Modified atmosphere for selective control of spider mites

2018.3

Biological Production Science

United Graduate School of Agricultural Science

Tokyo University of Agriculture and Technology

WANG, CHIH-HUNG

(王 至弘)

i  

Contents

Chapter 1 General Introduction ....................................................................................................... 1

Chapter 2 Anoxia treatment ............................................................................................................. 6

2.1 Introduction ....................................................................................................................... 6

2.2 Materials and methods ....................................................................................................... 7

2.2.1 Rearing of spider mites ........................................................................................... 7

2.2.2 Rearing of predatory mites ...................................................................................... 8

2.2.3 Anoxia test .............................................................................................................. 8

2.2.4 Data analyses ........................................................................................................ 12

2.3 Results ............................................................................................................................. 12

2.3.1 O2 concentration in the container........................................................................... 12

2.3.2 Egg hatchability .................................................................................................... 12

2.3.3 Adult female survivability ..................................................................................... 15

2.4 Discussion ....................................................................................................................... 20

Chapter 3 Hypobaric treatment ...................................................................................................... 25

3.1 Introduction ..................................................................................................................... 25

3.2 Materials and methods ..................................................................................................... 26

3.2.1 Cultures of mites ................................................................................................... 26

3.2.2 Hypobaric treatment .............................................................................................. 27

3.2.3 Data analysis ......................................................................................................... 31

3.3 Results ............................................................................................................................. 31

3.3.1 Egg hatchability .................................................................................................... 31

3.3.2 Adult female survivability ..................................................................................... 33

ii  

3.4 Discussion ....................................................................................................................... 36

Chapter 4 General Discussion ....................................................................................................... 40

Summary ...................................................................................................................................... 45

摘要 .............................................................................................................................................. 48

Acknowledgements ....................................................................................................................... 51

References .................................................................................................................................... 52

1  

Chapter 1 General Introduction

 Man has used pesticides for a long time in multiple areas to manage pests, which are unwanted 

organisms. The history of the use of pesticides in agriculture can be tracked back to 8,000 BC, but

large-scale use was apparent just in the most recent 100 years, particularly during the quick

development of chemical manufactures in World War II (Jones, 1973; Delaplane, 1996). Pesticides

have improved the quality and quantity of agricultural production that has been the backstage driving

force for the green evolution (Pingali, 2012).

One way of classifying pesticides is by vapour pressure (VP), and the pesticides with higher VP

gasify more easily (Zacharia, 2011). Fumigation is a pest control means that uses high-VP chemicals

(fumigants), which are usually manufactured and stored in solid or liquid form. Fumigants transit to

the gaseous phase and fill the atmosphere in a sealed room during application. Fumigation is a broad-

spectrum treatment against multiple pest categories that can effectively disinfect microorganisms and

disinfest invertebrates. There are several advantages in the use of fumigation. The diffusive properties

of gases enable fumigants to reach the inside of a commodity or shelter to disinfest hidden pests, in a

way that traditional spraying cannot accomplish (Thompson, 1966). The powerful penetrative ability

to infiltrate the surface of pests also increases their absorbing rate, and complete disinfection can be

achieved within 24 h of application (Bond and Monro, 1967; Zettler and Arthur, 2000).

Methyl Bromide (MeBr) was a commonly used fumigant. After Le Goupil (1932) first reported the

insecticidal value of MeBr, it had been widely spread and was used in plant quarantine in many fields.

One of the reasons that MeBr became a popular fumigant is that it is a non-flammable and

nonexplosive substance – no special fire prevention measures are required during its transport or

storage. It not only has a strong penetrative ability, quickly reaching deep inside commodities and

products at room atmospheric pressure and temperature compared to other fumigants, but the small

2  

molecular particle also dissipates rapidly without leaving any residue at the end of treatment (Bond,

1984). MeBr is also found in some living plants that can endure insecticidal concentration (Bond,

1984). Moreover, the cost and price of MeBr are low enough to be accepted by many applicators

(Sydorovych et al., 2008). All these advantages once made MeBr a popular fumigant.

However, there are some limitations to the use of fumigation in pest management. The high

effectiveness of fumigants implies that they are highly toxic. Fumigants may cause health concerns

to applicators, whether their exposure is acute or chronic (Akca et al., 2009). Moreover, some

fumigants, e.g., carbon tetrachloride and ethylene dichloride, decrease the commodity quality due to

undesirable prolongation of residue effects (Berck, 1974; Bond, 1984). Although there is no residue

after MeBr treatment, a severe problem has occurred: MeBr was confirmed as an important ozone-

depleting substance. The downgrade of MeBr releases the bromine atom into the atmosphere, where

it induces a chain reaction that recombines ozone to oxygen particles, thus attributes to the ozone hole

(Wofsy et al., 1975). Nowadays, the use of this kind of ozone-depleting substances has been strictly

restricted by the Montreal Protocol. This protocol listed MeBr and it was already phased out in 2015

(UNEP, 2016).

Restriction of the use of MeBr and some other fumigants increases the cost of agricultural

production and storage. Although there are some alternative fumigants available, their cost is higher

than that of MeBr and they cannot achieve the same effectiveness as MeBr (Soderstrom et al., 1984).

On the other hand, alternative treatments that physically disinfest pests have also been developed

(Schneider et al., 2003). Heat/cold treatment kills pests by raising/reducing temperature. Heat

treatment can completely disinfest a pest in a short time. However, it may cause adverse effects on

commodities, e.g., it may reduce contents and soften commodities, that reduce their value after

treatment (Lurie, 1998). Cold treatment also has been investigated and successfully eliminates pests,

but it takes long to achieve complete disinfestation and it may induce chilling injury (Sharp, 1993).

3  

The microwave is also used to control pests, but there is concern on commodity quality and it cannot

penetrate the surface of a commodity to cope with hidden pests (Birla et al., 2005; Ling et al., 2015).

The effectiveness of irradiation was also reported, but investment is costly and treatment doses vary

with target pest, which makes it difficult to make common rules (Schneider et al., 2003).

Although Boyle (1666) initially reported the effect of air composition on insects, the formal

development of controlled atmosphere/modified atmosphere (CA/MA) in pest disinfestation has been

investigated 250 years later (Back and Cotton, 1925). There are two main mechanisms in CA/MA:

(1) to increase the partial pressure of CO2 (pCO2) and (2) to reduce the partial pressure of O2 (pO2)

(Adler et al., 2000; Navarro, 2012; Navarro et al., 2012). Both mechanisms can be applied together

to improve effectiveness against insect pests (Cheng et al., 2013; Hashem et al., 2014; Imai, 2015).

CA/MA retains the advantages of fumigation. It is possible to completely disinfest pests within 24

h or shorter, depending on treatment conditions (Son et al., 2012; Wang et al., 2012; Suzuki et al.,

2015). This is important because some commodity cannot be stored for a long time and customers

want to receive fresh commodity as soon as possible after harvest. Traditional spray methods cannot

kill hidden pests because pesticides must get in direct contact with pests, but CA/MA can accomplish

this due to the diffusing property of gas (Hulasare et al., 2013; Jiao et al., 2013; Wang et al., 2016;

Athanassiou et al., 2017). Moreover, CA/MA has additional advantages that can improve the quality

of agricultural commodities and products and extend their shelf life (Gorris and Peppelenbos, 1992;

Sen et al., 2012; Imlak et al., 2017), and the residual effect continuously acts on the commodities to

maintain quality (Li and Kader, 1989). Because the killing mechanism of CA/MA is to modify air

composition, there is no concern of chemical residue on commodities and it does not cause any

environmental pollution (Adler et al., 2000; Navarro, 2006; Navarro et al., 2012). CA/MA can also

be used to cope with chemical-sensitive commodity, e.g., historical relics and specimens in a museum,

even books in libraries (Gilberg, 1990; Valentin, 1993; Zhang, 2013). Finally, acceptance of a new

4  

disinfestation method depends on the cost of involvement – the cost of CA/MA is low and may even

be lower than the cost of some alternative fumigants (Soderstrom et al., 1984). This implies that the

CA/MA has potential as a new effective and low-cost disinfestation method for pests.

Spider mites (Acari: Tetranychidae) are important economic agricultural pests in both field and

greenhouse. They reduce photosynthesis rates in plants due to leaf chlorosis, because they suck

contents of mesophyll cells (Sances et al., 1981). This results in reduction of fruit size and

productivity that cause up to 25% marketable yield losses (Sances et al., 1981; Nyoike and Liburd,

2013). Because spider mites of most species are small (< 0.5 mm) and difficult to detect, they can

easily hide in commodities to be transported and introduced to new places (Roques et al., 2009;

Navajas et al., 2010; Roderick and Navajas, 2016). The high reproductive ability also contributes to

their successful invasion and colony establishment (Bonato, 1999; Bounfour and Tanigoshi, 2001).

Spider mites also cause indirect losses in plants as they act as a vector of plant pathogens (Kitajima

et al., 2003; Adams et al., 2004). Moreover, they establish acaricide-resistant populations easily and

rapidly due to their short life-history, abundant progeny and high gene diversity (Van Leeuwen et al.,

2010; Dermauw et al., 2013). Because of these characteristics, they are very difficult to be completely

disinfested once they establish a colony. The best strategy for avoiding their invasion is to restrict

them before their entrance.

Nowadays, the effectiveness of CA/MA has been investigated and its potential as a new method

for disinfesting storage pests has been established. However, studies of field application and the

impact on natural enemies are less reported (Held et al., 2001; Wang et al., 2016). In the present study,

we evaluated the potential of CA/MA for disinfestation in spider mites, especially focusing on the

effect of pO2. We also assessed the impact of CA/MA on a predatory mite (Acari: Phytoseiidae) that

is used as a biological control agent of spider mites, in order to estimate the feasibility of CA/MA in

integrated pest management (IPM) applications.

5  

In general, the effectiveness of CA/MA has a negative correlation with pO2 (Navarro, 2012;

Navarro et al., 2012), i.e., lower pO2 can achieve better disinfestation effectiveness. Based on this

concept, we assumed that the maximum effectiveness of CA/MA against spider mites should appear

in anoxia treatment (pO2 = 0). In Chapter 2, we evaluated the effectiveness of anoxia on two important

agricultural spider mite pests, the two-spotted spider mite, Tetranychus urticae Koch, and the citrus

red mite Panonychus citri (McGregor), and its impact on a commercial biocontrol agent, the

predatory mite Neoseiulus californicus (McGregor). The experimental environment in traditional

CA/MA is usually created by flushing N2 or inert gases (Navarro, 2006; 2012; Navarro et al., 2012);

however, we chose deoxidants to absorb oxygen, thus removing all oxygen from the atmosphere.

Deoxidants are very simple to use, low-cost, fast and effective, and they also maintain a stable

experimental environment (Lambert et al., 1992; Grattan and Gilberg, 1994). These characteristics

met the laboratory limitations and requirements in the present study. A low pO2 environment can also

be created by hypobaria (low pressure) (Navarro, 2006, 2012; Navarro et al., 2012). Hypobaria per

se also attribute to disinfest pests in low pO2 environment that may enhance the effectiveness of

CA/MA (Galun and Fraenkel, 1961). In Chapter 3, we evaluated the effectiveness of hypobaria on

the same mite species that were tested in Chapter 2 plus an additional spider mite, the Kanzawa spider

mite, Tetranychus kanzawai Kishida, by using a vacuum pump. Finally, Chapter 4 discusses the

important discoveries in the present study and the further application of CA/MA against spider mites

and their side-effects on biocontrol agents.

6  

Chapter 2 Anoxia treatment

2.1 Introduction

For controlling agricultural pests, fumigants are widely used worldwide, particularly in developing

countries. MeBr has been used as a fumigant for controlling nematodes, arthropods and

microorganisms in agricultural products for more than five decades. Such broad-spectrum fumigants

are not only expensive, but there are also increasing concerns about the residues (Tebbets et al., 1983)

and human health (Akca et al., 2009). In addition, MeBr has been identified as the cause of significant

ozone depletion (Mellouki et al., 1992). Recently, the Montreal Protocol proposed phasing out the

use of MeBr as a general method for controlling pests in developed countries, and banning it by the

year 2015 in developing countries (UNEP, 2016). Therefore, developing environment-friendly

methods for pest control is crucial.

CA/MA is a potential pest control method that can avoid the problems of MeBr. Typical CA/MA

treatments for agricultural commodities involve physically changing the composition of the

atmosphere, such as by elevating the partial pressures of CO2 (pCO2), decreasing the partial pressures

of O2 (pO2) or combining both for a certain time at a certain temperature (Ke and Kader, 1992). These

treatments leave no chemical residues (Navarro, 2006; Navarro et al., 2012). The CA/MA techniques

affect the inside of an agricultural product by rapid diffusion of gases similar to fumigants (Hulasare

et al., 2013) and can prolong their shelf life (Kader, 1986). They are a potential alternative treatment

for postharvest pest control on fruits and vegetables (Liu, 2007; 2008b). However, elevated CO2 can

damage fruit and vegetables (Lipton et al., 1972), delay flowering and reduce survival in floricultural

plants (Held et al., 2001).

In the CA/MA techniques, a major cause of insect death is lack of O2 (Fleurat-Lessard, 1990). Low

pO2 (< 5 kPa) can induce mortality in several pests, including the fungus gnat Bradysia sp. (Diptera:

Sciaridae) (Held et al., 2001), two-spotted spider mite Tetranychus urticae Koch (Acari:

7  

Tetranychidae) (Held et al., 2001), currant-lettuce aphid Nasonovia ribisnigri (Mosley) (Homoptera:

Aphididae) (Liu, 2005), western black widow spider Latrodectus hesperus Chamberlin & Ivie

(Araneae: Theridiidae) (Liu et al., 2008), vine mealybug Planococcus ficus (Signoret) (Hemiptera:

Pseudococcidae) (Liu et al., 2010) and western flower thrips Frankliniella occidentalis (Pergande)

(Thysanoptera: Thripidae) (Liu, 2007; 2008a, b; 2011; 2012). Although a controlled atmosphere with

low pO2 was found to be effective for control of arthropod pests, little is known about the effect of

anoxia (pO2 = 0 kPa). Suzuki et al. (2015) showed that exposing eggs and adult females of T. urticae

and T. kanzawai Kishida to deoxidant-induced anoxia for 12 h at 25°C caused 100% mortality.

However, they did not examine other temperatures, or the effect of anoxia on phytoseiid predators,

which are biological control agents (Naranjo, 2001; Zhang and Swinton, 2009).

In the present study, we investigated the effects of deoxidant-induced anoxia on two economically

important mite pests, T. urticae and citrus red mite Panonychus citri (McGregor) (Acari:

Tetranychidae), and their natural enemy, the predatory mite Neoseiulus californicus (McGregor)

(Acari: Phytoseiidae), under different temperatures.

2.2 Materials and methods

2.2.1 Rearing of spider mites

Population of T. urticae was collected on 7 June 2013 at Hiraka, Akita, Japan and population of P.

citri was collected on 7 June 2010 at Nago, Okinawa, Japan. The populations were maintained at

25°C, 55–65% RH and with a photoperiod of 16:8 h L: D in an incubator (NCTE-1000T; Nihon

Freezer, Tokyo, Japan) using leaves of common bean (Phaseolus vulgaris L.) (Fabales: Fabaceae) for

T. urticae and citrus (Citrus unshiu Marcovitch) (Sapindales: Rutaceae) for P. citri. The leaves were

placed on water-saturated polyurethane mats in polystyrene Petri dishes (90 mm diameter, 20 mm

depth; one leaf per Petri dish). The edges of the leaves were covered with three-folded tissue papers

8  

to prevent the leaves from desiccating and to prevent the mites from escaping (Ehara and Gotoh,

2009). The leaves were replaced approximately every 7 days as they became damaged by feeding

mites.

2.2.2 Rearing of predatory mites

N. californicus was obtained from SPICAL EX (Arysta LifeScience, Tokyo, Japan) and was

maintained using all stages of T. urticae on bean leaf disc (∼16 cm2) placed on water-saturated

polyurethane mats in polystyrene Petri dishes at 25 ± 1°C, 55–65% RH and with a photoperiod of

16:8 h L:D in the incubator. The leaves were replaced whenever they appeared to dry out or to be

damaged by feeding mites.

2.2.3 Anoxia test

The experimental apparatus for collecting newly deposited eggs consisted of (from bottom to top)

a Petri dish bottom, a circular wet sponge, a bean or citrus leaf, a thin film of water, stretched Parafilm

(Parafilm M; Structure Probe, West Chester, PA) that was in close contact with the leaf and a circular

water-soaked filter paper with a 3 cm × 3 cm square hole in the middle that acted as the experimental

arena for the mites (Fig. 1). Fifty adult females of each spider mite species or 25 adult females of N.

californicus were separately transferred onto the experimental arenas with a fine brush and were

allowed to lay eggs for 24 h. Sufficient mixed stages of T. urticae were provided as food for N.

californicus. After 24 h of oviposition, adult females, prey T. urticae, webs and faeces were removed

from the experimental arena, leaving only the eggs on the Parafilm. The Parafilm and eggs were

transferred to a dry Petri dish for the following anoxia test.

The Petri dish was placed in a well-closed polycarbonate container (2.5 L; Mitsubishi Gas

Chemical, Tokyo, Japan) with 40 packages of an iron-based deoxidant consisting of iron, calcium

9  

Fig.

1. T

he e

xper

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tal a

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atus

that

was

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tudy

. Mit

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n a

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acka

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10  

chloride and silicon dioxide (AGELESS® FX-400; Mitsubishi Gas Chemical), a sensor for measuring

the O2 concentration (OXY-1; Jikco, Tokyo, Japan), a sensor for measuring the air temperature and

relative humidity (RH) (TR-77Ui; T & D, Nagano, Japan) and six filter papers (9 cm in diameter;

Advantec No. 2; Toyo Roshi, Tokyo, Japan) soaked with 20 mL distilled water (Suzuki et al., 2015).

The filter papers were used to increase the humidity, which accelerated the activity of the deoxidant.

A webcam (C920; Logicool, Tokyo, Japan) was used to capture images of the O2 sensor’s display at

1-min intervals (Fig. 1).

The container was placed in an incubator (MIR-254; Sanyo Electric, Tokyo, Japan) at temperatures

between 20°C and 30°C for times between 0 and 24 h of anoxia (Fig. 2). After each anoxia duration,

the container was opened and the deoxidant packages were removed. The container was closed again

when the O2 concentration inside the container returned to the ambient level (≈ 21%). Then, 24 h

after the first closing of the container of all treatments, the eggs on parafilms were removed onto the

detached leaves from the container and held at atmospheric pressure. The exposed eggs were observed

using a stereomicroscope and the number of hatched larvae was counted every day until no larvae

hatched or 2 days after the last control hatched. The anoxia test used three independent replicates in

each species using eggs that originated from different mothers to minimize maternal effects.

The same set-up and conditions (anoxia duration and temperature) were used in the anoxia test on

the adult females of T. urticae, P. citri and N. californicus. Mated females of 3-day old of both spider

mite species were transferred onto the experimental arena with a fine brush and these samples were

used in the anoxia test. To avoid cannibalism in N. californicus (Schausberger, 2003), we used a 1.5-

mL polypropylene tube and confined adult females (2- dayold after adult females emergence and

copulated) individually into the tube with the soft brush. The opening of the tube was covered with a

gas-permeable polytetrafluoroethylene (PTFE) membrane (Milli-Seal; Merck KGaA, Darmstadt,

Germany). For each replicate, tubes were washed and sealed with a fresh PTFE membrane. After 6,

11  

Fig. 2. The experimental container with mites was placed in an incubator with a recordable camera at temperatures 20, 25 and 30°C.

12  

12 or 24 h anoxia treatment, mortality was evaluated by gently probing with a fine brush. No

movement upon probing was judged to indicate death and response to the brush was judged to indicate

survival. Individuals that had impaired motor coordination but could still move were counted

separately.

 2.2.4 Data analyses

An arcsine square-root transformation was applied to normalize the hatchability and survival data

for eggs and adult females in each replicate prior to analysis. Three-way analysis of variance was

used for the cumulative hatchability of eggs and survivability for adult females to know the effect of

temperatures, anoxia duration and species. Means of egg hatchability and adult female survivability

were compared using Tukey’s honestly significant difference (HSD) test when significant F values

were obtained (p < 0.05). Impaired motor coordination in adult females was compared between

temperatures using a chi-square test. All the analyses were done by using IBM SPSS statistical

software version 22 (IBM, 2014).

2.3 Results

2.3.1 O2 concentration in the container

The deoxidant decreased the O2 concentration in the container to 0% in about 2 h, with the rate of

decrease increasing with increasing temperature (Fig. 3).

2.3.2 Egg hatchability

The egg hatchability was significantly influenced by temperature, anoxia duration and species (P

< 0.001) (Table 1). In addition, the interaction effects were observed in all combinations of the three

factors (P < 0.001). In T. urticae, the hatchability in the control (0 h anoxia) was 93.7 ± 2.6, 96.8 ±

13  

051015202530

020

4060

8010

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O2concertration (%)

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14  

FP

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. Thr

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sis

of v

aria

nce

on t

he e

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t of

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h in

depe

nden

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and

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he

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of e

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and

the

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15  

1.2 and 95.5 ± 1.1% at 20°C, 25°C and 30°C, respectively (Fig. 4). The egg hatchability after anoxia

for 15 and 18 h at 20°C was 1.8% and 0%, respectively (Table 2). No eggs hatched after anoxia for

≥18, ≥12 and ≥6 h at the respective temperatures. In P. citri, the hatchability in the control was 94.4

± 0.9, 94.5 ± 2.6 and 87.9 ± 2.8% at 20°C, 25°C and 30°C, respectively (Fig. 4). The egg hatchability

after anoxia for 15 and 18 h at 20°C was 0% for both anoxia treatments (Table 2). No hatched

individuals were observed after anoxia for ≥15, ≥12 and ≥6 h at the respective temperatures. In N.

californicus, the hatchability in the control was 99.0 ± 1.0, 99.5 ± 0.5 and 99.3 ± 0.7% at 20°C, 25°C

and 30°C, respectively (Fig. 4). About 40% of eggs hatched even after anoxia for 24 h at 20°C,

whereas all eggs died after anoxia for 24 h and ≥12 h at 25°C and 30°C, respectively. In addition, the

hatchability after anoxia for 15 and 18 h at 20°C was 97.1% and 52.7%, respectively (Table 2).

At 20°C, the duration of anoxia for 100% mortality of eggs was ≥18, ≥15 h and ND (not detectable)

for T. urticae, P. citri and N. californicus, respectively. At 25°C, the duration was ≥12, ≥12 and ≥24

h for the respective species (Fig. 4). At 30°C, the duration was ≥6, ≥6 and ≥12 h for the respective

species. Consequently, with anoxia for 18 h at 20°C and 12 h at 25°C, no eggs survived in either

spider mite species, whereas 53% and 72% of N. californicus eggs hatched at the respective

temperatures.

2.3.3 Adult female survivability

Survivability of adult females was significantly influenced by temperature, anoxia duration and

species (p < 0.001) (Table 1). In addition, the interaction effects were observed in all combination of

three factors (p < 0.001). In T. urticae, the survivability in the control was 97.2 ± 1.4, 93.2 ± 4.4 and

91.8 ± 1.9% at 20°C, 25°C and 30°C, respectively (Fig. 5). In addition, no females survived after

anoxia both for 15 h at 20°C and 8 h at 25°C (Table 3). Consequently, the survivability showed 0%

after anoxia for ≥15, ≥8 and ≥6 h at 20°C, 25°C and 30°C, respectively. In P. citri, the survivability

16  

Fig. 4. Hatchability of eggs of T. urticae, P. citri and N. californicus at three temperatures (20°C, 25°C and 30°C) at 10 days after anoxia treatment for 0 (control), 6, 12 and 24 h. Data are shown as mean ± SE (vertical lines). Numerals in each bar or parentheses show the number of eggs tested. Different letters above the bars for each temperature indicate a significant difference (analysis of variance followed by Tukey’s honestly significant difference test, p < 0.05).

17  

na%

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. Fin

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C)

Ano

xia

dura

tion

(h)

18  

Fig. 5. Survivability of adult females of T. urticae, P. citri and N. californicus at three temperatures (20°C, 25°C and 30°C) at 24 h after anoxia treatment for 0 (control), 6, 12 and 24 h. Data are shown as mean ± SE (vertical lines). Numerals in each bar or parentheses show the number of adults tested. Different letters above the bars for each temperature indicate a significant difference (analysis of variance followed by Tukey’s honestly significant difference test, p < 0.05).

19  

na%

Sur

viva

lbn

% S

urvi

val

2015

166

0c66

97.1

±2.

9a18

140

0c10

646

.9±

4.24

b25

815

30c

7495

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2.64

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189

0c94

48.6

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39b

a Num

ber

of fe

mal

es te

sted

.b D

ata

are

show

n as

mea

n±S

E. V

alue

s in

a c

olum

n fo

llow

ed b

y di

ffere

nt le

tters

are

sig

nific

antly

Tab

le 3

. Sur

viva

bilit

y of

T. u

rtic

ae a

nd N

. cal

ifor

nicu

s fe

mal

es a

t 1 d

ays

afte

r an

oxia

trea

tmen

t for

15

and

18 h

at 2

0°C

or

for

8 an

d 10

h a

t 25°

CT

empe

ratu

re(°

C)

Ano

xia

dura

tion

(h)

T. u

rtic

aeN

. cal

ifor

nicu

s

20  

in the control was 98.8 ± 0.6, 98.5 ± 0.2 and 99.1 ± 0.9% at 20°C, 25°C and 30°C, respectively (Fig.

5). The survivability was 0% after anoxia ≥6 h at all temperatures. In N. californicus, the survivability

in the control was 80.9 ± 1.1, 88.8 ± 4.8 and 95.8 ± 1.8% at 20°C, 25°C and 30°C, respectively. More

than 95% of females were alive after anoxia both for 15 h at 20°C and 8 h at 25°C (Table 3). The

survivability was 0% after anoxia for 24, 24 and ≥12 h at 20°C, 25°C and 30°C, respectively. The

survivability was not significantly different with the control even when the anoxia duration was for

15 and 8 h at 20°C and 25°C, respectively.

At 20°C, the duration for 100% mortality of adult females was ≥15, ≥6 and ≥24 h for T. urticae, P.

citri and N. californicus, respectively. At 25°C, the duration was ≥8, ≥6 and ≥24 h for the respective

species. At 30°C, the duration was ≥6, ≥6 and ≥12 h for the respective species. Consequently, with

anoxia for 15 h at 20°C and 8 h at 25°C, no adult females survived in either spider mites, whereas

>95% of adult females N. californicus females survived.

Impaired motor coordination was observed in adult females of T. urticae after anoxia for 6 h: 150

out of 169 survived females (88.7 ± 1.24%) and 4 out of 4 survived females (100%) at 20°C and 25°C,

respectively (χ2 = 225.33, p < 0.001). After anoxia for 12 h at 20°C, six out of eight alive adult females

(75%) of T. urticae also showed impaired motor coordination. No impaired motor coordination was

observed in P. citri at any anoxia condition at all temperatures.

In N. californicus, only three out of five alive individuals (60%) and two out of two alive

individuals (100%) showed impaired motor coordination after anoxia for 12 h at 25°C and 6 h at

30°C, respectively (χ2 = 0.38, p > 0.05). In additional experiments, none of the survived females

showed impaired motor coordination after anoxia for 15 and 18 h at 20°C as well as for 8 and 10 h

at 25°C.

21  

2.4 Discussion

Regardless of the possibility of antagonistic effects on some plants (Held et al., 2001), this work

indicates that practice of modified atmospheres using anoxia treatment has potential to eliminate

spider mites with less impact on predatory mites in a closed system. Both eggs and adult females of

T. urticae and P. citri were more susceptible to anoxia than those of N. californicus (Fig. 4, Fig. 5,

table 2 and Table 3). This result suggests that anoxia treatments under the above-mentioned conditions

can be useful for controlling spider mite pests while having little impact on their natural enemy, N.

californicus.

The anoxia tolerance in eggs may be related to the shell structures. It was reported that the waxy

outer layer and capsule wall of eggs restricted the diffusion of O2 in the estuarine gastropod

Crepipatella dilatata (Lamarck) (Segura et al., 2010). In the reduviid bug Rhodnius prolixus Stål, the

gas exchange proceeds from micropyles and pseudomicropyles that penetrate the shell and connect

to the inner layer at a specific zone (Tuft, 1950). It is possible that eggs with fewer micropyles and/or

pseudomicropyles lose less O2, which could increase hatchability even under anoxia. Furthermore,

the inner structure of insect eggs has a layer that could store air (Wigglesworth and Beament, 1950),

and that might supply O2 during periods of hypoxia. Differences in this layer among species could

account for differences in anoxia tolerance. Exposing eggs and adult females of T. urticae to hypoxic

conditions (0.2-1.4% O2 concentration) at 20°C for 24 h caused complete mortality (Held et al., 2001),

which suggests that O2 levels under a lethal threshold are as effective as the anoxia treatment used in

our study.

In the present study, eggs were more tolerant to anoxia than adult females (Fig. 4 and Fig. 5). This

is in agreement with Suzuki et al. (2015), who reported a longer LT50 (time required for 50%mortality)

in eggs than in adult females (non-diapausing) of T. urticae and T. kanzawai. Differences in anoxia

tolerance among developmental stages and species were also reported for house dust mites,

22  

Dermatophagoides farinae Hughes, D. pteronyssinus (Trouessart) and Tyrophagus putrescentiae

(Shrank). In D. farinae and D. pteronyssinus, 100% mortality of the eggs was induced by 48 h of

anoxia, but almost half of the eggs of T. putrescentiae hatched even after 14 days of exposure

(Kamezaki et al., 2007). In adults of D. farinae and D. pteronyssinus, 100% mortality was induced

by 48 h anoxia, but it was 72 h for T. putrescentiae (Kamezaki et al., 2005).

Anoxia causes death in arthropods by two main mechanisms: water loss (Linnie, 1992) and O2

deficiency (Suzuki et al., 2015). The diameters of spiracles in insects depend on the composition of

the ambient air. At extreme low pO2 levels, the spiracles open fully (Förster and Hetz, 2010), which

could increase the rate of water loss and gas exchange to induce quick death. In T. urticae, the

openings of the peritremes are controlled by adjustment of the stylophore position, which controls

the area available for vapour diffusion and respiration through the tracheal system, and adult females

are able to reduce the rates of vapour diffusion and respiration by withdrawing their peritremes

(McEnroe, 1961). Because the deoxidant packages used in the present study were water dependent

for scavenging O2, and vapour saturated conditions (100% RH) were maintained during treatment.

Therefore, the peritremes of adult females under these conditions might have been exposed to anoxia,

whereas no diffusion of vapour might have occurred; thus O2 deficiency alone might be a cause of

death in the present study.

The present study showed that the time required to kill by anoxia decreases with increasing

temperature (Fig. 4 and Fig. 5). This phenomenon was also reported in other arthropods: the currant-

lettuce aphid N. ribisnigri (Liu, 2005), the western flower thrips F. occidentalis (Liu, 2008a) and the

confused flour beetle Tribolium confusum J. du Val (Chiappini et al., 2009). Downes et al. (2003)

found that anoxia for 6 h at 20°C caused metabolic arrest and consequently caused death in the green

peach aphid, Myzus persicae (Sulzer). In general, the metabolic rate of arthropods and other

poikilotherms increases with increasing temperatures. However, the reverse effect was observed

23  

under anoxia environment: the metabolic rate declined with increasing temperature under anoxia

(Neven and Hansen, 2010; Neven et al., 2014), heartbeat and circulation of haemolymph stop rapidly

and nerve cell activity ceases leading to lethal level of ATP to cause quick death of insects (Wegener,

1993). Therefore, high temperatures could enhance the effectiveness of anoxia in the present study.

Adult females of N. californicus were more tolerant to anoxia than those of the spider mites (Fig. 3).

There are two possible mechanisms to explain the anoxia tolerance of N. californicus. First, N.

californicus can maintain metabolism more effectively to reduce the metabolic rate or produce more

ATP from anaerobic metabolism. Second, physical adaption, like advanced tracheal system that can

deliver oxygen more effectively or allow more air to reach the tissues, can also help to survive

(Hoback and Stanley, 2001).

At 20°C, adult females of T. urticae showed higher impaired motor coordination (88.7% from

survived females) after anoxia for 6 h, while such a disorder was not observed in surviving adult

females of N. californicus, even when the anoxia duration was as long as 18 h. In Drosophila, O2

storage plays a vital role in sustaining normal functioning (Krishnan et al., 1997), but this does not

appear to be the case with T. urticae because it rapidly losses coordination and muscle tone when

exposed to anoxic conditions. T. urticae exhibited a lack of coordination in a stereotypic way (e.g.

falling down and paralysis) after only a few minutes of anoxia. Our results are more consistent with

the results obtained in Drosophila (Krishnan et al., 1997; Bartholomew et al., 2015). Anoxia not only

reduces hatchability and survivability, but also impairs normal behaviours, such as locomotion and

mating.

Our results suggest that anoxic conditions with increasing duration at controlled temperatures is a

promising method for controlling egg hatchability and adult female survivability of T. urticae and P.

citri, whereas it is less detrimental to N. californicus. Its higher tolerance to anoxia makes N.

californicus a good candidate for insect pest management (IPM) against T. urticae and P. citri along

24  

with the application of anoxia treatment. Further studies on the effects of anoxia on plants and a

method for large-scale treatment of anoxia are needed to determine the practicality of using anoxia

for IPM.

25  

Chapter 3 Hypobaric treatment

3.1 Introduction

Hypobaria in an air-tight vessel containing a commodity has been used to kill arthropod pests for

more than 90 years (Back and Cotton, 1925; Johnson, 2010; Hulasare et al., 2013). It is the simplest

type of CA/MA treatment, in which the total pressure is decreased without any changes in the

proportional composition of the air. Hypobaric treatments have been shown to be effective in

controlling pest insects and mites, particularly those in stored products (Žďárková and Voráček, 1993;

Navarro et al., 2007; Hulasare et al., 2013; Jiao et al., 2013). This technique was also effective against

a sanitary insect pest, the bed bug Cimex lectularius L. (Heteroptera: Cimicidae) (Liu and Haynes,

2016). The insect mortality was attributed to low oxygen concentration rather than any physical effect

of low pressure or dehydration (Navarro and Calderon, 1979; Friedlander and Navarro, 1983).

To prevent invasive species from crossing quarantine boundaries, it is essential to disinfest

imported commodities with phytosanitary treatments. Some commercial treatments that have been

used so far include lethal heat and cold, vapour heat, fumigants, anoxia, and ionizing irradiation

(Heather and Hallman, 2008; Wang et al., 2016). Other experimental treatments include modified

atmosphere and CA/MA in the form of low oxygen and/or high carbon dioxide (Wang et al. 2016).

Except for one successful shipment of asparagus from New Zealand to Japan in 1990, these latter

methods have not been used commercially (Carpenter and Potter, 1994; Heather and Hallman, 2008).

Importing countries often require the use of chemical fumigants such as MeBr to prevent the entry

of invasive pests (Jiao et al., 2013). Although MeBr was once a commonly used fumigant, its use has

been limited since 2015 because of human health issues (USDHHS, 1978) and its ozone depletion

potential (UNEP, 2016). Therefore, the development of non-chemical alternatives for controlling

agricultural pests is urgently needed.

Moreover, CA/MA treatments are often used not only for controlling pests but also for storing

26  

agricultural products (Oliveira et al., 2015), especially those that are sensitive to chemicals (Selwitz

and Maekawa, 1998). In general, the effectiveness of CA/MA including hypobaric treatment is

positively related to temperature because elevated temperatures enhance the metabolic rate (Cotton,

1932). Increasing temperature can increase the lethal activity of hypobaric treatments based on three

mechanisms: hypoxia (decreased pO2) (Navarro, 2012), desiccation (Thornton and Sullivan, 1964),

and hypobaria (decreased total pressure) (Galun and Fraenkel, 1961). Hypobaria has also been

recommended as an alternative disinfestation treatment for agricultural products since it is organic,

residue-free, and environmentally sustainable (Davenport et al., 2006; Navarro et al., 2007; Johnson

and Zettler, 2009; Jiao et al., 2013). However, so far, little is known about the effects of hypobaric

treatments on spider mites and their natural enemies.

Therefore, the objective of this study was to evaluate the effectiveness of hypobaric treatments

under different temperature and water source conditions as a phytosanitary treatment on three

important spider mite species Tetranychus urticae Koch, T. kanzawai Kishida, and Panonychus citri

(McGregor) and one of their natural enemies, the predatory mite Neoseiulus californicus (McGregor).

3.2 Materials and methods

3.2.1 Cultures of mites

T. urticae was collected from apple Malus pumila Miller (Rosales: Rosaceae) in Hiraka, Akita,

Japan, in 2013; T. kanzawai was collected from red clover Trifolium ratense L. (Fabales: Fabaceae)

in Usu, Hokkaido, Japan, in 2008; and P. citri was collected from citrus (Citrus unshiu Marcovitch)

(Sapindales: Rutaceae) in Nago, Okinawa, Japan, in 2010. The predatory mite N. californicus was

obtained from commercial biological agent SPICAL EX (Arysta LifeScience, Tokyo, Japan) in 2007.

The collected populations were maintained using detached leaves of common bean (Phaseolus

vulgaris L.) (Fabales: Fabaceae) for T. urticae and T. kanzawai or citrus (Citrus unshiu Marc.)

27  

(Sapindales: Rutaceae) for P. citri. The detached leaves were placed on water-saturated polyurethane

mats in plastic Petri dishes (90 mm in diameter, 20 mm in depth) and renewed every 2 weeks. The

edge of a detached leaf was covered with a wet tissue paper for preventing leaf desiccation and

escaping mites (Ehara and Gotoh, 2009). The Petri dishes were kept at 25 ± 1°C and 60–70% RH

under a 16:8 h L:D photoperiod in an incubator (NCTE-1000T; Nihon Freezer, Tokyo, Japan). Mixed

stages of T. urticae were provided as food for N. californicus reared on bean leaves in the same setup

and environmental conditions with those for spider mites (Ullah and Gotoh, 2014).

3.2.2 Hypobaric treatment

A bean or citrus leaf placed on water-saturated cylindrical polyurethane mat in the Petri dish was

fenced with a water-soaked filter paper to make a square arena (3 × 3 cm). Thirty adult females of

each spider mite species or 20 adult females of N. californicus were transferred from each stock

culture onto the arena on each leaf with a fine brush to allow them to oviposit under the incubator

conditions for 24 h. For N. californicus, we transferred an ample number of mixed stages of T. urticae

to avoid cannibalism that might occur under insufficient food condition (Schausberger, 2003). After

24 h, all adult females, faeces and filter papers were removed from the surface of leaves leaving only

eggs and were used in the following hypobaric treatment.

The hypobaric system consists of a vacuum pump (DAP-6D, Ulvac Kiko, Miyazaki, Japan), a 1.3-

L acrylic container with a vacuum gauge (VXS; As One, Osaka, Japan) and an incubator (MIR-254-

PJ; Panasonic, Osaka, Japan). The container was connected to the vacuum pump by a rubber tube

(10/20 mm in inner/outer diameter) (Fig. 6). By using the system, the absolute atmospheric pressure

inside the container was maintained at 17 kPa where pO2 was ≈ 3.5 kPa using Dalton’s law and

temperature was maintained at 20°C, 25°C, or 30°C.

Petri dishes typically containing several hundred eggs were used for each treatment. The number

28  

Fig. 6. The experimental apparatus of hypobaric treatment in the present study. It consisted of a vacuum pump and an acrylic container that were connected by a rubber tube.

29  

of eggs used for each treatment is shown in Fig. 7. The Petri dishes including eggs laid on leaves

placed on water-saturated cylindrical polyurethane mat were transferred to the container and exposed

to the hypobaric condition for 0 (control), 6, 12, or 24 h. At the end of each hypobaric exposure time,

the pump was switched off and the lid of container was opened for recovering the atmospheric

pressure back to ambient pressure. Then the lid of the container was closed again by 24 h after the

beginning of each hypobaric exposure time. After that, the Petri dishes were transferred into an

incubator (NCTE-1000T) and the eggs were cultured at 25°C, 55–65% RH, and 16:8 h LD. The eggs

were observed every day using a stereomicroscope and hatchability was calculated. Observations

were terminated after 2 days when no more eggs hatched. Three replications in each treatment were

performed.

The susceptibility of adult females to the hypobaric treatment was also evaluated using the above

system but a tube was used to confine them. Female spider mites or N. californicus at 3 or 2 days,

respectively, after adult female emergence were used in the hypobaric test. In order to synchronize

the day-age of adult females, teleiochrysalid spider mites were transferred from stock cultures to new

fresh leaves with fine brush and cultured in the rearing condition for 150 24 h. Due to unobvious

chrysalis stage in N. californicus, eggs oviposited within 24 h were cultured for 7 days to synchronize

the adult female emergence. Synchronized adult females were then confined in the 1.5-mL micro

centrifuge tube (HydrologixTM, Molecular BioProducts, Inc., San Diego, CA) having a ventilation

hole (4 mm in 155 diameter) in the lid covered with a fine mesh (2 × 2 cm) with the fine brush (three

adults/tube for spider mites, one adult/tube for N. californicus). The tubes were transferred to the

container for the hypobaric treatment at 20°C, 25°C, or 30°C for 0 (control), 6, 12, or 24 h. After the

hypobaric treatment, survivability was judged based on the responses of the mites to being touched

with a fine brush. For each treatment, three replications were performed. In each replication, 30 adult

females were examined, i.e. 10 tubes for each spider mite species or 30 tubes for N. californicus were

30  

Fig. 7. Egg hatchability of T. urticae, T. kanzawai, P. citri, and N. californicus after hypobaric treatment at 20°C, 25°C, and 30°C for 0, 6, 12, and 24 h exposure time. The numbers of eggs in each treatment are shown on the bars. Different letters above the bars for each temperature indicate a significant difference (ANOVA followed by Tukey’s HSD test, P < 0.05).

31  

used.

We hypothesized that hypobaric treatment would be harsher for adult females than for eggs because

in the case of adult females, there was no source of water or water vapour (except the mites

themselves). On the other hand, the eggs were tested with leaves placed on a water-saturated

cylindrical polyurethane mat. To test this hypothesis, we additionally evaluated the effect of hypobaric

treatment on adult females confined in the vent tubes in which 300-μL-water-soaked cotton was

placed on the bottom as a source of water and water vapour.

3.2.3 Data analysis

Prior to the analyses, an arcsine square-root transformation was performed to normalize the

proportional data. Interactions between three factors (species, temperature, and exposure duration)

were compared by three-way analysis of variance (ANOVA). Means of the normalized egg

hatchability and adult female survivability among treatments were compared by Tukey’s honestly

significant difference (HSD) test. Means of the normalized adult female survivability under presence

or absence of water vapour were compared using Student’s t-test. The significance level (α) for all

tests was set at 0.05. Three-way ANOVA and Tukey’s HSD test were performed using IBM SPSS

version 24 (IBM, 2016) and Student’s t-test was performed using R version 3.3.1 (Venables et al.,

2016).

3.3 Results

3.3.1 Egg hatchability

Egg hatchability was significantly affected by species, temperature, and duration of exposure to

hypobaria (P < 0.001) and their interactions (P < 0.001) (Table 4). In general, hatchability decreased

with increasing temperature and duration of exposure to hypobaria (Fig. 7). No eggs of T. urticae

32  

FP

FP

FP

Cor

rect

ed m

odel

4731

8.09

< 0.

001

70.9

8<

0.00

18.

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0.00

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0.00

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221.

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0.00

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< 0.

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179.

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0.00

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re2

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0.00

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< 0.

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7<

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< 0.

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< 0.

001

Spec

ies

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0.00

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0.00

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< 0.

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ies

× T

ime

919

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< 0.

001

20.0

1<

0.00

15.

66<

0.00

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7<

0.00

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.09

< 0.

001

6.75

< 0.

001

Spec

ies

× T

empe

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re ×

Tim

e18

20.1

2<

0.00

120

.78

< 0.

001

7.88

< 0.

001

a 300

μl w

ater

was

add

ed

Tab

le. 4

Thr

ee-w

ay a

naly

sis

of v

aria

nce

on th

e ef

fect

of

each

inde

pend

ent f

acto

r an

d th

e in

tera

ctio

n ef

fect

s on

the

hatc

habi

lity

ofeg

gs a

nd th

e su

rviv

abili

ty o

f ad

ult f

emal

es in

hyp

obar

ic tr

eatm

ent.

Egg

Adu

lt w

ithou

t wat

er s

uppl

yA

dult

with

wat

er s

u ppl

ya

Sour

cedf

33  

hatched at 25°C or 30°C when exposed for 24 h, but a few eggs hatched at 20°C. No significant

difference in the hatchability of T. urticae was observed between 6- and 12-h exposure (>60% in both

duration) at 20°C. The hatchability of T. urticae was decreased to 58.0% and 10.6% at 25°C for 6-

and 12-h exposure, whereas it decreased to 39.4% and 6.1% at 30°C, respectively. No eggs of T.

kanzawai hatched after 24-h exposure at all tested temperatures. The hatchability of T. kanzawai was

decreased to 76.0% and 30.2% at 20°C for 6- and 12-h exposure of hypobaria, whereas it was 51.1%

and 6.2% at 25°C, respectively. A few eggs of T. kanzawai hatched for 6 and 12 h of exposure to

hypobaria at 30°C. The trend of hatchability in P. citri was similar to that in T. urticae. No eggs

hatched at 25°C or 30°C when exposed for 24 h. However, 21.3% eggs hatched at 20°C even when

exposed for 24 h. Interestingly, N. californicus eggs showed higher hypobaric tolerance than spider

mite eggs at all tested temperatures. All eggs hatched even when exposed to 24 h except at 30°C

where the hatchability slightly decreased to 91.7%. For spider mites, the hatchability of eggs was

negatively correlated with time of hypobaric treatment, while for N. californicus, a negative

correlation was observed only at 30°C (Table 5).

3.3.2 Adult female survivability

Adult female survivability was also significantly affected by species, temperature, and duration of

exposure to hypobaria (P < 0.001) and their interactions (P < 0.001) (Table 4.). The survivability

decreased with increasing temperature and duration of exposure to hypobaria (Fig. 8). When there

was no water supply, almost all adult females of T. urticae survived at 20°C and 25°C even when

exposed for 24 h, but the survivability at 30°C was 14.4% and 2.2% when exposed for 12 and 24 h,

respectively. Adult female survivability of T. kanzawai decreased significantly at 20°C, 25°C, and

30°C, when exposed for 24, ≥12, and ≥6 h, respectively (Fig. 8). Almost all adult females of P. citri

survived without water supply at 20°C and 25°C even when exposed for 24 h, whereas 0%

34  

Tu

Tk

Pc

Nc

Egg

s－

20−

0.97

3−

0.97

5−

0.94

0

－25

−0.

916

−0.

893

−0.

812

－30

−0.

873

−0.

689

−0.

683

−0.

878

Adu

lts－

20−

0.83

2−

0.87

8−

0.94

0

－25

−0.

946

−0.

965

−0.

680

−0.

915

－30

−0.

880

−0.

877

−0.

939

−0.

701

＋30

−0.

893

Tu,

Tk,

Pc,

and

Nc

indi

cate

Tet

rany

chus

urt

icae

, T

etra

nych

us k

anza

wai

, P

anon

ychu

s ci

tri,

and

Neo

seiu

lus

calif

orni

us,

resp

ectiv

ely.

Tab

le 5

. Con

ditio

ns u

nder

whi

ch s

urvi

val w

as n

egat

ivel

y co

rrel

ated

with

tim

e of

hyp

obar

ic tr

eatm

ent.

Mat

eria

lH

2O a

dded

Tem

pera

ture

(°C

)S

peci

es

35  

Fig. 8. Adult survivability of T. urticae (a), T. kanzawai (b), P. citri (c), and N. californicus (d) after hypobaric treatment at 20°C, 25°C, and 30°C for 0, 6, 12, and 24 h exposure time and the effect of water supply during treatments. Different letters above the bars for each temperature indicates a significant difference (ANOVA followed by Tukey’s HSD test, P < 0.05).

36  

survivability was observed at 30°C when exposed for 24 h. Contrary to the result of eggs, lower

hypobaric tolerance in adult females was observed in N. californicus than spider mites particularly

when no water was supplied. In N. californicus, only a few adult females survived at 20°C, 25°C, and

30°C when exposed for 24, 12, and 6 h, respectively. No adult females survived at 25°C and 30°C

when exposed for 24 and ≥12 h, respectively.

When water was supplied, almost all adult females of T. urticae, T. kanzawai and P. citri could

survive even at 30°C with 24-h hypobaric exposure. Although almost all adult females of N.

californicus survived at 20°C and 25°C even when exposed for 24 h, the survivability dropped to

6.7% at 30°C. For both spider mites and N. californicus, the survivability of adult females was

negatively correlated with time of hypobaric treatment, although when water was supplied, a negative

correlation was observed only at 30°C for N. californicus (Table 5).

3.4 Discussion

The increased miticidal effect of hypobaric treatment on eggs (Fig. 7) and adult females (Fig. 8) of

spider mites at higher temperatures appears to be due to hypobaric-induced desiccation. Hypobaria

appeared to affect adult mosquitoes and housefly pupae only when the pressure was lower than 13

kPa (Galun and Fraenkel, 1961), which was lower than the pressure used in the present study (17

kPa). Few eggs of T. urticae hatched even after 24-h exposure to total pressure of 1 kPa, where the

estimated pO2 was ≈ 0.2 kPa (Satou, 2012), which is lower than the lethal threshold (1.0 kPa pO2)

(Navarro, 2012). This inconsistency might have been caused by the low temperature of 2.1°C during

the hypobaric treatment (Satou, 2012). In the present study, pO2 decreased to ≈ 3.5 kPa by decreasing

the total pressure to 17 kPa with the vacuum pump. This level is higher than the lethal threshold (1.0

kPa pO2) shown by Navarro (2012), suggesting that hypobaric-induced desiccation rather than

hypoxia may be the main mechanism for killing the eggs in the present study.

37  

Eggs of the three spider mite species tested in the present study were completely killed by

hypobaric treatment at 25°C and 30°C for 24 h (Fig. 7). When anoxia (pO2 = 0 kPa) was produced by

using a deoxidant consisting of iron, calcium chloride, and silicon dioxide, mortality of spider mite

eggs was 100% (Suzuki et al., 2015; Wang et al., 2016). In the present study, it took 24 h of hypobaric

treatment to completely kill T. urticae, T. kanzawai, and P. citri eggs at 25°C (Fig. 7), which was

twice the time it took to kill the eggs by anoxia treatment at 25°C (Suzuki et al., 2015; Wang et al.,

2016). N. californicus eggs showed higher tolerance to hypobaria than anoxia at 20°C, 25°C, and

30°C (Wang et al., 2016). The hatchability of N. californicus eggs was almost 100% even when the

eggs were exposed to hypobaria for 24 h at all tested temperatures. On the other hand, hatchability

was almost 0% for 24 h at 25°C and for 12 h anoxia at 30°C. N. californicus eggs were more tolerant

than spider mite eggs under anoxia at three different temperatures (Wang et al., 2016). Therefore, egg

hatchability in Tetranychidae was more sensitive to hypobaric conditions at higher temperatures than

it was in Phytoseiidae. The outermost layer, the mite eggshells (which is waxy and highly

hydrophobic), and the exochorion both prevent water loss from the egg (Witaliñski, 1993). Water loss

also depends on the egg structure, i.e. whether micropyles, pseudomicropyles, or respiratory organs

are present. Although chorion structures may differ between species (Witaliñski, 1993), we did not

examine them in this study. Such differences might be responsible for the difference in hypobaria

tolerance that we observed between the spider mites and N. californicus.

Adult females of spider mites better tolerated to the hypobaric treatment than eggs of spider mites

and adult females of N. californicus even for 24 h exposure at 30°C when water was supplied (Fig. 7

and Fig. 8). In contrast to anoxia tolerance (Suzuki et al., 2015; Wang et al., 2016), adult females of

N. californicus were highly susceptible to hypobaric treatment particularly when water was not

supplied. When water was supplied, the adult female survivability of all spider mite species and N.

californicus increased substantially (Fig. 8). Therefore, the tolerance of adult females to long duration

38  

hypobaric condition was greater in Tetranychidae than in Phytoseiidae, which is the opposite of what

was observed with egg hatchability. The wet cotton might have kept the water vapour pressure in the

test tube at saturation and might have inhibited water loss from the mite body. Because N. californicus

can take in water directly, the wet cotton might be a source not only of vapour but also of water for

them.

Although the adults were more tolerant of hypobaria than eggs in the present study (Fig.7 and Fig.

8), immobile stages are generally more tolerant to CA/MA treatments than mobile stages due to their

low metabolic rate (Navarro, 2006). However, in some cases, adults are more tolerant than eggs

(Donahaye et al., 1996; Mitcham et al., 1997; Held et al., 2001). In these studies, controlled gases

were continuously supplied to test chambers during the treatment in which the evaporation rate might

be enhanced by the air current produced. Spiracles are the main pathway of water loss during

respiration in adult mites (McEnroe, 1961) and are open fully under extreme low pO2 conditions

(Förster and Hetz, 2010). Because the pO2 used in the present study was above the threshold at which

spiracles open fully (2.6 kPa pO2) (Förster and Hetz, 2010), water loss in adult spider mites might

have been moderated, leading to rather low susceptibility to hypobaric-induced desiccation.

Structural and functional difference in the gas exchange system among mites might underlie the

higher and lower susceptibilities to hypobaric-induced desiccation in eggs and adults observed in the

present study.

In the present study, pO2 was ≈ 3.5 kPa and the mortality of adult females was reduced by supplying

water, suggesting that desiccation accompanying the hypobaric treatment might induce high mortality.

Unlike traditional CA/MA treatments that often cause negative effects on seedlings (Held et al., 2001),

plants are tolerant to hypobaric hypoxia/desiccation without showing any obvious disorder (Paul et

al., 2004; Wheeler et al., 2011). This suggests that hypobaric treatments can be useful for controlling

pest mites while having a low impact on predatory mites and seedlings and can be an environmental-

39  

friendly means of replacing conventional fumigants. Finally, further studies of hypobaria should lead

to better phytosanitary treatments for commercially important fresh products and their effects on

quarantine insect and mite pests.

40  

Chapter 4 General Discussion

The effectiveness of CA/MA is dependent on several factors: temperature, partial pressure of each

gas, treatment duration, species and humidity (Adler et al., 2000; Navarro, 2006, 2012; Navarro et al.,

2012). In the present study, we focused on pO2 to examine the effect of total pressure, temperature

and treatment duration on CA/MA in three spider mite species and a predatory mite, one of their

natural enemies. Complete disinfestation was achieved in the spider mites, and a difference was

observed between the spider mites vs. the predatory mite.

In general, there is a positive correlation between CA/MA and temperature, because temperature

acts directly on metabolic rate (Neven et al., 2014; Boardman et al., 2016). The deleterious effect of

CA/MA on pests which are at low metabolic rate in low temperature still accumulates gradually, and

pests are doomed to die when accumulated adverseness reaches a threshold (Boardman et al., 2016).

In the present study, the treatment duration necessary for complete disinfestation was halved with

every 5°C temperature rise in anoxia treatment. A similar trend was observed in hypobaric treatment,

although treatment duration for complete disinfestation was longer than in anoxia treatment. In anoxia

treatment, the eggs of T. urticae and P. citri were completely killed within 24 h at 20°C. When

temperature raised to 25°C, 12 h treatment was enough to achieve complete disinfestation, and only

6 h was needed at 30°C (Fig. 4). Adult females were more sensitive than eggs; almost all T. urticae

and all P. citri were killed after 6 h anoxia at 25°C (Fig. 5). In hypobaric treatment, the eggs of T.

urticae, T. kanzawai and P. citri were all killed within 24 h at 25 and 30°C. Although only 6 h

treatment at 30°C was enough to kill all the eggs of P. citri, some eggs of T. urticae and a few eggs

of T. kanzawai still hatched (Fig. 7). Surprisingly, adult females had stronger hypobaric tolerance than

eggs, but temperature still affected their survivability. Hypobaric treatment was not effective at 20

and 25°C in T. urticae and P. citri, not even after 24 h treatment, but the survivability of T. kanzawai

reduced to ca. 50%. When treatment temperature was raised to 30°C, survivability of the three spider

41  

mite species started to decrease, but complete disinfestation failed even at 24 h treatment (Fig. 8).

Based on the above results, a difference in tolerance between spider mite species and stages was

observed in both treatments. Nevertheless, adult females of spider mites were more tolerant than N.

californicus in hypobaric treatment. On the other hand, although the tolerance of adult females of N.

californicus was weaker than the tolerance of eggs in anoxia treatment, the female predatory mites

were still stronger than the eggs of spider mites. The shortest treatment duration for complete

disinfestation in practical application is determined by the most tolerant stage. This implies that there

is a potential of anoxia treatment for selectively and simultaneously controlling both eggs and adult

females of spider mites. In order to verify this, we extended the treatment duration. As a result, both

stages of spider mites were completely killed after 18 h at 20°C (Table 2, 3), whereas both hatchability

and survivability of N. californicus still remained ca. 50%. This suggests that 18 h at 20°C of anoxia

treatment is the ideal condition for selective control of spider mites in practical use.

The deleterious effect of CA/MA is not only immediate death but it also induces sub-lethal effects.

The treated survivors present slower development, lower reproductive ability and shorter longevity

(Ofuya and Reichmuth, 2002), as well as behavioral changes (Krishnan et al., 1997). An animal’s

nervous system controls most muscle coordination. It is very sensitive to pO2 and appears to

dysfunction in a low pO2 environment (Rodgers et al., 2010). The sensitivity of the nervous system

to pO2 may vary among mite species. In the present study, T. urticae showed impaired motor

coordination in anoxia treatment after 6 h at 20°C, whereas N. californicus still walked normally. This

suggests that CA/MA can also be used as a support agent to increase predatory efficiency of N.

californicus by weakening spider mites.

Adenosine triphosphate (ATP) is an important energy transfer substance that carries energy to

maintain functionality and biochemical pathways in cells (Knowles, 1980). The main source of ATP

is the electron transport chain (ETC) in mitochondria during aerobic respiration (Inoue et al., 2003;

42  

Spees et al., 2006). O2 is the final electron receptor in ETC – the chain reaction of ETC inhibition

induces metabolic arrest in O2 deficiency. Interestingly, the metabolic arrest threshold/critical

concentration point (Pc) coincides with the upper pO2 of the insecticidal range in CA/MA (pO2 ≈ 5

kPa) (Fleurat-Lessard, 1990; Hoback and Stanley, 2001; Mitcham et al., 2006). Lowering the

metabolic rate to reduce ATP demand under low pO2 atmosphere is a survival strategy (Hochachka,

1986). However, the available ATP still cannot satisfy the reduced ATP demand if pO2 is lower than

the anaerobic compensation point (Pa) and rapid death starts (Mitcham et al., 2006). Pa was reported

at pO2 ≈ 1 kPa in several pests (Hoback and Stanley, 2001; Mitcham et al., 2006), and acute lethal

threshold/critical Pc was the same as this pO2 that killed the pest within 24 h (Fleurat-Lessard, 1990;

Navarro, 2012; Navarro et al., 2012); in other words, the anaerobic compensation point is the same

as the acute lethal threshold. Although the acute lethal threshold in most pests is at pO2 ≈ 1 kPa, there

is still some variation among species due to different physiological and structural adaptions (Navarro,

2006, 2012; Navarro et al., 2012).

In the present study, the eggs and adult females of N. californicus demonstrated better anoxia

tolerance than those of T. urticae and P. citri. The acute lethal threshold of N. californicus may be

higher than that of T. urticae and P. citri. More research is needed to clarify the underlying

mechanisms. Held et al. (2001) reported that T. urticae can be completely disinfested within 24 h at

pO2 = 0.2 - 1.4 kPa, i.e., lower than the acute lethal threshold. In the present study, T. urticae was also

completely killed within 24 h in anoxia treatment. This implies that there is not much difference in

the effectiveness of CA/MA once pO2 is below the acute lethal threshold. Intriguingly, Satou (2012)

reported an inconsistent case, in which there were still few eggs of T. urticae hatching after hypobaric

treatment at ≈ 2 kPa, i.e., lower than Pa – this may be explained by a protective effect of low

temperature at 2.1°C during the treatment.

Obviously, O2 deficiency was the main killing mechanism in the present anoxia study. However,

43  

the pO2 in hypobaric treatment was ≈ 3.5 kPa, which is higher than the acute lethal threshold, and the

exposure duration of hypobaric treatment for complete disinfestation was much longer than the anoxia

treatment. The different death patterns imply that there must be another killing mechanism involved

in hypobaric treatment. Although hypobaria per se has an insecticidal effect, atmospheric pressure

must be lower than 13 kPa (Galun and Fraenkel, 1961). Therefore, hypobaria per se was not the main

killing mechanism in the present hypobaria study.

Hypobaria induces dehydration due to vapor deficit, which leads to death (Thornton and Sullivan,

1964; Mitcham et al., 2006; Kumar et al., 2017). We verified this hypothesis by adding wet cotton as

a water source during treatment. The wet cotton not only supplied humidity to the air reducing water

loss from the body, but also served as a water source for drinking. Consequently, the survivability of

spider mites and N. californicus increased significantly even after 24 h treatment (Fig. 8).

In general, the tolerance for CA/MA is higher in immobile than in mobile stages due to their lower

metabolic rate (Navarro, 2006). Dehydration might also explain the unusual result that adult females

were more tolerant than eggs in hypobaric treatment. Adults are able to reduce water loss from the

spiracle by control over its opening, but eggs lack this control mechanism (McEnroe, 1961). “O2-

open” threshold is defined as the lowest pO2 at which spiracles can be closed, viz. at pO2 ≈ 2.6 kPa

(Förster and Hetz, 2010). Although spiracles open fully when environmental pO2 is lower than the

“O2-open” threshold, adults kept their spiracle control ability in the present hypobaric treatment at

which pO2 was ≈ 3.5 kPa. Moreover, adult females are also capable of locomotive behavior: they are

able to move away. There are several relatively safe places even in a single commodity during

treatment, as complex interactions between environment and commodity cause different

microenvironments (Athanassiou et al., 2017). The locomotive ability allows adult females to escape

to a relatively high humidity microenvironment where water loss is reduced, or even to seek a water

source to replenish.

44  

Besides the physiological adaption, structural and functional differences might also take part in

anoxia and hypobaria tolerance. The surface of the egg contains a highly hydrophobic waxy layer and

exochorion, that water and gas are hard to pass through (Witaliński, 1986; Witaliñski, 1993). Gas

exchange is carried out mainly through connective structures such as micropyles, pseudomicropyles

or a respiratory organ (Tuft, 1950; Dittrich and Streibert, 1969). The presence of a spongy layer that

can store air may also play a role (Wigglesworth and Beament, 1950). Further study is needed to

elucidate the mechanisms underlying the tolerance difference among the tested species.

In conclusion, the techniques of CA/MA that were studied in the present study have potential as a

novel selective pest disinfestation for IPM. These techniques can be used in agriculture to replace

recent chemical treatments, particularly for fumigation. They can also be used to increase the

predatory efficiency of natural enemies through weakening of the pests. Although large-scale

application of the disinfestation is impossible due to physical limitation, it is suitable for small-scale

application, especially for individual farmers and plant quarantine stations. Moreover, CA/MA is

inexpensive, special training is not needed, and it can be accepted easily by applicators in the future

management of spider mites.

45  

Summary

Man has used pesticides for disinfesting agricultural and storage pests since 8,000 BC, and more

recently their usage has increased incredibly, especially since World War II. Fumigation is one of the

derivative applications that copes with commodities by gaseous pesticides (fumigants). It has been

commonly used in transport activities, for instance in international trade. Because of the gaseous

diffusion property of fumigants, exposure duration for complete disinfestation is short, and

fumigation is especially effective against hidden pests. Methyl bromide was once a popular fumigant

because of its low price and high effectiveness. However, its use has been restricted since 2015 due

to human health and general environmental issues (it breaks down ozone). As a result, the cost of

agricultural production and storage has increased greatly.

Controlled Atmosphere/Modified Atmosphere (CA/MA) is an alternative that has received recent

attention. It kills pest by modifying atmospheric composition physically, especially by manipulating

the partial pressure of CO2 and/or O2 (pO2). CA/MA combines the advantages of fumigation with

being environmentally friendly. The effectiveness of CA/MA has been proven in several studies. In

the present study, we focused on the effects of pO2 in anoxia (no oxygen) and hypobaric (low

atmospheric pressure) treatment of important agricultural pests (several spider mite species) and one

of their natural enemies (a predatory mite).

In Chapter 2, we evaluated the effect of anoxia on two spider mite species, the two-spotted spider

mite, Tetranychus urticae Koch, and the citrus red mite, Panonychus citri (McGregor), and on one of

their natural enemies, the predatory mite Neoseiulus californicus (McGregor). The hatchability of

eggs and the survivability of adult females decreased with increasing temperature and treatment

duration, and differences in tolerance were observed among the species and stages. In short, N.

californicus could withstand anoxia better than the two spider mite species, and eggs were more

tolerant than adult females. The optimal durations for completely disinfesting the eggs of the two

46  

spider mite species were 12 h at 25°C or 6 h at 30°C, meanwhile the hatchability of N. californicus

was still ca. 70 and 65%, respectively. For adult females, in 12 h at 20°C and 6 h at 25°C the

survivability of T. urticae dropped to ca. 5% and all P. citri were killed, while, in contrast, ca. 80% of

N. californicus survived. To achieve complete disinfestation for eggs and adult females of spider mite

species – a prerequisite in many circumstances –, we extended the treatment duration in order to find

out the optimal condition that saves as many eggs and adult females of N. californicus as possible.

We successfully achieved that under treatment for 18 h at 20°C both hatchability and survivability of

N. californicus remained ca. 50%, while all stages of spider mites were killed. Interestingly, although

the survivability of T. urticae was ca. 90% when treated for 6 h at 20°C, ca. 80% of the adult females

showed impaired motor coordination, a behavioral effect not seen in N. californicus. Thus, anoxia

treatment has the potential to weaken spider mites, making them an easier prey, and to increase the

predatory rate of N. californicus.

In Chapter 3, we used a vacuum pump to create a low pO2 (≈ 2 kPa) environment by hypobaria

(atmospheric pressure ≈ 17 kPa). In addition to the two spider mites studied in the previous chapter,

a third species, the Kanzawa spider mite, Tetranychus kanzawai Kishida, was included. Similar to

anoxia treatment, the hatchability of eggs and the survivability of adult females decreased with

increasing temperature and treatment duration, and differences in tolerance were observed among

species and stages. However, for complete disinfestation in hypobaria, treatment had to be longer

than in anoxia. The eggs of the three spider mites were completely controlled after 24 h at both 25

and 30°C, while the hatchability of N. californicus stayed 100%, except after 24 h at 30°C, which

slightly reduced predator egg hatchability to ca. 90%. However, contrasting results were obtained

with adult females of the three spider mite species, who were more tolerant than N. californicus

females: few individuals of the three spider mite species survived treatment for 24 h at 30°C, but all

N. californicus had died after treatment for 24 h at 25°C or 12 h at 30°C. We added wet cotton as a

47  

water source to elucidate why the female adults died so rapidly. As a result, almost all mites survived,

and even some N. californicus survived after 24 h at 30°C. In brief, hypobaric treatment can only be

applied to selectively disinfest the eggs of spider mites, and the cause of death is dehydration.

In conclusion, the novel disinfestation CA/MA treatment in the present study that was achieved by

modifying pO2 has potential as an alternative for fumigation. It can also be applied in integrated pest

management because of its selective disinfestation. Moreover, we clarified the difference in killing

mechanism between anoxia and hypobaria, which were O2 deficiency and dehydration, respectively.

48  

摘要

農業害虫は農産物の生産および貯蔵に大きな被害を与え、これに対して人類は既に 8,000

BC から化学的防除を始め、そして第二次世界大戦後、その使用は飛躍的に伸びていった。

このうち、燻蒸法は蒸気圧の高い薬剤によって農産物に付着している害虫を防除する処理

法であり、国際貿易などの農産物を輸送する際によく用いられている。この方法では薬剤

が迅速に空気中に拡散するため、害虫を完全に防除するまでに必要な時間が短く、また農

産物の内部に隠れた害虫にも高い効果が得られる。特に臭化メチルは安価かつ効果的であ

るため、世界中で も用いられた燻蒸剤であった。しかし、人体に対する悪影響やオゾン

層破壊物質であることが確認され、2015 年以後の使用が禁止された。その結果、農産物の

生産および貯蔵コストが大幅に増加している。

その代替技術として空気制御法 (Controlled Atmosphere/Modified Atmosphere; CA/MA) が

近年注目されている。この方法は空気の組成を物理的に改変することによって、特に CO2

あるいは O2 (pO2) 分圧の調整によって害虫を防除するものであり、燻蒸法の利点を備えて

いると共に、生物や環境に及ぼす影響が少ない。現在、この方法による防除成功例が多く

報告されており、その防除効果は pO2 の低さに比例して高くなる。本研究では、pO2 に注目

して、脱酸素剤による O2 の除去と真空ポンプによる低 O2 状態の作出が、農業害虫である

ハダニとその天敵であるカブリダニに及ぼす影響を調査し、将来化学的防除法の代替法に

なる可能性について検討することを目的とした。

第 2 章では、脱酸素剤を用いて空気中の酸素をすべて抜き (pO2 = 0)、害虫ハダニのナミ

ハダニ Tetranychus urticae Koch (以下、ナミ)、ミカンハダニ Panonychus citri (McGregor) (以

下、ミカン) および生物農薬として販売されている天敵ダニのミヤコカブリダニ Neoseiulus

49  

californicus (McGregor) (以下、ミヤコ)の卵および雌成虫の無酸素耐性を調査した。その結

果、いずれの種でも処理温度の上昇と処理時間の増加に伴い、卵の孵化率と雌成虫の生存

率が低下した。ただし、種間と発育ステージ間に差が見られ、ミヤコはハダニより、また

卵は雌成虫より高い無酸素耐性をもつことが分かった。卵では、25°C／12 h と 30°C／6 h

が 適条件であり、ナミとミカンの孵化を完全に制御できた一方、ミヤコは約 70％と約

65％が孵化した。雌成虫では、ナミが 20°C／6 h で 90％ (このうち 80％は歩行困難であっ

た)、20°C／12 h と 25°C／6 h が共に 5％未満、そして 30°C／6 h が 0％、ミカンではこれら

いずれの条件においても 0％であった。これに対して、ミヤコではいずれの条件でも約 80％

が生存した。ハダニの卵と雌成虫を完全に制御し、しかもミヤコの卵と雌成虫を 大限に

温存できる条件を探るため、処理時間をさらに延長した。その結果、20°C／18 h において、

ハダニを完全に防除でき一方、ミヤコの孵化率と生存率が共に約 50％になる条件を見い出

した。さらに、無酸素処理は、ハダニの歩行困難による弱体化でミヤコの捕食率をさらに

向上させることができると考えられた。

第 3 章では、真空ポンプによって低 pO2 (≈ 2kPa) の真空環境 (大気圧 ≈ 17kPa) を達成

し、前章で述べたダニ種にカンザワハダニ T. kanzawai Kishida (以下、カンザワ) を加えて

真空耐性を調査した。その結果、第 2 章の無酸素処理と同様に処理温度の上昇と処理時間

の増加に伴い、卵の孵化率と雌成虫の生存率が低下した。しかし、すべてのハダニ種の雌

成虫を完全に防除できる処理条件がなかった一方、ミヤコの雌成虫はハダニより耐性が弱

かった。孵化率でみると、25°C／24 h と 30°C／24 h が 適条件であり、ナミ、カンザワと

ミカンを完全に防除できた。これに対して、ミヤコはほぼ影響を受けておらず、孵化率は

25°C／24 h で 100％、30°C／24 h でも約 90％であった。しかし、雌成虫は逆の結果となり、

50  

ハダニのどの種も 30°C／24 h の処理でわずかに生き残る個体がいた。一方、ミヤコは 25°C

／24 h と 30°C／12 h の処理ですべての個体が死亡した。このように雌成虫において無酸素

処理と異なる結果が得られた原因を解明するため、濡らせたコットンを水供給源として処

理容器内に入れた。その結果、いずれのダニ種の生存率も大幅に上昇した。30°C／24 h の

処理では、すべてのハダニ種の雌成虫が高い生存率を示した。ミヤコの雌成虫では、30°C

／12 h の処理でもほとんど影響を受けなかったが、30°C／24 h の処理では生存できる個体

が極めて少なかった。以上の結果から、真空処理が無酸素処理に比べて、雌成虫の死亡率

が高かった理由は、雌成虫の脱水によるものと推定された。

本研究では、空気制御によって害虫ハダニを防除できる一方、天敵を温存できる可能性

を示した。将来、天敵の利用を重視する総合的害虫管理 (Integrated Pest Management) にお

いて、空気制御が有効な手法の一つになるものと期待できる。さらに、無酸素処理と真空

処理の致死メカニズムが異なり、それぞれ酸素欠乏と脱水であることが示唆された。

51  

Acknowledgements

I would first like to express my appreciation of my supervisor, Prof. Dr. Tetsuo Gotoh, Faculty of

Agriculture, Ibaraki University, for his kind suggestions. Without his warm encouragements and

leadership, I might not have finished my research here. Moreover, his door was always open when I

faced problems and obstacles.

I would also like to thank my co-supervisor, Assoc. Prof. Dr. Yasuki Kitashima, Faculty of

Agriculture, Ibaraki University, for his suggestions on statistical analysis, and another co-supervisor,

Prof. Dr. Hideki Kawasaki, Faculty of Agriculture, Utsunomiya University, for his guidance and

support. I am grateful to Asst. Prof. Dr. Takeshi Suzuki, Graduate School of Bio-Applications and

Systems Engineering, Tokyo University of Agriculture and Technology, for his help in developing the

idea of my research. I also thank all my colleagues, graduate students and undergraduate students of

the Laboratory of Applied Entomology and Zoology, Faculty of Agriculture, Ibaraki University, for

their kindness and encouragements. I also thank Dr. Mohammad Shaef Ullah for reviewing my

doctoral thesis.

Finally, I want to express my gratitude to Honokai (報農会) and the Japan-Taiwan Exchange

Association (日本台湾交流協会) that offered the scholarships during my stay in japan.

52  

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