolecular weight porphyran, which has a mass of approximately 400 kDa (weight-average molecularweight), and small impurities, such as denatured proteins. Porphyran was reported to have highemulsifying activity and high emulsion stability, because it was presumed to adsorb to the surface ofoil droplets (Takahashi et al., 2000). In addition, higher molecular weight porphyran preparations had
higher emulsifying effects (Bhatia et al., 2010). Therefore, the dilution of SFF may have improved theuniformity of the solution, because porphyran was able to coat the surface of oil droplets in the SFF
solution. Solutions with high dispensability may facilitate the deposition of active ingredients, such as
the glycerides of fatty acids, onto mites. After being mixed with insecticides, some emulsifying
substances enhance the activities of the insecticides due to improvements to the viscosity, penetration,or tension of the solution (Hartzell and Wilcoxon, 1960; Wolfenbarger et al., 1967; Matsubara, 1968;
Imai et al., 1994). However, the mechanism of the effect of the porphyran in SFF on the mortality of
D. gallinae remains unclear.
Inert substances (e.g., diatomaceous earth, kaolin, and silicas) and oils (e.g., mineral oils and plantoils) have been reported to be physical acting substances against D. gallinae (Maurer et al., 2009;
Sparagano et al., 2014). However, these substances are difficult to apply in practical situations. For
example, D. gallinae becomes active during hot and humid periods in the poultry house (Nordenfors- 26 -- 26 -

and Höglund, 2000; Othman et al., 2012), and we also previously reported that the population of D.
gallinae reached a peak after the average temperature exceeded 24°C and the humidity was 70% to90% (Odaka et al.
(Kilpinen and Steenberg, 2009; Maurer et al., 2009). As for oils, the acaricidal effects of oils against
D. gallinae have been reported in vitro, but not in the poultry farm (Maurer et al., 2009). In addition,mineral oils, such as diesel, which was more toxic to phytoseiid mites than plant oils (Momen et al.,2006), are not recommended for use in poultry houses because of their unpleasant odor and the risk ofegg contamination (Hoop, 2008). Although our 200-fold diluted SFF solution smelled a little, theapplication of SFF was effective even under an environment of high humidity from June to July, when
the population of D. gallinae reached a peak.
The timing of treatment with acaricidal substances is important. After infestation of D. gallinae
prevailed in a poultry house, it is practically impossible to control the mites because of their highreproductive potential (Maurer and Baumgärtner, 1992). Therefore, we previously proposed using
non-parallel board traps to monitor the population level of the mites and to implement control
measures while the population is low (Odaka et al., 2017). In this study, we applied this method in thepoultry house and succeeded in controlling the population of the mites. As such, we recommend
monitoring of the mite population prior to the application of control methods.
In conclusion, SFF had an acaricidal effect by blocking the respiratory organs of D. gallinae andwas effective in controlling D. gallinae in the field. This SFF appears useful for controlling D. gallinae
in poultry farms.

b

Fig. 4. 1 The poultry farm used in this study and the design of the experiment.
a The arrangement of the test, control, and buffer sections in the poultry house is shown.
Treatment with a mixture of substances derived from food and food additives (SFF) covered
half of the buffer section as indicated by thick arrows.
b Information on the hens and traps used in the poultry house.

- 27 -- 27 -

Fig. 4. 2 Mortality rates of Dermanyssus gallinae.
a Mortality rates of D. gallinae with the application of SFF and SFF without the polysaccharide
extract (SFFWP). Asterisks indicate a significant difference (generalized linear model (GLM),
p < 0.01).
b Mortality rates of D. gallinae with SFF, which was serially diluted with distilled water, and two
insecticides, i.e., carbamate (Barizon®) and organophosphate (Premium Sumithion®). The
dilution rates are provided in parentheses. The mortality rate of mites exposed to distilled wateris also shown. Different alphabet letters indicate significantly different mortality rates (Tukey's
test, p < 0.01).

Fig. 4. 3 Scanning electron microscopic (SEM) observation of the respiratory organs of D. gallinae
after treatment with SFF.
a Blockage of both the peritreme and spiracle after treatment with SFF (1:200).
b Blockage of only the peritreme after treatment with SFF (1:400).
c No blockage of the peritreme and spiracle after treatment with distilled water.
d Ratios of D. gallinae with blockage of respiratory organs.

- 28 -

Fig. 4. 4 Changes in D. gallinae numbers and the proportions of blood-stained eggs during thefield trial.
a Changes in the numbers of D. gallinae per trap. The numbers of mites differed significantly
between the test and control sections (generalized linear mixed model, p < 0.01).
b Changes in the proportions of blood-stained eggs. The proportions of blood-stained eggs differedsignificantly between the test and control sections (GLM, p < 0.01).

- 29 -- 29 -

CHAPTER 5
General conclusion
Subject: Development of a trap for Dermanyssus gallinae
Sensitive monitoring method of the population of D. gallinae in the poultry house has beenrequired, because it is difficult for farmers to observe them visually and to understand the optimal
timing for control them. Therefore, we have constructed a novel trap, non-parallel board trap, basedon the behavior of the D. gallinae. The novel non-parallel board trap could capture more mites thanthe previous reported traps. This trap could be a useful tool for monitoring D. gallinae and studying
on the ecology of this mite in poultry farms.

Subject: Correlation between the proportion of stained eggs and the number of mites(Dermanyssus gallinae-

In this study, to clarify the indicator of optimal timing for treatment with acaricidal substances, themite population in poultry houses was examined using an established trap method, and the risk ofblood-stained eggs caused by the mites was assessed. The results showed that the number of mites
increased from April to May, and reached a peak around the beginning of June when the averagetemperature and humidity were >24°C and 70-90%, respectively. In the segmented model, thecorrelation between the proportion of blood-stained eggs and the number of mites was positive over a
threshold. In conclusion, our established trap method is useful for monitoring mites and can be used
to predict when poultry farms should be treated to prevent appearance of blood-stained eggs.

Subject: Efficacy of a novel mixture of substances derived from food and food additives forcontrolling Dermanyssus gallinae (Mesostigmata: Dermanyssidae)
In this study, a mixture of substances derived from food and food additives (SFF), including apolysaccharide extract from laver seaweed, Pyropia yezoensis (Bangiaceae), was examined foracaricidal effects as an alternative control measure. Treatment with SFF was more effective in killingmites by blocking their respiratory organs than SFF without the polysaccharide extract. In a field trial,
treatment with SFF reduced the density of mites in a poultry farm as well as the proportion of bloodstained eggs. This SFF appears useful for controlling the population of D. gallinae in poultry farms.

- 30 -

Conclusion
In this study, there are some novel findings.
1.

-

D. gallinae to monitor mite

population was developed.
-2.

when application of mite control measures may be implemented to prevent the appearance ofblood-stained eggs.
3. The novel mixture of substances derived from food and food additives (SFF) was effective incontrolling D. gallinae as a physical control substance.

- 31 -

ACKNOWLEDGEMENTSFirst of all, the author would like to express my great appreciation to Dr. Ken Maeda for accepting
me as a student and giving invaluable supports, advices and studies. The author learned everythingfrom Dr. Maeda.
The author is grateful thanks to Kazumasa Ogino for kindly giving invaluable advices. The author
is grateful thanks to Dr. Toshihiko Sunahara, Kazunori Matsuo, Kenji Takai for kindly expert advices
in statistical analysis. The author is grateful thanks to Michitaka Shikada, Takahiro Inoue, Yoshiyuki
Sakata for kindly giving invaluable advices and cooperation of field trials.
Grateful thanks are also due to the colleagues of all the members of Laboratory of Veterinary
Microbiology, Yamaguchi University, for their kind assistances.
Special gratitude is given to my all past, present superiors and seniors at the Fukuoka Agricultural
Research Center, Mr. Kenichi Asada, Mr. Syoujiro Kasa, Mr. Tsugumitsu Kamori and Dr. Futoshi
Koyama for giving elaborated guidance and invaluable advices.
Finally, I would like to thank my parents and my children for support to me throughout my study.

- 32 -

REFERENCES
1. Abdigoudarzi, M., Mirafzali, M. S., Belgheiszadeh, H. (2014). Human infestation withDermanyssus gallinae (Acari: Dermanyssidae) in a family referred with pruritus and skinlesions. J. Arthropod-Borne Dis. 8: 119 123.
2. Ali, W., George, D., Shiel, R., Sparagano, O., Guy, J. (2012). Laboratory screening of potential
predators of the poultry red mite (Dermanyssus gallinae) and assessment of Hypoaspis miles
performance under varying biotic and abiotic conditions. Vet. Parasitol. 187: 341-344.
3. Aulakh, R. S., Gill, J. P. S., Bedi, J. S., Sharma, J. K., Joia, B. S., Ockerman, H. W. (2006).
Organochlorine pesticide residues in poultry feed, chicken muscle and eggs at a poultry farm in
Punjab. India. J. Sci. Food Agric. 86: 741 744.
4. Axtell, R. C. (1999). Poultry integrated pest management; status and future. Integr. Pest Manag.
Rev. 4: 53 73.
5. Baker, E. W., Evans, T. M., Gould, D. J., Hull, W. B., Keegan, H. L. (1956). A manual of parasitic
mites of medical or economic importance. National Pest Control Association, New York, pp.
12-18.
6. Bartley, K., Wright, H. W., Huntley, J. F., Manson, E. D., Inglis, N. F., McLean, K., Nath, M., Bartley,Y., Nisbet, A. J. (2015). Identification and evaluation of vaccine candidate antigens from thepoultry red mite (Dermanyssus gallinae). Int. J. Parasitol. 45: 819-830.
7. Beugnet, F., Chauve, C., Gauthey, M., Beert, L. (1997). Resistance of the red poultry mite topyrethroids in France. Vet. Rec. 140: 577-579.
8. Bhatia, S., Namdeo, A. G., Nanda, S. (2010). Factors effecting the gelling and emulsifying
properties of a natural polymer. Sys. Rev. Pharm. 1: 86-92.
9. Busvine, J. R., Barnes, S. (1947). Observations on mortality among insects exposed to dry
insecticidal films. Bull. Entomol. Res. 38: 81-90.
10. Cafiero, M. A., Galante, D., Camarda, A., Giangaspero, A., Sparagano, O. (2011). Why
dermanyssosis should be listed as an occupational hazard. J. Occup. Environ. Med. 68: 628.
11. Chauve, C. (1998). The poultry red mite Dermanyssus gallinae (De Geer, 1778): current situationand future prospects for control. Vet. Parasitol. 79: 239-245.
12. Chirico, J., Tauson, R. (2002). Traps containing acaricides for the control of Dermanyssus gallinae.
Vet. Parasitol. 110: 109-116.
13. Chu, T. T. H., Murano, T., Uno, Y., Usui, T., Yamaguchi, T. (2014). Molecular detection of avian
pathogens in poultry red mite (Dermanyssus gallinae) collected in chicken farms. J. Vet. Med.
Sci. 76: 1583-1587.
14.

Dermanyssus gallinae invasion in battery-husbandry raised fowls.

- 33 -

Rev. Med. Vet. Toulouse 152: 89 96.
15. Davidson, N., Dibble, J., Flint, M., Marer, P., Guye, A. (1991). Managing insects and mites withspray oils. University of California Special Publication: 3347.
16. De Clerq, J., Nachtegaele, L. (1993). Dermanyssus gallinae infestation in a dog. Canine Pract. 18:
34-36.
17. de Ong, E. R., Knight, H., Chamberlain, J. C. (1927). A preliminary study of petroleum oil as an
insecticide for citrus trees. Hilgardia 2: 351-384.
18. Di Palma, A., Giangaspero, A., Cafiero, M. A., Germinara, G. S. (2012). A gallery of the key
characters to ease identification of Dermanyssus gallinae (Acari: Gamasida: Dermanyssidae)
and allow differentiation from Ornithonyssus sylviarum (Acari: Gamasida: Macronyssidae).
Parasit. Vectors. 5: 104.
19. Fiddes, M. D., Le Gresley, S., Parsons, D. G., Epe, C., Coles, G. C., Stafford, K. A. (2005).
Prevalence of the poultry red mite (Dermanyssus gallinae) in England. Vet. Rec. 157: 233-235.
20. Flochlay, A. S., Thomas, E., Sparagano, O. (2017). Poultry red mite (Dermanyssus gallinae)
infestation: a broad impact parasitological disease that still remains a significant challenge forthe egg-laying industry in Europe. Parasit. Vectors.10: 357.
21. George, D. R., Finn, R. D., Graham, K. M., Mul, M., Maurer, V., Valiente Moro, C., Sparagano,O. (2015). Should the poultry red mite Dermanyssus gallinae be of wider concern for veterinary
medical science. Parasit. Vectors. 8:178.
22. Gharbi, M., Sakly, N., Darghout, M. A. (2013). Prevalence of Dermanyssus gallinae(Mesostigmata: Dermanyssidae) in industrial poultry farms in North-East Tunisia. Parasite 20:
41.
23. Grant, D. I. (1989). Parasitic skin diseases in cats. J. Small Anim. Pract. 30: 250-254.
24. Gray, R. M., Barry, M. O. (2009). Mites (Acari). In: Gary, R., Lance, A. (eds). Medical and
Veterinary Entomology, 2nd edn. Academic Press, London, pp 433-492.
25. Guy, J. H., Khajavi, M., Hlalel, M. M., Sparagano, O. (2004). Red mite (Dermanyssus gallinae)
prevalence in laying units in Northern England. Br. Poult. Sci. 45: S15-16.
26. Hagimori, I., Senbo, S., Matsuda, A. (2004). Mite catcher and use of mite catcher. Patent nunmber:
4534671 (JP2006067810) (in Japanese).
27. Harrington, D., Canales, M., de la Fuente, J., de Luna, C., Robinson, K., Guy, J., Sparagano, O.
(2009). Immunisation with recombinant proteins subolesin and Bm86 for the control of
Dermanyssus gallinae in poultry. Vaccine 27: 4056-4063.
28. Harrington, D., Robinson, K., Guy, J., Sparagano, O. (2010). Characterization of theimmunological response to Dermanyssus gallinae infestation in domestic fowl. TransboundEmerg. Dis. 57: 107-110.

- 34 -

29. Harrison, I. R. (1963). Population studies on the poultry red mite Dermanyssus gallinae (DEG.).
Bull. Entomol. Res. 53: 657-664.
30. Hartzell, A., Wilcoxon, F. (1960). The importance of wetting agents as affecting the toxicity ofcertain insecticides. Contrib. Boyce Thompson Inst. 20: 421-424.
31. Hemingway, J., Small, G. J., Monro, A. G. (1993). Possible mechanisms of organophosphorus andcarbamate insecticide resistance in German cockroaches (Dictyoptera: Blattelidae) fromdifferent geographical areas. J. Econ. Entomol. 86: 1623-1630.
32. Höglund, J., Nordenfors, H., Uggla, A. (1995). Prevalence of the poultry red mite, Dermanyssus
gallinae, in different types of production systems for egg layers in Sweden. Poult. Sci. 74:
1793 1798.
33.
Zollikofen, pp 1-6 (in German).
34. Imai, T., Tsuchiya, S., Morita, K., Fujimori, T. (1994). Surface tension-dependent surfactant
toxicity on the green peach aphid, Myzus persieae (SuLzER) (Hemiptera: Aphididae). Appl.
Entomol. Zool. 29: 389-393.
35. Kaoud, H. A. (2010). Susceptibility of poultry red mites to entomopathogens. Int. J. Poult. Sci. 9:259 263.
36. Kaoud, H. A., El-Dahshan, A. R. (2010). Effect of red mite (Dermanyssus gallinae) infestation on
the performance and immune profile in vaccinated broiler breeder flocks. J. Am. Sci. 6: 72-78.
37. Katsumoto, T., Yanaginuma, D., Kawase, H. (2012). Insecticidal composition for agriculture andhorticulture. Japan Patent Kokai 2012-67083 (2012.4.5) (in Japanese).
38. Kayama, M., Imayoshi, J., Araki, S., Ogawa, H., Oofusa, T., Ueno, T., Saito, M. (1983). Changes

787-793 (in Japanese, with English abstract).
39. Kilpinen, O. (2001). Activation of the poultry red mite, Dermanyssus gallinae (Acari:
Dermanyssidae), by increasing temperatures. Exp. Appl. Acarol. 25: 859 867.
40. Kilpinen, O., Steenberg, T. (2009). Inert dusts and their effects on the poultry red mite
(Dermanyssus gallinae). Exp. Appl. Acarol. 48: 51-62.
41. Kitano, Y., Murazumi, K., Duan, J., Kurose, K., Kobayashi, S., Sugawara, T., Hirata, T. (2012).
Effect of dietary porphyran from the red alga, Porphyra yezoensis, on glucose metabolism indiabetic KK-Ay mice. J. Nutr. Sci. Vitaminol. 58: 14-19.
42. Koehler, P. G., Strong, C. A., Patterson, R. S., Valles, S. M. (1993). Differential susceptibility ofGerman cockroach (Dictyoptera: Blattellidae) sexes and nymphal age classes to insecticides. J.
Econ. Entomol. 86: 785-792.
43.

of Dermanyssus gallinae (poultry red mite) invasion on- 35 -

the plasma levels of corticosterone, catecholamines and proteins in layer hens. Pol. J. Vet. Sci.
12:231-235.
44. Lesna, I., Wolfs, P., Faraji, F., Roy, L., Komdeur, J., Sabelis, M. W. (2009). Candidate predatorsfor biological control of the poultry red mite Dermanyssus gallinae. Exp. Appl. Acarol. 48: 6380.
45. Lesna, I., Sabelis, M. W., van Niekerk, T. G. C. M., Komdeu, J. (2012). Laboratory tests forcontrolling poultry red mites (Dermanyssus gallinae) wi
cages. Exp. Appl. Acarol. 58: 371-383.
46. Levot, G. W. (1991). Chemical control of Dermanyssus gallinae (Acarina: Dermanyssidae) on
caged layer hens. Gen. Appl. Ent. 23: 49-52.
47. Lindquist, E.E., Krantz, G. W., Walter, D. E. (2009). A manual of acarology. In: Krantz, G. W.,
Walter, D. E. (eds). Classification, 3rd edn. Texas Tech University Press, Lubbock, pp 97-103.
48. Litwin, S. B. (1961). "Pigeon mites" causing a pruritic dermatitis. Report of a case. J. A. M. A.177:
714-716.
49. Lucky, A. W., Sayers, C., Argus, J. D., Lucky, A. (2001). Avian mite bites acquired from a newsource-pet gerbils report of 2 cases and review of the literature. Arch. Dermatol. 137: 167-170.
50. Lundh, J., Wiktelius, D., Chirico, J. (2005). Azadirachtin impregnated traps for the control of
Dermanyssus gallinae. Vet. Parasitol. 130: 337-342.
51. Marangi, M., Cafiero, M. A., Capelli, G., Camarda, A., Sparagano, O., Giangaspero, A. (2009).
Evaluation of the poultry red mite, Dermanyssus gallinae (Acari: Dermanyssidae) susceptibility-18.
52. Marangi, M., Morelli, V., Pati, S., Camarda, A., Cafiero, M. A., Giangaspero, A. (2012). Acaricide
residues in laying hens naturally infested by red mite Dermanyssus gallinae. PLoS One 7:
e31795.
53. Marangi, M., Cantacessi, C., Sparagano, O., Camarda, A., Giangaspero, A. (2014). Molecularcharacterization and phylogenetic inferences of Dermanyssus gallinae isolates in Italy within
an European framework. Med. Vet. Entomol. 28: 447-452.
54. Matsubara, H. (1968). Studies on the biological activity of surface-active agents part II. on theeffect of nonionic surface-active agents upon the lethal toxicity and knockdown speed of p, p'DDT emulsion against larvae of the common house mosquito, Culex pipiens pallens Coquillett.
-272 (in Japanese, with English abstract).
55. Maurer, V., Bieri, M., Folsch, D. W. (1988). Das Suchverhalten von Dermanyssus gallinae in
Hühnerställen. Host-finding of Dermanyssus gallinae in poultry -houses. Arch-furGeflügelkunde 52: 209-215.
56.

- 36 -

mite Dermanyssus gallinae (Acari: Dermanyssidae). Exp. Appl. Acarol. 15: 27-40.
57. Maurer, V. (1993). The dynamics of Dermanyssus gallinae (Acari: Dermanyssidae) populations
interacting with laying hens and the predatory mite Cheyletus eruditus (Acari: Cheyletidae).
Disseration ETHZ, No.10330, 104pp
58. Maurer, V., Perler, E., Heckendor
preparations against the poultry red mite Dermanyssus gallinae. Exp. Appl. Acarol. 48: 31-41.
59. Mignon, B., Losson, B. (2008). Dermatitis in a horse associated with the poultry mite
(Dermanyssus gallinae). Vet. Dermatol. 19: 38-43.
60. Miyata, M., Masuda, T. (2006a). Effects of some insecticides blocking spiracles of Tetranychus
urticae and Tetranychus kanzawai. Ann. Rept. Plant Prot. North Japan 57: 177-181 (inJapanese).
61. Miyata, M., Masuda, T. (2006b). Effects of insecticides blocking spiracle for aphids and powdery
mildew on strawberry. Ann. Rept. Plant Prot. North Japan 57: 182-184 (in Japanese).
62. Momen, F. M., Amer, S. A. A., Saber, S. A. (2006). Acaricidal potentials of some essential, mineraland plant oils against the predacious mite Neoseiulus cucumeris (Oudemans) (Acari:
Phytoseiidae). Acta Phytopathol. Entomol. Hung. 41: 383-393.
63. Morrice, L. M., McLEAN, M. W., Long, W. F., Williamson, F. B. (1983). Porphyran primarystructure. an investi-agarase I from Pseudomonas atlantica and 13C-NMR

spectroscopy. Eur. J. Biochem. 133: 673-684.
64. Moss, W. W. (1968). An illustrated key to the species of the acarine genus Dermanyssus(Mesostigmata: Laelapoidae: Dermanyssidae). J. Med. Entomol. 5: 67-84.
65. Mul, M., van Niekerk, T. G. C. M, Chirico, J., Maurer, V., Kilpinen, O., Sparagano, O. et al. (2009).
Control methods for Dermanyssus gallinae in systems for laying hens: results of aninternational seminar. Worlds Poult. Sci. J. 65: 589-599.
66. Mul, M. (2013). Fact sheet: The Poultry Red Mite, Dermanyssus gallinae (De Geer, 1778). A small
pest that packs a big punch.
67. Mumford, T. F., Miura, A. (1988). Porphyra as food: Cultivation and economics. In: Lembi, A.,
Waaland, J. R. (eds). Algae and Human Affairs. Cambridge University Press, Cambridge, pp
91-93.
68. Mungube, E. O., Bauni, S. M., Tenhagen, B. A., Wamae, L. W., Nzioka, S. M., Muhammed, L.,
Nginyi, J. M. (2008). Prevalence of parasites of the local scavenging chickens in a selectedsemi-arid zone of Eastern Kenya. Trop. Anim. Health Prod. 40: 101 109.
69. Murano, T. (2006). Red mite, Dermanyssus gallinae; ecology and latest problems in Japan. J. Jpn.
Soc. Poult. Dis. 42: 127-136 (in Japanese, with English abstract).
70. Murano, T. (2007). Red mite (Dermanyssus gallinae): current problem and trials for control in- 37 -

Japan. J. Jpn. Soc. Poult. Dis. 43: 23-30 (in Japanese, with English abstract).
71. Murano, T., Namiki, K., Shina, K., Yasukawa, H. (2008). The development of resistance byDermanyssus gallinae to commercial acaricides in Japan. J. Jpn. Vet. Med. Assoc. 61: 287-293(in Japanese, with English abstract).
72. Murano, T., Namiki, K., Shina, K., Yasukawa, H., Takahashi, Y., Tada, Y. (2013). The influence of
red mite (Dermanyssus gallinae) on laying hens in the poultry house. J. Jpn. Soc. Poult. Dis.
49: 274-280 (in Japanese, with English abstract).
73. Murano, T., Namiki, K., Shina, K., Yasukawa, H. (2015). Resistance development of Dermanyssus
gallinae against commercial acaricides in poultry farms in Japan. J. Jpn. Vet. Med. Assoc. 68:
509 -514 (in Japanese, with English abstract).
74. Nakamae, H., Kishi, S., Fujisaki, K., Oshiro, S., Furuta, K. (1997). Incidence of the parasitism ofchicken mite Dermanyssus gallinae parasitizing and propagating on chicken even in thedaytime and their life cycle. Jpn. Poult. Sci. 34: 240-247.
75. Nordenfors, H., Höglund, J., Uggla, A. (1999). Effects of temperature and humidity on oviposition,
molting and longevity of Dermanyssus gallinae (Acari Dermanyssidae). J. Med. Entomol. 36:
68-72.
76. Nordenfors, H., Höglund, J. (2000). Long term dynamics of Dermanyssus gallinae in relation tomite control measures in aviary systems for layers. Br. Poult. Sci. 41: 533-540.
77. Nordenfors, H., Chirico, J. (2001). Evaluation of a sampling trap for Dermanyssus gallinae (Acari:
Dermanyssidae). J. Econ. Entomol. 94: 1617-1621.
78. Nordenfors, H., Höglund, J., Tauson, R., Chirico, J. (2001). Effect of permethrin impregnatedplastic strips on Dermanyssu gallinae in loose-housing systems for laying hens. Vet. Parasitol.
102: 121-131.
79. Odaka, M., Fukuhara, E., Kaneko, K., Asada, K., Ogino, K. (2014). Development of a trap for
Dermanyssus gallinae. Jpn. J. Zootech. Sci. 85: 187-192 (in Japanese, with English abstract).
80. Odaka, M., Ogino, K., Shikada, M., Asada, K., Kasa, S., Inoue, T., Maeda, K. (2017). Correlation
between the proportion of stained eggs and the number of mites (Dermanyssus gallinae)--2083.

81. Øines, Ø., Brännström, S. (2011). Molecular investigations of cytochrome c oxidase subunit I(COI) and the internal transcribed spacer (ITS) in the poultry red mite, Dermanyssus gallinae,
in northern Europe and implications for its transmission between laying poultry farms. Med.
Vet. Entomol. 25, 402-412.
82. Oshio, Y. (1979). Pest of domestic animal. Japan Livestock Industry Association, Tokyo, pp 40-73(in Japanese)
83. Othman, R. A., Abdallah, J. M., Abu-Omar, J. (2012). Prevalence of the red mite (Dermanyssus- 38 -

gallinae) in layer flocks in four districts in northern West Bank, Palestine. Open J. Anim. Sci.
2: 106-109.
84. Potenza, L., Cafiero, M. A, Cucchiarini, L., Salandra, G. La., Giangaspero, A., Sparagano, O. etal. (2008). Dermanyssus gallinae mites collected from pigeon nests and laying hens: amolecular study based on ITS region. BSP Spring Trypanosomiasis/Leishmaniasis and Malaria
Meetings, March 30th, April 2nd, Newcastle upon Tyne, p 169.
85. Ramsay, G. W., Mason, P. C., Hunter, A. C. (1975). Chicken mite (Dermanyssus gallines) infestinga dog. New Zeland Vet. J. 23: 155-156.
86. Rosen, S., Yeruham, I., Braverman, Y. (2002). Dermatitis in humans associated with the mites
Pyemotes tritici, Dermanyssus gallinae, Ornithonyssus bacoti and Androlaelaps casalis in
Israel. Med. Vet. Entomol. 16: 442-444.
87. Roy, L., Chauve, C. (2007). Historical review of the genus Dermanyssus Dugés, 1834 (Acari:
Mesostigmata: Dermanyssidae). Parasite 14: 87-100.
88. Ruff, M. (1999). Important parasites in poultry production systems. Vet. Parasitol. 84: 337 347.
89. Sahibi, H., Sparagano, O., Rhalem, A. (2008). Dermanyssus gallinae: Acari parasite highly
aggressive but still ignored in Morocco. BSP spring trypanosomiasis/leishmaniasis and malaria
meetings, March 30th, April 2nd, Newcastle Upon Tyne, p 173.
90. Schulz, J., Berk, J., Suhl, J., Schrader, L., Kaufhold, S., Mewis, I., Hafez, H. M., Ulrichs, C. (2014).
Characterization, mode of action, and efficacy of twelve silica-based acaricides against poultry
red mite (Dermanyssus gallinae) in vitro. J. Parasitol. Res. 113: 3167 3175.
91. Sikes, R. K., Chamberlain, R. W. (1954). Laboratory observations on three species of bird mites.
J. Parasitol. 40: 691-697.
92. Sommer, D., Heffels-Redmann, U., Köhler, K., Lierz, M., Kaleta, E. F. (2016). Role of the poultry
red mite (Demanyssus gallinae) in the transmission of avian influenza A virus. Tierärztliche
Praxis Grosstiere 44: 26 33.
93.

et al. (2009).
Prevalence and key figures for the poultry red mite Dermanyssus gallinae infections in poultry
farm systems. Exp. Appl. Acarol. 48: 3-10.

94. Sparagano, O., George, D., Harrington, D., Giangaspero, A. (2014). Significance and control ofthe poultry red mite, Dermanyssus gallinae. Annu. Rev. Entomol. 59: 447-466.
95. Stadler, T., Buteler, M. (2009). Modes of entry of petroleum distilled spray-oils into insects: areview. Bull. Insectol. 62: 169-177.
96. Sugano, A., Sawano, M., Inoue, S. (2009). The new extracting method of functional material
porpyran from discoloration laver. Bull. Soc. Sea Water Sci. Jpn. 63: 377-380 (in Japanese).
97. Sumi, T., Tuge, K., Yoshimura, T., Nishio, S., Abe, S. (2005). Extraction and production of- 39 -

porphyran. Japan Patent Kokai 2005-120193 (2005.5.12) (in Japanese).
98. Sylejmani, D., Musliu, A., Ramadani, N., Sparagano, O., Hamidi, A. (2016). Associations between
the level of biosecurity and occurrence of Dermanyssus gallinae and Salmonella spp. in layer
farms. Avian Dis. 60: 454 459.
99. Takahashi, K., Hirano, Y., Araki, S., Hattori, M. (2000). Emulsifying ability of porphyran preparedfrom dried nori, Porphyra yezoensis, a red alga. J. Agric. Food Chem. 48: 2721-2725.
100. Tavassoli, M., Allymehr, M., Pourseyed, S. H., Ownag, A., Bernousi, I., Mardani, K.,
Ghorbanzadegan, M., Shokrpoort, S. (2011). Field bioassay of Metarhizium anisopliae strains
to control the poultry red mite Dermanyssus gallinae. Vet. Parasitol. 178: 374-378.
101. Thind, B. B., Ford, H. L. (2007). Assessment of susceptibility of the poultry red mite
Dermanyssus gallinae (Acari: Dermanyssidae) to some acaricides using an adapted filter paper
based bioassay. Vet. Parasitol. 144: 344-348.
102. Tomasz, C. (2003). Prevalence of Dermanyssus gallinae in poultry farms in Silesia region in
Poland. Bull. Vet. Inst. Pulawy 47: 465-469.
103. Tucci, E. C., Prado, A. P., Araújo, R. P. (2008). Development of Dermanyssus gallinae (Acari:
Dermanyssidae) at different temperatures. Vet. Parasitol. 155: 127-132.
104. Valiente Moro, C., Chauve, C., Zenner, L. (2005). Vectorial role of some dermanyssoid mites(Acari, Mesostigmata, Dermanyssoidea). Parasite 12: 99-109.
105. Valiente Moro, C., Thioulouse, J., Chauve, C., Normand, P., Zenner, L. (2009). Bacterial taxa
associated with the hematophagous mite Dermanyssus gallinae detected by 16S rRNA PCR
amplification and TTGE fingerprinting. Microbiol. Res. 160: 63-70.
106. van Emous, R. (2005). Wage war against the red mite! Poult. Int. 44: 26-33.
107. van Emous, R., van Niekerk, T. G. C. M., Mul, M. (2005). Red mites in theory and practice.
Praktijkrapport Pluimvee #17. Animal Science Groupe, Lelystad (in Dutch, with English
abstract).
108. van Emous, R. (2017). Verwachtte schade bloedluis 21 miljoen euro. Pluimveeweb. nl. (in Dutch)
109. Vincent, C., Hallman, G., Panneton, B., Fleurat-Lessard, F. (2003). Management of agricultural
insects with physical control methods. Annu. Rev. Entomol. 48: 261 281.
110. Wang, F. F., Wang, M., Xu, F. R., Liang, D. M., Pan, B. L. (2010). Survey of prevalence andcontrol of ectoparasites in caged poultry in China. Vet. Rec. 167: 934-937.
111. Wolfenbarger, D. A., Lukefahr, M. J., Lowry, W. L. (1967). Toxicity of surfactants and surfactantinsecticide combinations to the bollworm, tobacco budworm, and pink bollworm. J. Econ.
Entomol. 60: 902-904.
112. Yamamura, K. (1999). Transformation using (x + 0.5) to stabilize the variance of populations.
Res. Popul. Ecol. 41: 229-234.

- 40 -

113. Yamamura, K. (2016). Estimation of the predictive ability of ecological models. Commun. Stat.Simul. C. 45: 2122-2144.
114. Zenner, L., Bon, G., Chauve, C., Nemoz, C., Lubac, S. (2009). Monitoring of Dermanyssus
gallinae in free-range poultry farms. Exp. Appl. Acarol. 48: 157 166.

- 41 -

- 42 -

第 3 章非平行板トラップを用いたワクモの鶏舎内モニタリングによるダニ数と汚卵発生率の相関関係
鶏舎内に“非平行板トラップ”を設置し、モニタリングしたワクモ数とワクモによって発生する汚卵の関係を調査した。その結果、卵受けに設置したトラップ内のワクモ数と汚卵率は特定の閾値以上で正の相関関係にあることが明らかになった。すなわち、汚卵が発生するワクモの最低密度が明らかになった。以上のことから、“非平行板トラップ”を用いて鶏舎でワクモ数をモニタリングすることによって、駆除を開始する適期を把握でき、ワクモによる汚卵の発生を防止できることが示唆された。

第4章

食品および食品添加物から構成された混合物のワクモに対する制御効果

SFF は、in vitro においてワクモに対して殺虫効果を示し、その機序はワクモの呼吸器の閉塞であることが立証された。また、P. yezoensis 由来の多糖類抽出物は SFF のワクモに対する殺虫効果を増強することが明らかとなった。さらに、養鶏場における効果実証試験では、SFF を散布すると何も散布をしない対照区と比較して、鶏舎内のワクモ数と汚卵率を減らすことが明らかとなった。このことから、SFF は養鶏場におけるワクモの防除方法として有用であると考えられた。

結論として、本論文では以下のような幾つかの新たな知見が得られた。
1.

ワクモに対する高い捕獲能力と鶏舎内でワクモ数をモニタリングするための実用性を兼ね備えた新しい“非平行板トラップ”を開発した。
2.

“非平行板トラップ”を用いて鶏舎でワクモ数をモニタリングすることによって、駆除を開始する適期を把握でき、ワクモによる汚卵の発生を防止できることが示唆された。

3.

食品および食品添加物から構成された混合物（SFF）はワクモに対して物理的機序による殺虫効果を示し、ワクモを防除する手法の一つとして有効と考えられた。

- 43 -

福岡県農林業総合試験場特別報告第12号

Studies on a novel control system ofDermanyssus gallinae

発行

令和２年 ３月

福岡県農林業総合試験場〒818-8549

福岡県筑紫野市大字吉木 587
TEL 092-924-2971

著

者

印刷所

小高

真紀子

秀英社印刷株式会社〒818-0052

福岡県筑紫野市武蔵3-2-6
TEL 092-923-3154