THE EFFECTS of NATURAL and ANTHROPOGENIC FACTORS on the PHYSIOLOGICAL STATE of INSECTS IRJA KIVIMÄGI a Thesis for Applying

THE EFFECTS OF NATURAL AND ANTHROPOGENIC FACTORS ON THE PHYSIOLOGICAL STATE OF INSECTS

LOODUSLIKE JA ANTROPOGEENSETE FAKTORITE MÕJU PUTUKATE FÜSIOLOOGILISELE SEISUNDILE

IRJA KIVIMÄGI

A thesis for applying for degree of Doctor of Philosophy in Entomology

Väitekiri filosoofiadoktori kraadi taotlemiseks entomoloogia erialal

Tartu 2012

EESTI MAAÜLIKOOL ESTONIAN UNIVERSITY OF LIFE SCIENCES

THE EFFECTS OF NATURAL AND ANTHROPOGENIC FACTORS ON THE PHYSIOLOGICAL STATE OF INSECTS

LOODUSLIKE JA ANTROPOGEENSETE FAKTORITE MÕJU PUTUKATE FÜSIOLOOGILISELE SEISUNDILE

IRJA KIVIMÄGI

A thesis for applying for degree of Doctor of Philosophy in Entomology

Väitekiri filosoofiadoktori kraadi taotlemiseks entomoloogia erialal

Tartu 2012 Institute of Agricultural and Environmental Sciences Estonian University of Life Sciences

According to verdict No 119 of October 9, 2012, the Doctoral Committee of the Agricultural and Natural Sciences of the Estonian University of Life Sciences has accepted this for thesis for the defence of degree of Doctor of Philosophy in Entomology.

Opponent: Prof. Habil. Vincais Būda Institute of Ecology Vilnius University Lithuania

Supervisors: Dr. Luule Metspalu Institute of Agricultural and Environmental Sciences Estonian University of Life Sciences

Dr. Angela Ploomi Institute of Agricultural and Environmental Sciences Estonian University of Life Sciences

Defence of the thesis: Estonian University of Life Sciences, Karl Ernst von Baer House, Veski 4, Tartu, on November 15, 2012 at 10.00.

The English language was edited by Dr. Ingrid H. Williams and Dr. Eha Kruus, and the Estonian by Dr. Luule Metspalu.

Publication of this thesis is granted by the Estonian University of Life Sciences and by the Doctoral School of Earth Sciences and Ecology created under the auspices of European Social Fund.

LIST OF ORIGINAL PUBLICATIONS ...... 7 ABBREVIATIONS ...... 9 1. INTRODUCTION ...... 10 2. REVIEW OF THE LITERATURE ...... 13

2.1. Respiration measured by CO2 release ...... 13

2.2. The pattern of O2 uptake ...... 14 2.3. Muscular ventilation (pumping) ...... 16 2.4. The origin and functions of discontinuous gas exchange .. 17 2.5. Respiratory failures as indicators of chemical stress ...... 20 2.6. Effects of food plants on the development and diapause in insects ...... 21 3. AIMS OF THE STUDY ...... 23 4. MATERIALS AND METHODS ...... 24 4.1. Insects ...... 24 4.2. Treatments ...... 24 4.2.1. Pieris brassicae (I) ...... 24 4.2.2. Pterostichus niger (II) ...... 24 4.2.3. Platynus assimilis (III-IV) ...... 25 4.2.4. Mamestra brassicae (V) ...... 26 4.3. Measuring methods ...... 27 4.3.1. Coulometric respirometry (I, III, V) ...... 27

4.3.2. Flow-through CO2 respirometry (I-V) ...... 29 4.3.3. Infrared probe actography (I-IV) ...... 30 4.3.4. Calorimetry (V) ...... 31 4.4. Data acquisition and statistical analysis ...... 32 5. RESULTS ...... 33 5.1. Oxygen convective uptakes in gas exchange cycles in early diapause pupae of Pieris brassicae (I) ...... 33 5.2. Effects of humidity conditions on the gas exchange patterns on Pterosticus niger (II) ...... 35 5.2.1. Discontinuous gas exchange ...... 35 5.2.2. Cyclic gas exchange ...... 35 5.3. The gas exchange patterns and the effects of sublethal doses of pyrethroid on gas exchange in Playunus assimilis (III, IV) ... 37 5.4. Effects of larval food plants on diapause of the cabbage moth, Mamestra brassicae (III) ...... 39 6. DISCUSSION ...... 42 6.1. Cyclic gas exchange with Passive Suction Inspiration (PSI) in diapausing pupae of Pieris brassicae (I) ...... 42 6.2. Gas exchange in the carabid beetle Pterostichus niger in low and high humidity conditions (II) ...... 44 6.3. Interaction between circulation and gas exchange ...... 46 6.4. Gas exchange in the carabid beetle Platynus assimilis before and after treatment with sublethal doses of pyrethroid ..... 47 6.5. The influence of food plants on development and on dormancy of Mamestra brassicae (V) ...... 49 7. CONCLUSIONS ...... 52 REFERENCES ...... 54 SUMMARY IN ESTONIAN ...... 67 ACKNOWLEDGEMENTS ...... 71 PUBLICATIONS ...... 73 CURRICULUM VITAE ...... 124 ELULOOKIRJELDUS ...... 128 LIST OF PUBLICATIONS ...... 131

6 LIST OF ORIGINAL PUBLICATIONS

The present thesis is a summary of the following research papers, which are referred to by their Roman numerals in the text.

I Jõgar K., Kuusik A., Ploomi A., Metspalu L., Williams I.H., Hiiesaar K., Kivimägi I., Mänd M., Tasa T., Luik A. 2011. Oxygen convective uptakes in gas exchange cycles in early diapause pupae of Pieris brassicae. The Journal of Experimental Biology, 214, 2816 – 2822.

II Kivimägi I., Kuusik A., Jõgar K., Ploomi A., Williams I.H., Met- spalu L., Hiiesaar K., Sibul I., Mänd M., Luik A. 2011. Gas exchange patterns of Pterostichus niger (Carabidae) in dry and moist air. Physi- ological Entomology, 36, 62 – 67.

III Kivimägi I., Ploomi A., Metspalu L., Švilponis E., Jõgar K., Hiie- saar K., Luik A., Sibul I., Kuusik A. 2009. Physiology of a carabid beetle Platynus assimilis. Agronomy Research, 7 (special issue I), 328 – 334.

IV Kivimägi I., Kuusik A., Ploomi A., Metspalu L. Jõgar K., Williams I.H., Mänd M., Sibul I., Hiiesaar K., Luik A. Gas exchange patterns in Platynus assimilis (Coleoptera, Carabidae): respiratory failure induced by a pyrethroid. European Journal of Entomology. (Accepted August 17th 2012).

V Metspalu L., Kruus E., Jõgar K., Kuusik A., Williams I.H., Veromann E., Luik A., Ploomi A., Hiiesaar K., Kivimägi I., Mänd M. Larval food plants can regulate the cabbage moth, Mamestra brassicae L. population. Bulletin of Insectology. (Submitted).

The papers are reproduced by kind permission of the corresponding journal or publisher.

7 Authors’ contribution to each article Idea and Analysis Manuscript Paper Data collection study design of data preparation I KJ, IK, AP, LM, IK, KJ, AK, TT, IK, KJ KJ, IK, AP, IHW, AK, IHW, AK, KH, MM, AP, LM KH, LM, TT, AL AL, TT II IK, KJ, AP, KH, AK, IK, AK, AP, LM, IK, KJ IK, AK, KJ, AP, LM, LM, MM, AL KJ IHW, IS, KH, AL III IK, KJ, AP, KH, AK, IK, KJ, AK, AP, IK IK, AP, LM, EŠ, KJ, LM, AL AL, KH, LM IS, KH, AK AL IV IK, LM, AP, AK, KJ, IK, AP, KJ, AK, IK IK, AK, AP, LM, KH, AL, MM LM IHW, KH, KJ, IS, AL V LM, KJ, IK, EŠ, AP, LM, KJ, IK, AP, LM, IK LM, EŠ, KJ, IK, EV, AL, KH, MM, AK KH AP, IHW, KH, MM, AK, AL AK – Aare Kuusik, AL – Anne Luik, AP - Angela Ploomi, EŠ – Eha (Švilponis) Kruus, EV – Eve Veromann, IK – Irja Kivimägi, IS – Ivar Sibul, IHW – Ingrid H. Williams, KH – Külli Hiiesaar, KJ – Katrin Jõgar, LM – Luule Metspalu, MM – Marika Mänd, TT – Tea Tasa

8 ABBREVIATIONS a.i. active ingredient CGE cyclic gas exchange

CO2 carbon dioxide C-phase closed phase DGE discontinuous gas exchange F-phase flutter phase

H2O water IR infrared IRGA infrared gas analyser L litre

O2 oxygen O-phase open phase PSI passive suction inspiration PSV passive suction ventilation RH relative humidity RMR resting metabolic rate SMR standard metabolic rate

VCO2 rate of CO2 emission

VO2 rate of O2 emission WLR water loss rate µV m W rate of heat flow

9 1. INTRODUCTION

The physiological state of the insect is influenced throughout by environmental factors, such as temperature and humidity, by anthropogenic toxicants such as pesticides and by insect food plants. Insects respond to these factors with adequate changes in physiological state. To describe changes in insect physiological state it is necessary to understand responses to any influencing factors in the insect. Changes of respiratory physiology show the level of activity and vitality of the insect. The main difference between inactive and active insects reflects metabolic levels and gas exchange patterns. There is data about the influence of temperature on insect physiology (Contreras and Bradley, 2009, 2010), but only few data (Terblanche et al., 2008) are available concerning the effects of humidity on physiology and respiratory metabolism.

During the second half of the last century, agricultural intensification (pesticides, fertilization, and high-yielding crop varieties) has increased food production, but has also changed the function of the agroecosystem. Agricultural intensification decreases the proportion of the ratio between pest and predator.

Pesticides have a direct lethal effect on species and can cause major population declines, gradually accumulate in the food web and are taken up by vertebrates and top predators, such as mammals or raptors. Arthropod predators including several species of beetle may serve as indicator species for the overall health of the insect ecosystem (Cherry and Gilbert, 2003). The use of insecticides has a negative effect on biodiversity and reduces the biological control potential (Geiger et al., 2010). More than 515 tons of pesticides are produced annually for application in the agroecosystem to control insect pests, diseases and also weeds in Estonia alone. But, the application of pesticides to control target organisms can also influence surrounding non-target ecosystems. The most important are interactions and balance in the food web. In more intensive agriculture, arthropod populations are lowest (Ruby et al., 2010).

The respiration and transpiration systems in insects are the most vulnerable targets. However, to date, sublethal effects of contemporary insecticides are poorly studied. These effects are known as delayed or after-effects. Effects on metabolic rate reflect the physiological state of

10 the insect and are commonly measured via respiratory rates. There is literature concerning the effects of various chemical compounds on oxygen consumption in insects (Keister and Buck, 1974), which have been measured by volumetric-manometric techniques, where activity periods cannot observe (Warburg apparatus). Modern flow-through O2 and CO2 respirometry does not allow recording of oxygen convective uptakes (passive suction inspiration or PSI). Obviously there must be used also a volumetric-manometric respirometry system which is able to separate the activity and inactivity periods.

Standard metabolic rate is defined as a value when an insect is motionless and not digesting food, whereas resting metabolic rate is used when an insect is motionless but there is no information available about its digestion (Withers, 1992). The physiological state of an insect is characterised not only by its respiratory rates, but also by its patterns of gas exchange: Discontinuous Gas Exchange (DGE), Cyclic Gas Exchange (CGE) and continuous respiration.

Many studies have suggested and even demonstrated that DGE is a mechanism to restrict water loss in insects (Ligthon, 1994, 1996; Chown, 2011). However, several other theories exist to explain the origin and function of DGE (Chown et al., 2006; Terblanche et al., 2008). When DGE plays a role of a water conserving mechanism in insects, then it may suggest that in high humidity conditions the CGE is lost and continuous respiration must appear. The knowledge of the role of humidity on gas exchange cycles is poor and controversial data are available. According to all hypotheses about the origin of DGE, this gas exchange pattern may have several functions depending on insect species, its biology and its environmental conditions.

A specific physiological state is diapause. Diapause is considered a dynamic process consisting of several successive phases: induction, preparation, initiation, maintenance, termination and post-diapause (Koštál, 2006). Succession of phases during diapause development, i.e. initiation, is based on the interplay between endogenous (genotype-driven) and exogenous (environmental factors-driven) changes in physiology. In the initial phase, direct development (morphogenesis) ceases, usually followed by regulated metabolic suppression. Mobile diapause stages may continue accept- ing food, building energy reserves and seeking a suitable microhabitat. Physiological preparations for the period of adversity may take place and

11 the intensity of diapause may increase (Koštál, 2006). With arresting of development, the most general feature, a decrease in metabolic rate takes place during the initiation phase (Grodzicki and Walentynowicz, 2011). Physiological processes are strongly influenced especially by changes in the environment. Specific physiological measurements help separate important physiological parameters such as metabolic rate, changes in respiration and gas exchange patterns of respiration in the initial phase.

For herbivorous insects, the availability of different host plants plays an important role in triggering population outbreaks (Singh and Parihar, 1988). The investigation of the effects of food quality on the physiology of insects is essential for understanding host suitability of plant-infesting insect species (Xue et al., 2010). The food plants and food quality may influence insect development and reproduction (Awmack and Leather, 2002). Furthermore, food quality may influence diapause induction in lepidopteran pupae (Morris, 1967) and larval diapause (Hunter and McNeil, 1997). High quality larval food plants provide a better preparation for pupal diapause, which is essential for survival during winter months (Denlinger, 2011). On the other hand, factors which inhibit the induction of pupal diapause or prevent diapause reduce the survival of insects in winter (Metspalu, 1976). Taking this into account, it is important to investigate whether development of insect larvae on less acceptable food plants has potential for decreasing population size in following years. To assess the influence of food plant quality on diapausing pupae the characteristics of standard metabolic rate and gas exchange patterns need to be recorded.

Two carabid beetles were selected as beneficial and two lepidopteran species as pest insects for this work. The two model carabid beetles were Pter- ostichus niger Schaller and Platynus assimilis Paykull due to their predatory polyphagous nutrition as natural pest control agents. Pieris brassicae Lin- naeus, representing an oligophage, overwinters as a pupa over snow cover. The pupae in their initiation phase have more frequent periodic release of CO2 than those in deep diapause and therefore are favourable subjects for the study of cyclic gas exchange. The fourth species was Mamestra brassicae Linnaeus, a polyphagous insect, which hibernates underground as a pupa. Both lepidopteran species are widespread and serious agricultural pests.

12 2. REVIEW OF THE LITERATURE

Insects are influenced throughout their lives by several normal and anthropogenic factors. It is very important to study the effects not only of temperature but also of humidity on the physiology on insects. Among anthropogenic factors it is important to investigate the effects of sublethal doses of pesticides on physiological state.

2.1. Respiration measured by CO2 release

Discontinuous gas exchange as typical periodic breathing is one of the most striking aspects of external gas exchange in insects. Overviews of periodic emissions of CO2 in insects have been reported in the literature for many years, starting from Schneiderman (1956) to Gibbs and Johnson (2004) and Chown (2011). External gas exchange in insects has important consequences for the measurement of insect metabolic rates; it also helps understanding of the functioning of a respiratory system.

Discontinuous gas exchange has been identified using the gas exchange flow-through infrared CO2 analyser (Lighton, 1994; Duncan et al., 2002; Chown and Nicolson, 2004; Chown et al., 2006; Lighton, 2008). The classical DGE in moth pupae consists of three periods or phases, originally termed CFO cycles according to spiracular activity: closed (C- phase), flutter (F-phase), and opening (O-phase) (Schneiderman, 1960).

The term, CFO used by CO2 burst occurred without active or muscular ventilation is not supported by active ventilation or pumping movements of the abdomen (Miller, 1981; Hadley, 1994; Kestler, 1985, 2003). Dur- ing the O-phase, CO2 is released in a burst. The terms were deduced from the spiracular activity observed during CO2 release.

Besides DGE, cyclic CGE has also been described in insects. This is a gas exchange pattern showing regular bursts of CO2, but instead of discrete phases between the bursts there is an interburst period, during which a small level of CO2 is released. Characteristically the C-phase is absent and thus the recording trace never descends to the zero line (Gibbs and Johnson, 2004). According to Marais et al. (2005), CGE is likely to be the ancestral pattern of respiratory gas exchange in insects at rest. The periods between bursts of CO2 release are termed the interburst periods.

13 In the CGE pattern mostly the C-phase and F-phase cannot be separated (see Duncan and Byrne, 2002; Lighton and Joos, 2002). Simultaneous recording of tracheal pressure and CO2 emission may be necessary to detect the different phases exactly (Levy and Schneiderman, 1966; Kest- ler, 1971, 1985; Wobschall and Hetz, 2004). The exact length of the

F-phase by CGE cannot be measured either by CO2 respirometry or by flow-through O2 respirometry, if no significant diffusive component exists. The inward bulk flow of air into the tracheal system is functionally equivalent to a minute and probably undetectable reduction in the flow rate through the respirometer chamber (Lighton, 1988, 1994). Thus, single microopenings of the spiracles and air (oxygen) convective uptake into the tracheae cannot be detected by flow-through respirometry.

On the other hand, the most commonly measured physiological variables, showing physiological state in insects, are standard metabolic rate or SMR. The terms were deduced from the spiracular activity observed during CO2 release. The SMR is defined as a value measured at a particular temperature, when the insect is quiet (at rest) and inactive and is not digesting a meal, exposed to any stress (Withers, 1992), or the energetic cost of living under specific environmental conditions (Chown and Gaston, 1999). Often the term resting metabolic rate or RMR is used, assuming that RMR is a synonym for SMR. However, the term RMR was originally used, when the inactivity was estimated by visual observation and not with special apparatus, such as the infrared optical actograph or other types of activity detectors (see Withers, 1992).

The metabolic rate may depend on the season (Tauber et al., 1986). Many insect species survive the winter by entering diapause which is a dynamic physiological process (Koštál, 2006) including changes in energy metabolism. For example, insect metabolism is greatly lowered at diapause compared to that of a non-diapausing stage (Hahn and Denlin- ger, 2007; Koštál et al., 2008).

2.2. The pattern of2 O uptake

The O2 uptake during the O-phase together with the release of CO2 during the DGE cycle is a common phenomenon in the adult and pupal stages of many insects. A discontinuous O2 consumption during the O-phase has been demonstrated in some carabid beetles and in the

14 pupae of Hyalophora cecropia Linnaeus, using heat conductivity detectors or diaferometers (Punt et al., 1957). Simultaneous measurements of

O2 uptake and CO2 release by flow-through respirometry in Psammodes striatus Fabricius, showed peak of O2 consumption at the beginning of the O-phase, together with a burst of CO2 release (Lighton, 1988). Oxy- gen uptake also peaked during the release of CO2 in the Macropanesthia rhinoceros Saussure (Woodman et al., 2007).

The F-phase in DGE cannot be detected by flow-through O2 respirometry without a significant diffusive component, because the inward bulk flow of air into the tracheal system is functionally equivalent to a minute and probably undetectable reduction in the flow rate of air through the respirometer chamber (Lighton, 1988, 1994). Thus, single microopenings of the spiracles and air convective uptakes into the tracheae cannot be detected by flow-through respirometry. Special techniques are required to record bulk air inflow into the spiracles during the F-phase or, in other words, to record the Passive Suction Ventilation or PSV during the microopening of the spiracles. Schneiderman (1960) used cannulated spiracles to measure partial pressure and thus described the rhythms of passive air uptake in H. cecropia pupae. Sláma (1984, 1988) recorded a saw tooth pattern of abdominal retractions with contact transducers in lepidopteran pupae. This pattern was caused by the microopening of the spiracles and passive inspirations. Thus, instead of the term PSV, Sláma (1984, 1988) used the new and more proper term Passive Suction Inspiration (PSI). A similar pattern of passive inspirations was recorded by Sláma and Neven (2001) in young pupae of Cydia pomonella Linnaeus. Hetz et al. (1994) used miniaturized amperometric sensors to make direct O2 measurements within the tracheal system of lepidopteran pupae. Wobschall and Hetz

(2004) recorded O2 uptake directly in diapausing Attacus atlas Linnaeus pupae by simultaneous measurements of tracheal pressure and volume changes (plethysmometry) in the tracheal system, while combining CO2 measurements by flow-through respirometry. Coulometric (volumetric- manometric) respirometry has been used to directly record O2 convective uptakes in diapausing 2 – 5 month old pupae of M. brassicae (Jõgar et al., 2007), P. brassicae (Jõgar et al., 2004, 2005, 2008) and in the initiation phase of diapause (early diapause) (see Kostál, 2006; Belozerov, 2009).

15 2.3. Muscular ventilation (pumping)

Gas exchange through the spiracles is usually regulated by muscles which control their opening and closing. Muscular or ventilating movements commonly occur in the abdomen of both large and small insects where they take the form of dorsoventral or longitudinal pumping strokes. In many insect species, expiration is muscular while inspiration is dependent on cuticular elasticity. Pumping movements act on the compressible region of the tracheal system, driving air in and out of open spiracles. Muscular ventilation may occur continuously or periodically. For example, the flies Megaselia spp. pump incessantly when they are inactive, or in other words at rest (for reviews about pumping see Mill, 1972; Miller, 1974, 1981).

In coleopteran species, pumping may be a sign of stress lasting for some hours until normal respiratory rhythms are restored. In Leptinotarsa decemlineata Say long periods of muscular ventilation are precursors for normal cyclic release of CO2 (Vanatoa et al., 2006). However the regular pumping is not a symptom of activity, while activity can be triggered by apparatus and handling stress which results in struggling behaviour or the insect exhibiting irregular body movements. It is a very difficult task to restrain the insect for respiratory measurements. When an insect is fixed in the respiratory chamber, the forced immobility periods last a short time and are interrupted by much longer activity periods (Kestler, 1971; Sibul et al., 2004a, 2004b, 2008).

Very little is known about the metabolic cost of the pumping movements. According to Sibul et al. (2008), in Hylobius abietis Linnaeus, O2 consumption rises about twofold during pumping, suggesting a significant metabolic effect. By contrast, in Tenebrio molitor Linnaeus pupae, the energetic cost of ventilatory movements is negligible (Harak et al., 1999).

To determine metabolic rates and respiratory patterns the individual insect must be motionless. Many adult insects are very active at room temperatures, and it is often a very complicated task to force them to stop struggling and to stay motionless in the insect chamber of a respirometer and the activity periods of immobility or resting state are commonly too short for the study of SMR and respiratory patterns.

16 Several methods have been proposed to immobilise the insect. The use of the anaesthetic enflurane is recommended to ensure immobility in ants (Hymenoptera, Formicidae) and other small insects during the determination of SMR (Holm-Jensen et al., 1980). However, the anaesthetic cannot be used to quantify respiratory patterns (Duncan and Newton, 2002). Decapitation has been demonstrated as a useful measure for immobilizing small insects for the measurement of resting gas exchange, while movement is minimal and gas exchange patterns are more regular and stereotyped (Lighton, 1992; Quinlan and Lighton, 1999). However, this method is not used nowadays because, together with the head, the obligatory abdominal contractions are eliminated. The breathing measurements at low temperatures also suppress the activity and gas exchange pattern are clearly recorded, e.g. in Apis mellifera Linnaeus (Lighton and Lovegrove, 1990), in L. decemlineata (Vanatoa et al., 2006); in Bombus terrestris Linnaeus foragers (Karise et al., 2010).

2.4. The origin and functions of discontinuous gas exchange

Several adaptative hypotheses have been proposed to account for the origin of DGE. The Hygric hypothesis is the oldest interpretation of the origin and function of DGE, assuming that it provides a means by which insects restrict respiratory transpiration i.e. respiratory water loss or RWL (Buck et al., 1953; Buck and Keister, 1955; Levy and Schneider- man, 1966); many other authors also support this idea (Kestler, 1985; Lighton, 1994, 1996; Vogt and Appel, 2000; Duncan et al., 2002). For natural selection to take place, several conditions must be met, primarily between-individual variation in gas exchange patterns. This has been documented in many studies; it should be regarded as the outcome of the adaptive processes (Lighton, 1988; Duncan and Dickman, 2001). All phases in DGE may serve to restrict water losses. Besides the C-phase when no gas exchange occurs, the F-phase considerably hinders water evaporation from the spiracles, air then being sucked passively into the trachea serving as an anti current for CO2 and water release from the spiracles (Kestler, 1980, 1982). The opened spiracles during the O-phase have been regarded as the main avenue for water loss together with CO2 emission. But when active ventilation or muscular contractions support the escape of CO2 from the spiracles then the O-phase shortens while the C-phase may be longer. Kestler (2003) proposed that a specific strategy for water retention is the replacement of the diffusive O-phase (CFO

17 cycle) by a convective V-phase (CFV cycle) whenever possible to avoid the higher diffusion of water vapour. The modern gas analyzers allow simultaneous measurements of CO2 emission and water vapour release in insects. Numerous studies have found that respiratory water loss (respiratory transpiration) accounts for a small fraction of total water loss, and that insects stop performing DGE at times when this pattern should be most useful (see Quinlan and Lighton, 1999; Gibbs and Johnson, 2004; Gray and Chown, 2008).

The chthonic hypothesis states that DGE primarily facilitates gas exchange in hypoxic (low O2) and hypercapnic (high CO2) environments, i.e. in underground conditions (Lighton and Berrigan, 1995). An alternative theory is oxygen damage suggesting that DGE may serve to reduce the supply of O2 to the tissues during periods of low metabolic rate. Oxygen is toxic to cells, even ambient concentrations, and the insect may use

DGE to reduce oxidative damage during periods of reduced O2 demand (Bradley, 2000; Hetz and Bradley, 2005).

The emergent property hypothesis is based on the idea that DGE originated as a consequence of interacting feedback systems. According to this theory, DGE is a nonadaptative outcome of interactions between the O2 and CO2 set points that regulate spiracle opening and closing. The nonadaptative hypothesis postulates that the brain relinquished control of gas exchange to the segmental ganglion, an interaction between peripheral

CO2 sensing and central O2 sensing sets in, leading to the discontinuous pattern of gas exchange (see Chown et al., 2006; Förster and Hetz, 2010; Chown, 2011).

Nevertheless, it has been demonstrated that DGE confers a fitness benefit. Schimpf et al. (2011) demonstrated that Nauphoeta cinerea Semoniyk using DGE live longer when food and water are restricted, than those using the continuous respiratory mode. Besides, the hygric hypothesis recently received support from works by Marais et al. (2005) and White et al. (2007). Schimpf et al. (2009) demonstrated that the longer exposure to low levels of relative humidity (RH) results in a reduction in duration of the O-phase whereas the duration of the CF-phase was unaf- fected. This change in the duration of the O-phase is inconsistent with the explicit prediction that stems from the hygric hypothesis. The duration of the O-phase was longest following acclimation for 5 – 6 days to high levels of humidity. However, switching humidity from high to low

18 or from low to high had no effect on the gas exchange pattern, and, at high humidity, diapausing pupae of Samia cynthia Drury did not lose their DGE pattern (Terblanche et al., 2008). This did not support the hygric hypothesis. It may be concluded that particular humidity conditions must last for longer to modify or change the gas exchange pattern (see Schimpf et al., 2009).

The conductance constraint hypothesis proposed by Schilman et al. (2008) states that some insect species, especially tenebrionid beetles (Coleoptera, Tenebrionidae), cannot exchange respiratory gases discontinuously because of a morphological constraint (small tracheal or spiracular conductance). This theory may help to explain the otherwise puzzling phylogenetic patterns of continuous versus DGE observed in tracheate arthropods (see Schilman et al., 2008). Other morphological aspects must also be taken into account. Diapausing lepidopteran pupae with extremely low metabolic rates, such as P. brassicae, must have special mechanisms to restrict water loss to survive the long winter period. Cuticular water loss is assumed to be small compared with respiratory water losses, because of the hard (sclerotized) cuticule. The flow-through

CO2/H2O analyzer is useless in this case (Sláma, 2008, 2010). The pupa of P. brassicae release CO2 only once per day, and CO2 and water release per hour is too small to be recorded by the analyzer (Sláma, 2008, 2010). Other methods must be used. One of the best is the continuous recording of body mass. Such a gravimetric method was used by Machin et al. (1991) to quantify water loss during different phases of DGE in the cockroach Periplaneta americana Linnaeus. Machin et al. (1991) demonstrated that water loss rate during the O-phase (burst of CO2 release) was substantially greater than during the CF-phase.

The emergent property hypothesis, which states that DGEs are a nonadaptative outcome of interactions between the CO2 and O2 set points that regulate spiracle opening and closing (Chown, et al., 2006; Chown,

2011), is a nonadaptative hypothesis, with both CO2 and O2 involved in regulating spiracle behaviour. Partial pressure of the CO2 causes opening of the spiracle by the spiracle muscle. The O2 partial pressure threshold is sensed in the segmental ganglia of the central nervous system (Förster and Hetz, 2010; Chown, 2011). Separate CO2 and O2 systems as a nonadaptative explanation for DGE behaviour does not contradict several adaptative explanations (Förster and Hetz, 2010; Chown, 2011).

19 2.5. Respiratory failures as indicators of chemical stress

Many insects react sensitively to anthropogenic changes to habitat quality and are affected by intensive agricultural cultivation. They can be influenced by tillage as well as by crop treatment with pesticides (Kromp, 1990). In conventional farms, pesticides present risks to non-target carabid beetles living in both treated and untreated areas, because carabid beetles move relatively fast. They may contact pesticides directly, by contact with soil (Acikkol et al., 2012) or feed on chemically-treated seeds and pests (Kromp, 1999). Basedow (1987) considers pesticide application to be the main factor in the reduced carabid beetle numbers in conventionally-farmed wheat fields. Pesticide use on conventional farms may also cause ecological damage to neighbouring organic farms by killing carabid beetles.

Insect respiration is an established index of the stress imposed by insecticide (Kestler, 1991). To date, the sublethal and delayed effects of toxicants on insect respiration have been poorly studied. Kestler (1991) demonstrated that topical application of chlorpyriphos resulted in elimination of the normal DGE pattern in the resting cockroach, P. americana, conclud- ing that the physiological cause of death may well be respiratory failure. Appel et al. (1997) demonstrated that exposure to contact insecticides eliminates the DGE pattern in Diploptera punsctata Blattaria, and Sole- nopsis invicta Buren, making the insects more susceptible to desiccation and possibly easier to control. The L. decemlineata lost its normal DGE pattern after treatment with Neem-Azal T/S (a.i. azadirachtin) (Kuusik et al., 2001), and elimination of DGE was discovered in H. abietis (Sibul et al., 2004b). Treatment of young P. brassicae pupae with sublethal doses of the extract of Tanacetum roseum Adams resulted in loss of the typical DGE pattern and failure of adults to emerge (Harak et al., 1999). Similar DGE loss was described in P. brassicae pupae after treatment with Neem EC (Jõgar et al., 2008). The direct neurotoxicity of sublethal doses of insecticides was also tested on insects lacking the CGE, such as in adult T. molitor, which is characterized by regular respiratory contractions (Zafeiridou and Theophilidis, 2006).

Pyrethroids are currently the most commonly used insecticides in the world (Horton, 2011), and alpha-cypermethrin the most widely used active ingredient in synthetic pyrethroid insecticides used to control insects on fruit, vegetable crops and also to kill fleas, cockroaches and

20 other insects in houses, stores, warehouses etc. (Cox, 1996). These pesticides are highly toxic to insects and aquatic organisms (Mueller-Beil- schmitdh, 1990; Solomon et al., 2001; Karise et al., 2007) but have relatively low toxicity to terrestrial vertebrates (Solomon et al., 2001; Yarkov et al., 2003). The mechanisms of pyrethroid toxicity in pests and non-target organisms are similar to the molecular targets present in insects being analogous to those in mammals (Marrs and Ballantyne, 2004). Pyrethroids are primarily functional toxins targeting the nervous system (Narahashi et al., 1998). Thus, they owe their insecticidal potency to a rapid functional disruption of the insect neuromuscular system and the secondary consequences of this, rather than to any direct cytotoxicity (Ray and Fry, 2006). Pyrethroids may bioconcentrate through the food web (Solomon et al., 2001).

Treatments with chemical pesticides never kill all the insects within a population and may evoke poorly studied sublethal and delayed effects in the survivors. Several methods have been used to assess the effects of insecticides on the physiology of beetles. Sláma and Miller (1987) used a hydraulic transducer to record the neurotoxic effects of a pyrethroid on pupae of T. molitor. Zafeiridou and Theophilidis (2006) used a force displacement transducer attached to the second abdominal segment of the dorsal surface of the abdomen T. molitor and respiratory contractions were monitored before and after sublethal poisoning with pyrethroid. The normal respiratory contractions are de-regulated by toxicant.

Determination of the normal physiological state of an insect is highly important before studying pathological effects. The physiological state of an insect has most often been estimated by SMR. There are data recording the effects of the toxicants on the SMR (Keister and Buck, 1974). Gas exchange patterns are also used to characterize the physiological state of insects.

2.6. Eﬀects of food plants on the development and diapause in insects

For polyphagous insects, the availability of different host plants plays an important role in triggering population outbreaks (Singh and Pari- har, 1988) and study of the effects of food quality on the biology of insects is important for understanding host suitability of plant infesting

21 insect species (Xue et al., 2010). Food plants affect insect development and reproduction and food quality is a key determinant of the fecundity of herbivorous insects (Awmack and Leather, 2002). Furthermore, food quality may interact in responses to influence diapause induction, as demonstrated in pupal diapause of Hyphantria cunea Drury (Morris, 1967), Helicoverpa armigera Hübner (Liu et al., 2009, 2010) and larval diapause in Choristoneura rosaceana Harris (Hunter and McNeil, 1997).

Some information is available on induction and development of this diapause, showing the major role of day length and temperature (Goto and Hukusima, 1995; Hodek, 1996) but, in spite of its economic importance, little information exists on the nutritional indices of this pest on different food plants. However, the effects of larval food plants on pupal DGE phases, haemolymph circulation and water loss of diapausing pupae have been dealt with in earlier publications (Metspalu et al., 2003; Jõgar et al., 2004, 2005; Jõgar, 2006). Similar studies, performed in recent years by Liu et al. (2007, 2009, 2010) on H. armigera showed, that high quality larval food plants provided a better preparation for diapause, which appeared to be a prerequisite for successful overwintering and increased survival. Denlinger (2011) found that diapause is such an important aspect of the life cycle that disruption of its timing, e.g. by making the insects go into diapause at the wrong time or by breaking them out too early when no food is available, has potential as an effective tool for pest control. Thus, factors inhibiting the induction of diapause or preventing diapause from becoming more intense will, at the same time, reduce the survival of insects in winter (Metspalu, 1976).

22 3. AIMS OF THE STUDY

Insects are influenced throughout life by several natural and anthropogenic factors. The effects of these factors are well reflected in their physiological state, and commonly characterised by changes in respiratory patterns and metabolic levels. The effects of temperature are relatively well studied, but few studies have been made on the effects of humidity on physiology. There have been many studies on the lethal doses of insecticides on pests, but few have investigated the effects of sublethal doses on contemporary insecticides. The effects of insecticides on beneficial insects, for example, on carabid beetles, have been little studied. To investigate the details of the physiological state of insects, novel systems and methods were needed. The role of larval host plants on pupae during the overwintering period is still unclear. Such knowledge is essential to understanding the overwintering biology of insects.

1. To elaborate a sensitive respiratory method which allows recording of oxygen uptakes (passive suction inspiration) and associated body movements and is able to monitor the smallest changes caused by natural and anthropogenic factors in the physiological state of insects (I).

2. To compare metabolic rate and gas exchange patterns in dry and in moist environmental conditions (II).

3. To measure the standard metabolic rate (SMR), resting metabolic rate (RMR), water loss rate (WLR), to characterise gas exchange patterns and to investigate the effects of sublethal doses of Fastac 50 EC on RMR, on patterns gas exchange and on water loss (III, IV).

4. To assess the influence of food plant quality on the physiological state of larvae and diapausing pupae (V).

23 4. MATERIALS AND METHODS

4.1. Insects

Experiments were carried out with two carabid beetle and two lepidopteran species. The carabid beetles are key indicators to assess human- altered abiotic conditions, such as pesticide use in agro-ecosystems (Koivula, 2011). The model carabid beetles P. niger and P. assimilis were selected. The P. brassicae larvae are one of the greatest oligophagous pests on cruciferous plants; they can destroy a significant part of the crop. In Estonia, P. brassicae has two full generations and in some years a partial third generation. The diapausing pupae of P. brassicae overwinter in covered locations high above the snow surface. The M. brassicae larvae are serious polyphagous pests of a wide range of plant species throughout the world. In Estonia has one full generation and partially two generation during summer. They overwinter as diapausing pupa in the soil.

4.2. Treatments

4.2.1. Pieris brassicae (I)

Eggs of P. brassicae (second generation) were collected from cabbage fields during July and August 2009. They were reared in a laboratory under short-day conditions (12h:12h light:dark) at 21 °C and ambient air humidity (55 – 65% relative humidity). On hatching, the larvae were fed on white cabbage (Brassica oleracea Linnaeus, variety capitata) leaves. After pupation, each pupa was placed in an Eppendorf tube and kept in laboratory conditions. For the experiments, at least two week old (14 ± 2 days) diapause pupae in the initation phase were used. Young pupae are considered favourable objects for study of the dynamics of cyclic gas exchange as they have relatively short gas exchange cycles lasting only a few hours.

4.2.2. Pterostichus niger (II)

Adult carabid beetles P. niger were collected in December 2008 and Janu- ary 2009. They were kept in 5-L bins half-ﬁlled with soil, maintained at room temperature (20 – 23 °C) and at over 80% relative humidity (RH),

24 and supplied with water and cat food (Friskies Junior 1; Purina, Nestlé S.A., Switzerland) ad libidum. The test beetles were tested within two weeks of collection. Before the experiment each beetle was starved 24 h. During this starvation period, they were held in moist conditions (over 80% RH) and given access to water, because they are very susceptible to dry conditions and cannot be dehydrated for longer than 15 – 20 h (our unpublished data).

Test insects (30 individuals) were divided into two groups by random selection. In one group (n = 15), environmental conditions were changed from dry (5 – 7% RH) to moist (90 – 97% RH) air, and, in the other group (n = 15), from moist to dry air. Each test beetle was placed in the respiratory chamber and left undisturbed for 1 h before the start of the measurement period, in the humidity conditions that would be used in the test. Tests with each individual were made on the same day. Each recording lasted at least 3 h in the dry and 3 h in the moist conditions. Of the 30 beetles, 16 showed continuous respiration and were excluded from further analysis.

4.2.3. Platynus assimilis (III-IV)

Treatment by feeding method (III) Test material collected from hibernation sites (52 individuals in total), was separated into two groups: treatment (24 individuals: 13 female and 11 male beetles) and control (28 individuals: 13 female and 15 male beetles). Fastac 50 EC was diluted in distilled water according to dose recommendations for the field. The beetles were fed with cat food (Frisk- ies Junior 1; Purina, Nestlé S.A., Switzerland) every fourth day. Feeding laboratory-reared ground beetles with cat food is suggested also by Tréfás et al. (2001) and Ploomi (2004). For exposure to insecticide, food pieces were dipped in the emulsion of alpha-cypermethrin (treated) or in distilled water (control group) for 10 seconds. The experiment lasted 14 days, while individual beetles were kept in plastic boxes (0.5 L) on wet tissue at room temperature (22 ± 1 °C).

The respiration rate of P. assimilis was measured on the first day before treatment and two days after the last feeding (15th day after the onset of the experiment). CO2 releases and abdominal movements were measured on P. assimilis before and after treatment.

25 Treatment by dipping method (IV) Adult P. assimilis beetles were collected in January 2010 from their hibernation sites (tree stumps) in Tartu County, Estonia. They were kept for a day in the laboratory in plastic boxes filled with moistened moss without food before the experiment. For the experiments, 43 beetles (40 – 42 mg) were selected and placed individually in Petri dishes. Experiments were performed at 22 ± 1 °C in a thermostat. Respiratory measurements in a beetle lasted at least 3 hours before and after treatments. During these, temperature and humidity conditions were recorded by means of Tem- perature and Humidity Display Instruments for digital HygroClip probes (HygroPalm, Rotronic Company). Water loss estimated in 17 beetles showing DGEs by the weighing method (OHAUS Digital Explorer Balances, Pine Brook, New Jersey) per day.

The pyrethroid Fastac 50 EC with a commercial formulation of alpha- cypermethrin (a.i. 50g L–1) was used in the experiments on beetles showing only DGE. One ml of formulation (a 5% emulsion) was diluted in 100 mL distilled water (0.05%, field solution of formulation) and different concentrations (0.01%, 0.001% and 0.005%) were prepared. Preliminary tests showed that concentrations of 0.05% and 0.01% had lethal toxicity for carabid beetles, so these concentrations were not used in the study. Thus, the treatments were carried out with two concentrations: 0.001% and 0.005%. Also, our preliminary studies revealed that topical application using acetone as solvent administrated on the thorax or abdomen evoked strong toxic and metabolic delayed effects. For this reason, we used another contact method; the dipping of beetles into an aqueous emulsion of the pyrethoid for 10 s. Dipping is used as an alternative contact method for bioassays with several insect species (Van der Stern, 2001; Cetin et al., 2006; Wanyika et al., 2009). The beetles for control were dipped in distilled water without pesticide.

4.2.4. Mamestra brassicae (V)

For laboratory experiments, egg clutches of M. brassicae were collected from an experimental field of the Estonian University of Life Sciences in 2009. All clutches containing at least 100 eggs collected within one week. After hatching M. brassicae larvae from the same egg clutch were divided onto each replicate of the food plant species (five replications, each 20 larvae) in Petri dishes (15 cm diameter and 2 cm deep), one replication in each dish. Larvae were fed on one of the following food plants: Brassica

26 oleracea Linnaeus, variety capitata Linnaeus, variety ‘Krautman’, Pisum sativum Linnaeus, variety ‘Aamisepp’, Beta vulgaris Linnaeus, variety ‘Bordoo’, Allium cepa Linnaeus, variety ‘Peipsiäärne’ and Brassica napus Linnaeus, variety napobrassica Linnaeus Rchb, variety ‘Kõpu’. The food plants were selected according to their importance as cash crops as well as their known associations with M. brassicae. On reaching the 3rd instar stage, the larvae were placed in groups of five in 1 L breeding vessels covered with net. The food plant was replaced daily. Larvae were reared in environmental test chambers “Sanyo” with a short-day light cycle (LD 12:12 h), at 21 °C and 75% RH. Before pupation, a 10 cm deep layer of peat was placed on the bottom of the vessels. To ensure that the pupae reached a stable diapause state, they were kept in the peat for one month, before collection by hand-sorting. The duration of DGE cycles was determined on 3 month-old pupae (five male and five female) from each treatment. The calorimeter was sufficiently sensitive to record carbon dioxide release by bursts and abrupt air intakes into the tracheae of the pupae. Respirometry was conducted on 3 month-old pupae in deep diapause.

4.3. Measuring methods

4.3.1. Coulometric respirometry (I, III, V)

The coulometric respirometer (a volumetric-manometric system) is based by a continuously O2-compensating system (Kuusik, 1977; Kuusik et al., 1996; Vanatoa et al., 2006) (Fig. 1). The main measurement principles of coulometric respirometry systems are also described by Lighton (2008).

These respirometers ascertain continuous and respective replacement of consumed O2 with electrolytically created O2. The functional part in this self-regulating system is the insect itself. The rates of O2 production and O2 –1 consumption by the insect are represented on graphs as VO2 (mL h ). The system also records temporary changes in the rate of release of CO2. The coulometric respirometer allowed simultaneous recording of O2 consumption and abdominal pumping movements (Figs. 2, 3). In the respirometer, the electrolysis current was directly connected with a photoelement. High sensitivity of the respirometer to pressure changes in the respiration chamber has a very small photosensitive area (approximately 0.5 mm2). It enables detection of the smallest movement in the meniscus of ethanol inside the U-shaped capillary, reflected as a signal on the recording trace.

27 Figure 1. Continuously recording coulometric respirometer design. 1, plexiglass block; 2, removable cover; 3, insect chamber; 4, current amplifier; 5, light source; 6, phototransistor; 7, compensating vessel.

��

� ��

��

� � ��

Figure 2. An example of gas exchange cycles recorded by coulometric respirometer in

adult Platynus assimilis. Each burst of CO2 release is actively ventilated by pumping movements of the abdomen. Upper trace is a detail with high resolution of pumping

movements during CO2 burst. 28 0.80,8 77

0.70,7 66

0.60,6 55

) 0.50,5 44 -1

1 min . . 0.40,4 33 (ml h (ml Volts 2

VO 0.30,3 22

0.20,2 11

0.10,1 00

0,00 -1-1 60010 90015 12001 10 1500115 1800120 2100125 24001 30 2700135 . Time (min)

Figure 3. A typical pattern of continuous respiration associated with unbroken pumping movements recorded with coulometric respirometer in adult Platynus assimilis; an activity period is noted by the horizontal bar. Middle trace, the horizontal bar (1 min) is a detail of pumping movements. The upper trace is a simultaneous recording by infrared opto-cardiograph showing heartbeats between pumpings.

4.3.2. Flow-through CO2 respirometry (I-V)

An infrared gas analyser (IRGA) was used to record the CO2 signals. –1 Metabolic rates (VCO2 mL h ) and gas exchange patterns were measured in a flow-through system by a differential gas analyser (I-V). A flow rate of 200 ml min–1 was used (IV). Air flow rate was commonly 60 mL min–1, by which the rate of carbon dioxide release was measured –1 (VCO2 mL h ) (III). The CO2 channel was calibrated with commercially available span gas (Eesti AGA AS, Estonia; Linde AG, Höllriegelskreuth, Germany). The flow-through respirometry was combined with infrared (IR) opto-cardiographic measurements for entomological research. The flow- through respirometer was calibrated at different flow rates by means of calibration gases. (I-V)

29 11 11

5 2 5 + 12 V

12 10 13 5 1 5

Pt 9

7 8

Cu 3 4

6 14

Figure 4. Design of the electrolytic continuously O2-compensating differential volumetric-manometric closed-system respirometer. 1, Insect chamber with pupa; 2, empty insect chamber; 3, vessel with potassium hydroxide solution; 4, electrolysis unit, CuSO4 solution with platinum (Pt), and copper (Cu) electrodes; 5, taps for switching from volumetric-manometric respirometry to flow-through CO2 respirometry; 6, glass capillary half-filled with ethanol; 7, light source; 8, photo transistor; 9, infrared (IR) emitter diode; 10, IR sensor diode; 11, connections to the flow-through respirometry system; 12, compensating vessel; 13, current amplifier; 14, microsyringe.

In our system (Fig. 4), the air flow-through respirometry system was switched with tapes on the coulometric respirometry system. This ena- bled the two respirometry systems to be used alternately.

4.3.3. Infrared probe actography (I-IV)

The IR-actograph recorded heartbeats and also all other abdominal contractions, including muscular ventilation. An IR-emitting diode was placed on the side of the insect chamber near the ventral side of the insect abdomen, while the IR-sensitive diode (BP104) was placed on the opposite side of the chamber. The light from the IR-diode was modulated by abdominal contractions. The level of the output voltage reflected the vigour of the muscular contractions of the insect (Hetz et al., 1999;

30 Karise et al., 2010). The coulometric (electrolytic) respirometer and the flow-through respirometer were combined with an IR insect cardiograph (opto-cardiography). The level of output voltage reflected the vigour of the muscular contractions of the insect.

For simultaneous measurements of CO2 emission and water loss rates

(WRL) we used an infrared CO2 and H2O analyzer (LI-7000, Li-Cor, Lincoln, NE, USA) (IV). We compared total water loss as determined from the analyzer recordings with gravimetrically (Quinlan and Lighton, 1999) estimated values; no differences were found between these calibration methods.

4.3.4. Calorimetry (V)

Calorimetry is the method for continuous recording of DGE for weeks in individuals without evoking stress by handling and adjusting the apparatus. A simple twin differential calorimeter was constructed of vessels made from copper foil (0.1 mm) connected with copper-constantan thermocouples, while a micro-nano-voltmeter and recorder were used (Kuusik et al., 1994; Harak et al., 1999; Jõgar et al., 2005).

The volume of both the insects and reference vessels was 0.5 mL and the sensitivity of the calorimeter was 50 µV m W–1 with a detection limit of 4 µW. The calorimeter was calibrated electrically by the Joule effect (Hem- minger and Höhne, 1984). The duration of DGE cycles was determined in pupae (see Fig. 5).

Figure 5. An example of calorimetric recording of heat production in a diapausing long- cycle pupa. The peaks are caused by the bursts of carbon dioxide release. The exothermic (upward) spikes recorded at the beginning of bursts transformed into endothermic signals due to the cooling effect of transpiratory water vapour (Jõgar et al., 2005).

31 4.4. Data acquisition and statistical analysis

Computerized data acquisition and analysis were performed using an analog-to-digital converter and TESTPOINT software with a sampling rate of 10 Hz (DAS 1401; Keithley- Metrabyte, Keithley Instruments Inc., Cleveland, Ohio). The differences of means of metabolic rate were calculated automatically using a statistical program.

In the statistical analyses the software packages STATISTICA versions 5.6 (II), 8 (I, III, V), 9.1 (V) was used. Statistical comparisons were made with one-way ANOVA (analysis of variance) (I, II, III, V); two-way ANOVA (V), Wilcoxon Matched Pairs Test (II, III), Chi-squared (χ2) test (II); Student t-test (III, IV). Significant differences were followed with the Fisher’s LSD (least significant difference) test (I, II, IV, V). The significance level was set at P ≤ 0.05.

32 5. RESULTS

5.1. Oxygen convective uptakes in gas exchange cycles in early diapause pupae of Pieris brassicae (I)

At the initiation phase of diapause (14 ± 2 days old), P. brassicae pupae displayed DGEs lasting 40 – 70 min, whereas the duration of CO2 release by burst was 2 – 6 min (3.1 ± 0.1). Recordings by flow-through respirometry showed a typical pattern. After the O-phase CO2 emission had ceased, the

C-phase began, followed by the F-phase with small bursts of CO2 release. No true C-phase was found by flow-through respirometry. Shortly after the end of the O-phase, coulometry revealed convective O2 uptake. Dur- ing this time, CO2 release was suppressed by the inward directed PSV.

This convective O2 uptake indicates an earlier beginning of the F-phase than detectable with the flow-through system (Fig. 6). Thus the F-phase lengthened on account of the C-phase. At each microopening of the spiracles and passive convective oxygen uptake event, signals of abdominal lengthening were simultaneously recorded by coulometric respirometry and infrared actograph. Recordings of coulometric respirometry showed clear, gradually shortened signals due to convective oxygen uptake.

* 0.12 * * 1 * ) 0.10 * * -1 * 0.95 *

0.08 0.9 .

0.06 0.85 Volts

consumption (ml h (ml consumption 0.04 0.8 2 O

0.02 0.75

0 0.7 0 30 60 90 120 150 180 . Time (sec)

Figure 6. Typical pattern of convective O2 uptake by coulometric respirometry recorded in a Pieris brassicae pupa at the time that corresponded to the closed phase by the flow- through CO2 respirometer. The spikes are gradually shortened due to the pressure rises inside the tracheae. Asterisks (upper trace) indicate the extensions (lengthening) of abdominal segments. Arrow indicates a CO2 release by miniburst.

33 Each of the two to three first micro-openings and O2 uptakes lasted less than 0.5 s. Oxygen convective uptakes during the interburst period increased metabolic rate by 5 – 6% compared with the metabolic rate when these uptakes were absent. Flow-through measurements made synchronously with infrared actography also indicated abdominal lengthening concurrent with the small bursts of CO2 during flutter (Fig. 6).

Between two large CO2 bursts, a series of small bursts of CO2 were recorded by coulometric respirometry. Each small burst started with a brief uptake of air into the tracheae, recorded by the IR actograph as a sudden extension of the abdomen, indirectly indicating air (O2) uptake.

Between two consecutive small CO2 bursts, a series of air uptakes (miniature inspirations) were recorded, which we considered as ‘miniflutter’. These uptakes were irregular with respect to spike height and interval.

During such miniflutters, no emissions of CO2 were recorded.

Simultaneous recording with the IR actograph during flow-through CO2 respirometry indicated that pupae differed in the type of body movement associated with the respiratory patterns of CO2 release. In some pupae,

CO2 bursts were always associated with abdominal ventilating movements (CFV cycles) (n = 9), whereas in others (n = 10), only some CO2 bursts were concurrent with abdominal ventilating movements. In a few pupae (n = 6), CO2 bursts occurred without active ventilation (CFO cycles). Ventilating movements (amplitude 1 – 2 V) associated with CO2 bursts were visible externally as twisting abdominal movements.

Active ventilation during the bursts of CO2 showed individual variation in the vigour of contractions and their number (from one to 15). In pupae with only one to five muscular (active) ventilating movements accompanying the burst, as well as in those lacking active ventilation, a relatively low level of CO2 release was observed.

In contrast, pupae with vigorously ventilated bursts showed a significantly higher level of CO2 release. Each burst lasted 3 – 6 min in the ‘always’ group of pupae, but 2 – 2.5 min in the ‘occasionally’ and ‘never’ groups.

Statistical comparison of CO2 release frequency (F2,24 = 41.8; P > 0.05) did not show a significant difference. The energy cost of muscular ventilation during a burst was not studied.

Abdominal two-phase regular contractions (5 min–1) of low amplitude

34 (0.2 – 0.3 V) (referred to as abdominal pulsations) occurred periodically; these were not visible externally. In some pupae showing no active ventilation during CO2 release by bursts, very regular low amplitude (0.1 – 0.2 V) pulsations (57 – 70 min–1) were recorded; these we interpreted as heartbeats.

5.2. Eﬀects of humidity conditions on the gas exchange patterns on Pterosticus niger (II)

In the current experiment, ten beetles (four moist to dry, six dry to moist) displayed cyclic gas exchange and four beetles (three moist to dry, one dry to moist) showed discontinuous gas exchange.

5.2.1. Discontinuous gas exchange

A minority (n = 4) of the P. niger that were tested showed a clear pattern of discontinuous gas exchange in dry air, as well as in moist air. The pattern of gas exchange in these four individuals was characterized by a closed (C) phase, when CO2 emission went to zero or close to zero, and a flutter phase (F). The open (O) phase of the discontinuous gas exchange was associated with vigorous and regular pumping movements of the abdomen i.e. with active ventilation. This pattern of discontinuous gas exchange was recognized as constriction–flutter–ventilation cycles. The duration of a ventilation period contributed approximately 60 – 80% of the total length of a cycle. The peak of the CO2 burst lasted only 1 – 2 min, after which the level of CO2 emission remained high for a longer −1 time. The maximum VCO2 (mean ± SD) was 0.58 ± 0.039 mL h and 0.48 ± 0.028 mL h−1 in moist and dry conditions, respectively (t = 6.82, d.f. = 3, P < 0.05; Student’s t-test). These results revealed that DGE pattern was not lost in high humidity conditions.

5.2.2. Cyclic gas exchange

A cyclic gas exchange pattern was studied in ten of the 14 beetles at rest.

The bursts of CO2 release during cyclic gas exchange were associated with weak abdominal movements. Very weak movements of the abdominal tergites were designated as abdominal pulsations. These very regular movements were recorded by the infrared probe actograph, although they were barely visible by eye, even under a stereomicroscope. A characteristic

35 feature of this cyclic gas exchange was a ‘long burst’ period, which started with a short peak of CO2 release, lasting 3 – 5 min (4.2 ± 0.5 min). After the CO2 emission peak, a very even level of CO2 emission was recorded. The long burst period was followed by a short interburst period, during which the pulsations stopped for a short time (3 – 5 min), until the next burst of CO2 release. When the pulsations stopped, CO2 release decreased abruptly but never reached baseline. After switching from dry to moist air flow in the insect chamber, the general pattern of cyclic gas exchange did not alter, although short periods of activity appeared (1 – 2 per h), in which abdominal pulsations were coincident with the CO2 bursts. The standard metabolic rate was higher in the beetles displaying cyclic gas exchange than in individuals showing discontinuous gas exchange (F1,13 =

82.28 in dry air; F1,13 = 58.6 in moist air, P < 0.001; Fisher’s least significant difference test P < 0.0001 (Tab. 1). However, statistical comparison using merely a frequency test of ratios (χ2 test) showed no significant difference between tested groups (χ2 = 1.40, P = 0.2367, d.f. = 1).

Vertical comparison was performed by one-way analysis of variance (different superscript letters indicate statistically significant differences, P < 0.05) and horizontal comparison by Wilcoxon matched pairs test. SMR, standard metabolic rate.

Table 1. Summary statistics and results from paired tests of gas exchange in Pterosti- cus niger in dry and moist air (mean ± SD) and metabolic rate between different gas exchange patterns: discontinuous gas exchange (DGE) and cyclic gas exchange (CGE) n Dry air Moist air t P

DGE VCO2 4 0.07 ± 0.0056b 0.07 ± 0.0063b 0.0344 0.970 (mL h−1) (SMR) DGE burst (h−1) 4.00 ± 0.17 4.00 ± 0.15 −0.0229 0.983

CGE VCO2 10 0.11 ± 0.012a 0.11 ± 0.017a −0.3455 0.752 (mL h−1) (SMR) CGE burst (h−1) 7.50 ± 0.64 7.50 ± 0.72 0.1031 0.920

36 5.3. The gas exchange patterns and the eﬀects of sublethal doses of pyrethroid on gas exchange in Playunus assimilis (III, IV)

Our results with 43 beetles revealed that 23 of them used only the DGE mode of respiration, 13 displayed CGE and seven used continuous respiration. The beetles that used CGE showed a short O-phase contributing 40 – 50% of the whole cycle. The O-phase was followed by a short C-phase whilst the F-phase was absent or at least not separated from the C-phase. The DGE was characterized by a relatively long O-phase (burst of CO2) contributing 80 – 90% of the whole cycle while C- and F-phases were not separated from each other

In both patterns of DGE and CGE the burst of CO2 release was always accompanied by active (muscular) ventilation (V) or pumping. The mean durations of the DGE and CGE were 444.8 ± 8.1 s and 491.9 ± 5.7 s, respectively (F1,34 = 0.17; P ≤ 0.05). No differences were found in resting –1 –1 metabolic rates between DGE and CGE: 0.94 ± 0.01 (VCO2 mL h g ) –1 –1 and 0.93 ± 0.01 (VCO2 mL h g ), respectively (F1,34 = 0.17, P = 0.68).

Continuous respiration of P. assimilis was characterised by continuous pumping movements recorded by coulometric respirometry. Between the pumping movements frequent pulsations were recorded which we interpreted as heart beats. Individuals using continuous respiration had higher –1 –1 resting metabolic rates (1.03 ± 0.02 VCO2 mL h g ) than those using –1 –1 DGE (0.94 ± 0.01 (VCO2 mL h g ) (F1,28 = 11.46; P = 0.029).

After treatment with sublethal doses (0.005%, 0.001%) of the pyrethroid alpha-cypermethrin the beetles stopped displaying DGE and switched to a form of continuous gas exchange. In the treated beetles short activity periods (15 – 20 min) occurred with a frequency of 1 – 2 per hour. Dur- ing each activity period an increase in CO2 emission was recorded. The activity periods may be easily confused with CO2 release by burst. These activity periods (struggling) were also confirmed by visual observations under a stereomicroscope. The metabolic rate was measured between the activity periods. The mean resting metabolic rate of beetles treated with 0.005% and 0.001% of Fastac 50 EC were 1.140 ± 0.015 (n = 9) and –1 –1 1.104 ± 0.016 (n = 8) VCO2 mL h g , respectively. Thus the mean resting metabolic rate significantly increased after the treatments of both groups –1 –1 0.948 ± 0.016 (0.005%) and 1.140 ± 0.016 (0.001%) VCO2 mL h g , respectively (see also Tab. 2).

37 –1 –1 Table 2. The individual data of the metabolic rates (VCO2 mL h g )of Platynus assimilis before and after treatment with two concentrations of Fastac 50 EC and distilled water as control –1 –1 Treatment Individuals Metabolic rate (VCO2 mL h g ) Before After 1 0.854 1.171 Fastac 50 EC 2 0.976 1.244 3 0.951 1.121 4 0.878 1.098 5 0.976 1.145 6 0.927 1.122 7 0.878 1.120 8 1.122 1.146 9 1.002 1.122 t = 0.000 d.f. = 8 P = 0.007 0.001% 1 0.927 1.098 Fastac 50 EC 2 0.976 1.073 3 0.878 1.070 4 1.002 1.171 5 0.927 1.121 6 0.976 1.122 7 0.925 1.049 8 0.951 1.120 t = 0.000 d.f. = 7 P = 0.012 Distilled water 1 0.878 0.902 2 0.976 0.927 3 0.927 0.925 4 0.879 0.976 5 0.979 1.002 6 0.925 0.952 t = 6.00 d.f. = 5 P = 0.345

Our measurements with the CO2/H2O analyzer (LI-7000) showed that respiratory water loss constituted only a small part (< 10%) of total water loss in P. assimilis beetles (n = 5) which displayed the DGE pattern. Nevertheless, the pattern of water loss (< 10%) was similar to the pattern of the phases of the DGE (Fig. 7). The water loss estimated by weighing beetles using DGEs before treatment was 47.0 ± 7.9 (mg day–1 g–1) and in beetles treated with pyrethroid (eliminated DGEs) was 91.4 ± 11.0 (mg day–1 g–1) (t = 9.2; d.f. = 14; P = 0.001).

38 0.122,7 -1,160.2

0.09-1,1 -1,200.15 ) ) -1 -1

-1,24 lh 0.1  (ml h (ml O( 2 0.06-4,9 2 VH

VCO E -1,280.05

0.03-8,7 -1,320

-12,50 0 4 8 12 16 20 24 Time (min)

Figure 7. Simultaneous recording of CO2 emission (thick trace) and water vapour release (thin trace) during discontinuous gas exchange in adult Platynus assimilis (42 mg). E shows the traces from the empty chamber.

5.4. Eﬀects of larval food plants on diapause of the cabbage moth, Mamestra brassicae (III)

The SMR of diapausing pupae was significantly affected by the food plant of the larvae (F4,37 = 8.50, P < 0.0001). SMR, measured as the rate of O2 production, was lowest on B. oleracea (mean 0.038 ± 0.006 mL O2 –1 –1 g h ; n = 12) and highest on P. sativum (mean 0.067 ± 0.01 mL O2 g–1 h–1; n = 7) with a statistically significant difference between the two (LSD-test, P < 0.05). The SMR of pupae from larvae fed on B. oleracea was significantly lower from those fed B. vulgaris (mean 0.048 ± 0.01 mL –1 –1 –1 –1 O2 g h ; n = 8) and A. cepa (mean 0.054 ± 0.01 mL O2 g h ; n = 8).

No significant differences in O2 consumption were found between male and female pupae.

The time lapse between DGE bursts of M. brassicae pupae was significantly affected by the food plant of the larvae (F4,45 = 17.58; P < 0.0001). Overall, the time lapse between bursts lasted longest in pupae from larvae reared on B. oleracea (mean 18.8 ± 2.2 h; n = 10) which differed from all

39 0.09

c 0.08

b 0.07

-1 0.06 h -1 ab ml g 2

O 0.05 a

0.04

0.03 Mean Mean±SE Mean±SD

0.02 B. oleracea A. cepa B. napus B. vulgaris P. sativum

–1 –1 Figure 8. Standard metabolic rate (O2 mL g h ) in three month old Mamestra brassicae pupae from larvae fed on Brassica oleracea, Allium cepa, B. napus, Beta vulgaris and Pisum sativum. Columns with different letters were significantly different (P < 0.05, LSD-test).

22 a Mean Mean±SE 20 Mean±SD b b 18 b

12 c

10 Time-lapse between DGE (hours) 8

4 B. oleracea A. cepa B. napus B. vulgaris P. sativum

Figure 9. Mean duration of DGE cycles in hibernating pupae of Mamestra brassicae on different larval food plants: Brassica oleracea, Allium cepa, B. napus, Beta vulgaris and Pisum sativum. Columns bearing the same letter were not significantly different (ANOVA, LSD, P < 0.05).

40 other food plants. Statistically significantly shortest periods between the DGE bursts occurred on P. sativum (mean 9 ± 2.3 h; n = 10).

Pupae from larvae fed on B. napus (mean 15.1 ± 2.9 h, n = 10), B. vulgaris (mean 15.2 ± 2.5 h, n = 10) and A. cepa (mean 14.2 ± 2.5 h; n = 10) did not show any statistically significant difference within the group, but only in comparison with B. oleracea and P. sativum. Pupal mass (Tab. 3) of M. brassicae was significantly affected by food plant (Two-way ANOVA:

F4,60 = 3.2, P = 0.017), and the interaction of gender and food plant

(Two-way ANOVA: F4,60 = 2.97, P = 0.026) but gender alone was not significant (Two-way ANOVA: F1,60 = 3.073, P = 0.08). The pupae from larvae fed on B. oleracea resulted in a female-biased sex ratio, whereas those fed on A. cepa, B. napus, P. sativum and on B. vulgaris resulted in a male-biased sex ratio.

Pupal mass loss was not significantly affected by food plant or gender, but the interaction of gender and plant was significant. In male pupae, food plant appeared a significant factor (F4,30 = 9.37, P < 0.0001); the largest pupal mass loss of 26.1% occurred on B. napus which was significantly higher (all values P < 0.05) than on other food plants: 6.4% on P. sativum, 4.8% on B. oleracea, 4.5% on A. cepa, and 4.4% on B. vulgaris.

Food plant had no significant effect on mass loss of female pupae (F4,30 = 1.362, P = 0.27); the highest mass loss appeared on A. cepa (12.2% of the initial weight), followed by P. sativum (7.5%), B. vulgaris (4.8%), B. oleracea (4.6%) and B. napus (4.4%).

Table 3. Pupal mass (Mean ± SE, mg) and sex ratio of Mamestra brassicae larvae reared on five different species of food plants Food plants Pupal mass Pupal mass Pupal sex ratio Female Male Female : Male Allium cepa 417.0 ± 13.9 ab 424.8 ± 11.58 a 1 : 1.23 Brassica oleracea 461.0 ± 10.85 b 415.9 ± 13.0 ab 1 : 0.52 Beta vulgaris 399.3 ± 34.2 ab 465.0 ± 9.95 ad 1 : 1.4 Pisum sativum 346.3 ± 47.0 a 367.0 ± 42.3 b 1 : 1.32 Brassica napus 403.9 ± 16.19 a 474.5 ± 18.4 d 1 : 1.31 F 2.84 3.77 d.f. 4 4 P 0.04 0.01 Means within columns followed by different letters are significantly different at P ≤ 0.05 (LSD- test).

41 6. DISCUSSION

6.1. Cyclic gas exchange with Passive Suction Inspiration (PSI) in diapausing pupae of Pieris brassicae (I)

The results showed that, in the initiation phase of diapause, P. brassicae pupae display relatively short DGE cycles (40 – 70 min), with CO2 bursts lasting 2 – 6 min. This contrasts with earlier studies, using P. brassicae pupae more than 2 months old; these displayed longer DGE cycles (8

– 23h) (Harak et al., 1999; Jõgar et al., 2004, 2005) with CO2 bursts lasting 13 – 18 min (Harak et al., 1999; Tartes et al., 1999). The young pupae we used, with their short DGE cycles, were convenient for studying flutter events. They had a relatively high metabolic rate; in 2 – 3 month old pupae metabolic rate is at least twofold lower (12 – 28 mL –1 –1 O2 g h ) (Kuusik, 1977; Jõgar et al., 2004, 2005).

Commonly, after the O-phase, a period with no CO2 release occurs

(C-phase); later, the CO2 level was marginally elevated (F-phase) (Chown et al., 2006). The present study revealed that in young P. brassicae pupae, the DGE measured with flow-through respirometry differed from the F-phase measured with coulometric respirometry. In the recordings of flow- through respirometry the C-phase showed no O2 uptakes. Nevertheless, a serious O2 convective uptake was recorded by coulometric respirometry. Thus, the C-phase in those pupae was not as closed as previously thought by measurements of flow-through respirometry. Between two large CO2 bursts, almost regular small CO2 bursts were recorded. Each small burst started with sudden uptake of air into the tracheae (PSI). The main finding in the present study was a series of irregular microopenings of the spiracle(s) with convective O2 uptakes (miniflutter) found between small bursts. During the mini-flutter, no recordable CO2 emission occurred. There are several examples where the interburst period consists of discrete small CO2 bursts. Such bursts were described by Lighton (1988) in P. striatus where each burst was accompanied by active abdominal movement. Discrete CO2 emissions during the F-phase have also been reported by Duncan et al. (2002) in Pimelia grandis Fabricius and by Kovac et al. (2007) and Lighton and Lovegrove (1990) in resting A. mellifera. How- ever, spiracle openings within the F-phase were commonly observed to be irregular with respect to frequency and amplitude, if inferred from the

CO2 release pattern (e.g. Wobshall and Hetz, 2004).

42 The flow-through CO2 measurements showed no CO2 release for a short time after the O-phase in P. brassicae pupae. The coulometric respirometry and IR actographic recordings showed rapid and clear uptakes of air shortly after the O-phase, indicating the beginning of the F-phase. An earlier study by Tartes et al. (2002) revealed that air convective uptakes began immediately after the O-phase. Air convective uptakes, shortly after large CO2 bursts, also occurred in old diapausing M. brassicae pupae (Jõgar et al., 2007). The result suggests that, at the beginning of the flutter, air uptakes were convective but later were diffusive-convective. These results concur with the plethysmometry flow-through measurements of

Wobschall and Hetz (2004), revealing that the convective uptakes of O2 dominate at the beginning of the F-phase but in the later F-phase, diffusion takes over from convection as the chief mechanism of O2 uptake. One may suppose that water is conserved only at the beginning of the

F-phase when clear convective O2 uptakes (PSI) occur, but not later when diffusion is the dominating mechanism of the fluttering period. Wob- schall and Hetz (2004) showed that, in diapausing pupae of A. atlas the uptake of air into the tracheal system at the beginning of the F-phase along the negative hydrostatic pressure gradient may initially inhibit CO2 release from the tracheae. However, in the present study with P. brassicae pupae, the measurements demonstrated that, CO2 was inhibited not only at the beginning of the F-phase but also at the later period of the F-phase.

The duration of the F-phase may be underestimated, as the F-phase may start before the CO2 measurements can detect it (Hetz et al., 1994; Wob- schall and Hetz, 2004). The present study revealed that in P. brassicae pupae, the duration of the F-phase was longer than estimated by the flow-through system, as far as CO2 release was prevented at the beginning of the F-phase by convective air uptakes. Wobschall and Hetz (2004) showed that small volume and pressure decreases occurred between the microopenings in the F-phase. This confirmed a small but significant contribution of suction ventilation during each microopening (see also Kestler, 1985). In the present measurements in P. brassicae pupae, each miniature inspiration was synchronised with rapid extension of the abdomen, confirming that a convective component was always present in O2 uptakes. In the present study of P. brassicae we observed a relatively longer F-phase compared with that in some other insects, such as in P. niger (I) and B. terrestris (Karise et al., 2010).

43 Manometric O2 respirometry methods have been criticized and their readings mistrusted because these methods usually do not allow separation of active and resting metabolism (see Van Voorhies et al., 2008). Nevertheless, some volumetric manometric methods, including coulometric respirometry, are regarded as useful (Klok and Chown, 2005). In the present investigation we demonstrated that, in gas exchange studies with insects, coulometric respirometry supplemented by the flow- through method, has clear advantages. Lighton (2008) considered that coulometric continuously recording respirometry deserves to be more widely used.

In this study, we showed that the pattern of gas exchange in P. brassicae pupae may be effectively investigated by the combined use of coulometric respirometry and flow-through CO2 systems by switching the same respiration chamber from one system to the other without disturbing the insect. By combining IR actography in parallel with both types of respirometry, it was possible to record rapid air uptakes. Thus, on flow- through recording traces the patterns of microopenings of the spiracles were clearly indicated by simultaneous actographic measurements.

6.2. Gas exchange in the carabid beetle Pterostichus niger in low and high humidity conditions (II)

Under the experimental conditions examined, the metabolic rate and respiration pattern, as well the type of body movement, varied between individuals in all tested insects. A few P. niger carabid beetles showed a true pattern of DGE, with distinct Closed (C) and F-phases. In individuals showing DGE, vigorous abdominal contractions (i.e. pumping movements) accompany each burst of CO2 expulsion. The metabolic rate of individuals with CGE is higher than in individuals with DGE. Gibbs and Johnson (2004) showed that metabolic rate varies with gas exchange patterns, being lowest in individuals using DGE, intermediate for individuals using CGE and highest for individuals using continuous respiration.

We recorded long burst periods (O-phase) from individuals exhibiting both DGE and CGE patterns. The long burst phase is also described in dung beetles (Davis et al., 1999; Duncan and Byrne, 2002; Duncan et al., 2009). A common pattern of gas exchange in P. niger is CGE associated

44 with very weak and uniform body movements or abdominal pulsations associated with the O-phase and these pulsations stop for just a short time before the next burst. This pattern without clear C and F-phases does not differ between dry and moist air. The overall pattern differs from the intracyclic activity in the study by Kestler (1991), and also differs from the interburst activity in the cockroach Perispaeria spp. (Marais and Chown, 2003) and in B. terrestris (Karise et al., 2010). P. niger has a mode of breathing that is different from another member of the same genus, Pterostichus stygicus Say. The latter shows rapid cycles of collapse and then reinflation of the tracheal tubes that are synchronous with convective expulsion of CO2, although larger bursts of CO2 release, which are characteristic of DGE and CGE, are absent (Socha et al., 2008).

Gas exchange patterns vary with metabolic rate (Contreras and Bradley, 2009) and with the state of hydration of the insect (Chown, 2011). To date, there have been only a few studies concerning a direct effect of environmental humidity on gas exchange in insects. Sláma et al. (2007) showed a clear pattern of DGE in the Cuban subterranean termite Pro- rhinotermes simplex Hagen in a dry respirometer chamber but, in a moist chamber, the same species displays CGE. Terblanche et al. (2008) showed that, in pupae of S. cynthia, DGE is not abandoned in conditions of high atmospheric O2, or in high or low gas moisture levels, thus supporting the oxidative damage hypothesis. In this present study, we examined respiratory gas exchange in P. niger adults under different moisture conditions. The species is eurytopic (i.e. able to withstand a wide range of environmental conditions) and is common in a variety of open habitats, occurring also in almost every type of forest community (Witzke, 1976; Šustek, 1994).

In the present investigation, we observed bursts of CO2 release in dry as well as in humid air in P. niger. Thus the hygric hypothesis for the origin of DGE is not supported, at least in this species. Multiple gas conditions are not tested simultaneously in the current experiments; therefore, there is no evidence to support or challenge the alternative oxidative damage theory. Until recently, DGE has been considered as the main mechanism for conserving water in several species of insect (Lighton, 2007; Nes- polo et al., 2007; White et al., 2007). However, CGE may also help to conserve water (Gibbs et al., 2003). Insects may alter their gas exchange patterns to cope with changes in atmospheric moisture over longer time scales. For example, Drosophila sp. selected for desiccation resistance show

45 an increased presence of CGE (Gibbs et al., 2003). However, it needs to be considered that DGE may have originated in insects living in hypercapnic (high CO2) and hypoxic (low O2) conditions to aid in the release of CO2 (chthonic hypothesis) described by Lighton (1996).

6.3. Interaction between circulation and gas exchange

Beside active ventilation, some other types of body movements, differing in frequency and amplitude, were observed in the present study. The pulsations with low amplitude and high frequency are interpreted as cardiac pulsations (heartbeat) (Sláma, 1984, 1999).

In this investigation with P. niger carabid beetles the heartbeats were continuous, without inactivity periods (I); in P. assimilis no correlations were found between gas exchange cycles and heart activity (II). In some beetles e.g. Thermophilum hexmaculatum heartbeat periods coincide with the

O-phase CO2 burst of the DGE (Wasserthal, 1996).

In P. brassicae pupae, we interpreted high-frequency but low amplitude signals, accompanied by CO2 release in bursts, as heartbeats. In a previous study using thermographic measurements, we demonstrated heartbeat reversal, correlated with gas exchange cycles and twisting abdominal movements in diapausing P. brassicae pupae (Jõgar et al., 2005). In pupae of P. brassicae we found that the burst of CO2 (2 – 6 min) coincided with heart activity. Earlier studies demonstrated that, during the burst, heartbeat peristalsis is directed forward but that during the interburst period reversal occurs and peristalsis is directed backward (Tartes et al., 2002; Sláma, 2003). Heartbeat reversal correlated with gas exchange cycles has also been reported in saturnid moth pupae (Wasserthal, 1996; Hetz et al., 1999; Sláma, 2003).

The pattern of heartbeats, especially the periodicity of heart pulsations may be used to estimate the physiological state of insects. In diapausing pupae of P. brassicae the periods of heart activity alternate with periods of inactivity, while the length of the latter depends of the intensity of diapause (Metspalu et al., 1982). The shortening or absence of pause periods indicates breaking of diapause by certain cause (internal or external factors) (Kuusik et al., 1995).

46 The heart pulsations may be easily confused with weak abdominal contractions, or extracardiac pulsations (see Sláma, 2000). Therefore, simultaneous recording of the rhythm of abdominal contractions and heartbeats is essential. In the present study (I; II) we showed that, on the recordings of the IR actograph or IR optocardiograph, body contractions show larger amplitudes than those of heartbeats, while heartbeats have higher frequencies than body contractions. The same differences between heart pulsations and abdominal ventilating movements were described by Sláma (2000).

6.4. Gas exchange in the carabid beetle Platynus assimilis before and after treatment with sublethal doses of pyrethroid

For adult P. assimilis the between-individual variability in their gas exchange patterns was conspicuous. Variability within and between individuals in physiological characteristics, including gas exchange patterns, is regarded as a normal phenomenon in insects (Chown, 2001; Chown et al., 2002; Marais and Chown, 2003). Essential between-individual variation was found in the patterns of gas exchange, chowing DGE, CGE and continuous gas exchange. Our results indicated that, in P. assimilis, the metabolic rate did not differ between DGE and CGE, in contrast to the literature in which DGE is commonly recorded with a lower metabolic rate than CGE. However, P. assimilis exhibited an uncommon pattern of DGE: the O-phase (burst) was extraordinarily long compared with the whole cycle. This pattern of gas exchange was similar to that described by Duncan and Dickman (2001) in carabids Cerotalis sp. and Carenum sp. and also in our datas with P. niger (I). In P. assimilis beetles we recorded a DGE pattern with a very long O-phase, contributing 80-90% of the whole cycles, a clear but short C-phase but no evident F-phase. The O-phase (burst) in all beetles coincided with the appearance of muscular ventilation. The other common gas exchange pattern in P. assimilis beetles was CGE, which characterised 45% of the whole cycle. The minority of resting P. assimilis exhibited a pattern of continuous respiration, where ventilation by muscular abdominal pumping occurred continually.

Breathing by muscular abdominal pumping is well known in insects (Miller, 1974, 1981). The continuous respiration in adult T. molitor is also characterised by abdominal pumping or respiratory contractions occurring continually or periodically (Zafeiridou and Theophilidis, 2004,

47 2006). Weak abdominal movements, such as extracardiac haemocoelic pulsations were described also by Sláma (2008, 2010). However, handling and instrument stress may be expressed as continuous respiration via uninterrupted pumpings, regarded as a precursor for CGE e.g., in L. decemlineata (Vanatoa et al., 2006) and in H. abietis (Sibul et al., 2004a).

We found that standard metabolic rate in beetles with continuous active ventilation was significantly higher than in individuals using DGE. Gibbs and Johnson (2004) have reported that metabolic rate varies with the gas exchange pattern, and is lowest for individuals that use DGE and highest for individuals using continuous gas exchange.

Our measurements with the infrared CO2/H2O analyzer showed that respiratory water loss contributed only a small fraction of total water loss in P. assimilis exhibiting DGE. However, according to Schimpf et al. (2011), DGE exhibition significantly lengthens survival during food and water shortage, suggesting DGE confers a fitness benefit by reducing water loss. Nevertheless, respiratory water loss may contribute only a small part of total water loss. Several studies reveal that respiratory water loss comprises < 15% of total water loss, even when the spiracles are open (e.g. Quinlan and Lighton, 1999; Gibbs and Johnson, 2004).

Treatment of adult P. assimilis with concentrations of 0.005% and 0.001% alpha-cypermethrin resulted in elimination of their DGE and change to a pattern of continuous respiration via pumping. However, the pumping in treated beetles had higher frequencies and amplitudes than in untreated beetles characterized by continuous gas exchange. Kestler (1991) demonstrated that mild desiccation, sublethal doses of toxicant and handling stress lead to a similar release pattern of CO2, and commonly the DGE was lost and the pattern of continuous respiration appeared.

The abolishing of the gas exchange cycles may be regarded as the earliest symptom of poisoning by toxicant in insects. It may be suggested that disturbances of DGE cycles are due to the paralyzed opening-closing mechanisms of spiracles. However, it is also possible that the pesticide increases metabolic rate due to uncontrolled muscle activity and it is this which causes them to abandon the DGE cycle and adopt a continuous mode of gas exchange. However, there also exist other factors abolishing the normal DGEs. The mechanism by which insects lose their DGEs

48 may either be autointoxication or/and release of neurohormones, or by an influence on the O2/CO2 thresholds for the control or on the CO2 capacitance due to changes in the acid-base status (Kestler, 1991). We suggested that Fastac 50 EC as a neurotoxic substance may cause similar actions.

Metabolic rate and body mass loss in treated individuals of P. assimilis were significantly higher than in untreated individuals. The elevation of metabolic level in treated beetles may also be caused by the metabolic cost of vigorous pumping. According to Chown and Holter (2000) and Sibul et al. (2008) an increase in metabolic rate may be due to a small increase in metabolic cost of the convective ventilation caused by the muscular contractions.

Carabid beetles are important agents of biological control in organic farming (Kromp, 1990). Our results indicate that the respiratory system of P. assimilis is vulnerable to pyrethroids evoking respiratory failure. When pyrethroids are applied to agricultural land their physiological impact on predaceous beetles should not be ignored.

6.5. The inﬂuence of food plants on development and on dormancy of Mamestra brassicae (V)

This study demonstrated significant differences in some biological parameters of M. brassicae larvae and pupae reared on different food plants. Overall, food plants influenced larval development rate, larval and pupal mass, mass loss and the intensity of diapause.

Developmental time of larvae varied with food plant; that on B. oleracea was significantly shorter than on A. cepa, B. napus, B. vulgaris or P. sativum. For normal growth and development of larvae the proportions of nutritional elements in the food plant are of primary importance (Awmack and Leather, 2002; Syed and Abro, 2003). Faster development may allow a short life cycle, high reproductivity, and rapid population growth (Singh and Parihar, 1988; Liu et al., 2004). Generally, slower development or digestion and lower fertility rate in herbivorous insects are caused by lower food quality (Chen et al., 2004). To compensate for deficiency of essential nutrients, insects may start feeding voraciously, extend their feeding period or apply both strategies. Additionally, they

49 may enhance feeding efficiency by extending the time food is in the alimentary canal or by activating digestive enzymes (Barbehenn et al., 2004). Quality food plants may give rise to the second full generation in individuals with a faster development cycle in northern regions, increasing crop damage. Food quality also affected the viability of M. brassicae.

Body mass is an important fitness indicator in insect population dynamics (Liu et al., 2004). Pupal body mass varied with food plant. Again, the lowest mean pupal body mass was in P. sativum fed larvae. Body mass is directly dependant on reserves stored at the larval stage, and pupae with small body mass appear when growing conditions, including food quality in the larval stage are unfavourable. Pupal mass is important, since heavier female pupae lay more eggs when adult (Kramer, 1959; Haukioja and Neuvonen, 1985) consequently affecting potential growth rate of the population. Insects cannot clear the hurdle of food quality and the nutritional features are directly reflected in the abundance of progeny (Ruo- homäki et al., 2000). Larval food plants affected potential hibernation success of M. brassicae pupae; they affected not only diapause induction (Hunter and McNeil, 1997) but also the intensity of pupal diapause.

Diapause intensity (see Koštál, 2006; Belozerov, 2009) is characterised by SMR, which in lepidopteran pupae may decrease to very low levels –1 –1 – 0.01–0.04 mL O2 g h (Keister and Buck, 1974; Jõgar et al., 2005, 2007). On the contrary, at the initiation of pupal diapause in P. brassicae, –1 –1 a SMR 0.07 – 1.2 mL O2 g h may be observed (Jõgar et al., 2004, 2005). Similar SMR was found in M. brassicae pupae when the larvae were reared on less suitable food plants. These results suggested that the most suitable food plant was B. oleracea, as judged by the lowest level –1 –1 of SMR in diapausing pupae (0.04 mL O2 g h ). Such a low level of SMR points to a deep diapause which favours overwintering of the pupae (Fourche, 1977). On P. sativum, the significantly higher SMR in –1 –1 the pupae (0.067 mL O2 g h ) was a sign of an abnormally decreased intensity of diapause.

Diapause intensity of M. brassicae pupae in the present experiment was characterized by DGEs with large outbursts of CO2 lasting 15 – 20 minutes. Time lapses between outbursts were long, occurring only once or twice per 24 hours. However, in larvae reared on P. sativum, the DGE cycles were shorter (4 – 5 minutes) and occurred more frequently, 3 – 4 times per 24 h. As a rule, lower metabolic rate is associated with fewer

50 DGE cycles per day (Sláma, 2010). Besides short gas exchange cycles and relatively high metabolic rates were found in diapausing pupae of M. brassicae, when measured caterpillars were fed on less favourable food plants (e.g. P. sativum). Resulting pupae were characterized by frequent gas emission cycles, higher respiration rate and body mass loss. This suggests that pupal diapause had not developed normally and such a physiological state is probably unfit for the overwintering period.

It may be concluded from this study that one of the reasons for the great decrease in the abundance of M. brassicae following mass reproduction is the reduced viability of caterpillars growing on lower quality food plants. The pupae were underweight, diapause was not as deep as expected and, in most cases, the pupae perished during winter. Different food plants obviously play an important role in triggering population increases and outbreaks.

51 7. CONCLUSIONS

1. A novel O2 respirometry system termed coulometric respirometry was developed able to record oxygen convective uptakes (passive sucction inspiration) during the F-phase. It is not possible to meas-

ure and record these O2 uptakes using a flow-through respirometry system. It was found that the F-phase begins immediately after the

end of O-phase. The convective O2 uptakes at the beginning of the

F-phase did not allow the emission of CO2 and water vapour. It was

demonstrated that when coulometric O2 respirometry and flow-

through CO2 respirometry are used alternately more information can be obtained than from each system separately (I).

2. This work demonstrated that, in P. niger, the DGE pattern is typical in a dry environment (5% RH), as well as in conditions of high humidity (95% RH). The main prediction of the hygric hypothesis is that, in moist air, DGE is eliminated. In this way, the hygric theory was not supported in this carabid species. Therefore, we give more

support to the well-known alternative hypothesis: the O2 damage hypothesis (II).

3. The results showed that sublethal doses of pyrethoid Fastac 50 EC influence the physiology of beneficial insects. The main effect of pyrethroid was to eliminate cyclic gas exchange of P. assimilis and the pattern of continuous gas exchange appeared. After this transforma- tion of gas exchange pattern, water loss increased due to the higher frequency and amplitude of the continuous respiratory contractions (III, IV).

4. The present work demonstrated significant differences in some biological parameters of M. brassicae larvae and pupae reared on different food plants. Food plants influenced larval developmental rate, larval and pupal body mass, mass loss and the intensity of pupal diapause. Developmental time of larvae was shorter when reared high quality of food plants. Poor quality of larval food plant influenced diapause intensity of pupae. The weakening diapause of pupae caused higher winter mortality (V).

52 From the results it may be concluded that the effect of the pesticides should not be observable in a short time frame since there is a chance the insect survival is affected subsequently. This is the reason why pest monitoring is required before pesticide application.

The elaborated O2 respirometry system termed coulometric respirometry allows to measure even smallest differences caused by numerous factors influencing the physiological state of insect. By this method it is possible to carry out more detailed research about long-term effects of sublethal doses of pesticides including biopesticides on insect.

Together with physiological research field studies on availability and quality of host plants are needed in order to accumulate basic knowledge for planning crop layouts within a field and successional cropping/rota- tional practices from year to year to minimise the success of pest.

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66 SUMMARY IN ESTONIAN

LOODUSLIKE JA ANTROPOGEENSETE FAKTORITE MÕJU PUTUKATE FÜSIOLOOGILISELE SEISUNDILE

Putukate tundlikkus keskkonnatingimuste muutuste suhtes peegeldub ka nende füsioloogilises seisundis. Putukate füsioloogilise seisundi hindamiseks kasutakse peamiselt hingamis-ainevahetuse taseme määramist koos hingamis-mustrite kirjeldamisega. Ainevahetuse taset määratakse kas süsihappegaasi (CO2) eraldumise või hapniku (O2) tarbimise hulga alusel. Katkendlik gaasivahetus on hingamistüüp, mis iseloomustab pal- jude putukaliikide hingamist nende rahuoleku ajal. Selle all mõistetakse

CO2 tsüklilist eraldumist ja samuti O2 tsüklilist absorbeerimist. Tsüklid koosnevad kolmest faasist vastavalt hingamisavade olekule: suletud faasi (C-faas) ajal gaasivahetust ei toimu, flatteri faasi (F-faas) ajal hingamisavad avatakse vaid pilukile murdosa sekundi vältel ja siis suletakse. Sellise vahelduva protsessi vältel hapnik (õhk) imetakse trahheedesse seal oleva alarõhu tõttu (võrreldes atmosfäärsega) ning väikesed kogused süsihap- pegaasi eraldatakse läbi hingamisavade väliskeskkonda. Kolmanda faasi ajal (O-faas) hingamisavad avatakse maksimaalselt ja CO2 eraldub nii kudedest kui trahheedest. Ühe kõige enam levinud hüpoteesi põhjal katkendlik gaasivahetus on füsioloogiline adaptatsioon mis kahandab vee väljaaurumist putuka kehast, sest veeaur eraldub koos CO2-ga vaid selle lühikese aja jooksul mil hingamisavad on lahti. Kirjanduse põhjal toimub katkendlik gaasivahetus vaid kuivas keskkonnas ja katkeb niiskes ning seejärel läheb üle pidevale gaasivahetusele. Siiani kasutatud läbivoolule tuginev respiromeetria ei võimalda F-faasi ajal jälgida välisõhust trahheedesse toimuvaid O2 konvektiivseid sissetõmbeid. Läbivoolusüsteemi respiromeetriat kasutades saab F-faasis registreerida vaid CO2 väljumis- rütme selle faasi vältel.

Putukapopulatsioonide arvukust vähendavad insektitsiidide letaalsete dooside kõrval ka nõrgemad, otseselt mittesurmavad doosid, mis mõju- vad populatsioonidele kahjulikult järeltoime kaudu. Insektitsiidide järel- toimet putukate füsioloogilisele seisundile on seni vähe uuritud. Putukate hingamissüsteem on väga tundlik välismõjutuste, sealhulgas toksiliste ühendite suhtes. Füsioloogilise seisundi hindamiseks määratakse ainevahetuse tase ja hingamismustrid enne ja pärast insektitsiidiga töötlemist. Käesolevas teadustöös uuriti põllumajanduses laialt kasutatavat insektit-

67 siidi püretroid Fastac 50 EC, mille toimeaineks on alfa-tsüpermetriin.

Mitmetoidulistele putukatele erinevate toidutaimede olemasolu ja kät- tesaadavus mängib tähtsat osa populatsiooni arvukuse dünaamikas, selle kahanemises ja järsus suurenemises. Uurimused toidu kvaliteedist ja selle mõjust kahjurputukate bioloogiale on olulised, kuna toidutaimedest sõl- tub putuka areng, paljunemine, viljakus ning diapausi sügavus. Diapausi intensiivsust iseloomustab ainevahetuse tase.

Lähtudes nendest probleemidest oli käesoleva doktoritöö eesmärki- deks:

1. Välja töötada väga tundlik respiromeetria, mis võimaldaks salves-

tada O2 sissetõmbeid (passiivne imev-ventilatsioon), et registreerida ka väiksemaid muutusi putukate füsioloogilises seisundis. See respiromeetria funktsioneerib ka aktograafina, registreerides hinga- misliigutuste rütme ja võimaldab eristada aktiivsusperioode (I).

2. Võrrelda ainevahetuse taset ja hingamismustreid nii kuivades kui niisketes keskkonnatingimustes (II).

3. Määrata standardainevahetus, puhkeoleku ainevahetus, veekadu ning hingamismustrid enne ja pärast insektitsiidi Fastac 50 EC subletaalsete doosidega töötlemist (III, IV).

4. Hinnata toidutaimede kvaliteedi mõju vastsete ja diapauseeruvate nukkude füsioloogilisele seisundile (V).

Mudelobjektidena kasutati kasulikke röövtoidulisi jooksiklasi suur-süsi- jooksikut Pterostichus niger ja süsi-ketasjooksikut Platynus assimilis ning kahjurputukatest kapsaöölast Mamestra brassicae ja suur-kapsaliblikat Pieris brassicae.

Käesolevas töö tulemusena töötati välja ja rakendati elektrolüütiline respiromeeter-aktograaf, mis võimaldab registreerida hingamisavade mikro-avanemisi ja õhu (hapniku) passiivseid imev-sissetõmbeid. Vara- jases diapausis oleva suur-kapsaliblika P. brassicae nukuga tehtud katsed näitasid, et gaasivahetuse tsükli F-faas on seniarvatust märksa pikem ja sageli algab vahetult peale O-faasi. Selgus, et F-faasi alguses toimuvad

O2 imev-sissetõmbed trahheedesse ilma CO2 eraldumiseta, sest trahhee-

68 desse järsult sissevoolav ehk sisseimetav õhk takistab CO2 ja vee väljumist trahheedest. Kasutatud volumeetriline-manomeetriline respiromeetriline süsteem näitas veel seda, et kogu F-faasi vältel O2 siseneb trahheedesse konvektiivselt, mis tähendab, et trahheedes küll rõhk pidevalt suureneb, kuid alarõhk atmosfäärse rõhu suhtes säilib ka F-faasi lõpus. Siit järeldus, et F-faasil on vaieldamatu tähtsus vee kokkuhoiu suhtes. F-faas ei koosne vaid hingamisavade kaootilisest mikro-avanemistest-sulgumistest vaid mikro-avanemised on diskreetsed ja selgelt omavahel eraldatud. Kuna hingamisavade iga mikro-avanemine põhjustab nuku tagakeha passiivseid liigutusi (kontraktsioon-ekstensioon) siis neid saadi registreerida infrapuna-aktograafiga. See tähendab, et läbivoolu-respiromeetria üles- kirjutustel saab infrapuna-aktograafi ja gaasianalüsaatori üheaegsel raken- damisel jäädvustada ka O2 konvektiivseid sissetõmbeid. Gaasivahetuse tsüklite ja kehas toimuvate liigutuste üheaegne registreerimine suur-kapsaliblika varases diapausis oleval nukul näitas, et südame (selgmise soone) aktiivsusperioodid toimuvad samaaegselt tsüklitega. Nuku diapausi alg- staadiumis hemolümfi ringluse suunamuutusi (reversioone) ei tuvastatud (I), kuid südamelöökide reversioone on registreeritud mitmes varasemas töös, mis käsitlevad suur-kapsaliblika diapausi hilisemaid perioode. See- vastu uuritud mõlemal jooksiklase liigil olid südamelöögid pidevad ja mitte perioodilised (II, IV).

Selles töös tõestati, et jooksiklase (P. niger) valmik hingab katkendlikult nii niiskes õhus (90% RH) kui ka kuivas õhus (5% RH) (II). Seega seati kahtluse all teooria, et katkendlik gaasivahetus on kohastumine veeka- dude kahandamiseks putuka kehast, kuigi tsüklilisel gaasivahetusel on sõltuvalt liigist ja keskkonnatingimustest erinevad funktsioonid, on see igal juhul vajalik kohastumine ning selle katkemine võib kahandada ka putuka eluiga.

Füsioloogiliste katsete tulemusel selgus, et Fastac 50 EC subletaalsed doosid kutsuvad esile olulisi gaasivahetuse hälbeid: tsükliline gaasivahetus muutub pidevaks. Selle hälbe oletatavaks põhjuseks on hingamisavade sulgurlihaste paralüseeritus. Gaasivahetuse tsüklilisuse kadumine tõi kaasa ka veekao kiirenemise putuka kehast. Süsihappegaasi eraldumise ja vee väljaaurumise üheaegne mõõtmine gaasi ja vee analüsaatori LI- 7000 abil näitas, et respiratoorne veekadu moodustas väikese osa (alla 10%) üldisest veekaost (III-IV). Katsetulemused jooksiklastega (P. niger ja P. assimilis) lubavad arvata, et CO2 katkendlik eraldumine putukast ei pruugi töötada vett säästva mehhanismina, samuti niiskes keskkonnas

69 putuka tsükliline hingamine ei katke (II-IV). Võib arvata, et jooksiklase P. niger kehamassi kiirenenud kahanemine ja ühtlasi vee kiirenenud väljaaurumine tulenes pideva gaasivahetusega kaasnevatest katkematutest respiratoorsetest kontraktsioonidest. Tsüklilise gaasivahetuse korral need kontraktsioonid toimuvad ainult hingamisavade avatud faasi ja seega

CO2 väljumise ajal (II).

Kapsaöölase (M. brassicae) erinevad toidutaimed mõjuvad erinevalt nii tema arengule kui talvitusfüsioloogiale ja muuhulgas ka diapausi süga- vusele, millest sõltub talvitumise edukus. Vastsete kvaliteetse toidu puhul kujunesid normaalsed, sügava diapausiga nukud. Neid iseloomustas madal ainevahetuse tase, hõredad gaasivahetuse tsüklid, harvad südame aktiivsusperioodid. Vastupidiselt, ebasobivamad röövikute toidutaimed põhjustasid nukkudel kõrgema ainevahetustaseme, tihedamad gaasivahetuse tsüklid ja olulise suremuse. Röövikud, kes kasvavad kvaliteetsel toidul suudavad paremini ette valmistuda talviseks diapausiks. Ja vastupidiselt, röövikud, kes toituvad vähem kõlbulikel toidutaimedel, nende diapausi induktsioon on pidurdatud ning äärmisel juhul diapaus jääb ära, või katkeb. See toob aga kaasa talvise suurenenud surevuse (V).

70 ACKNOWLEDGEMENTS

This thesis would not have been possible without the help, support and patience of my wonderful supervisors Dr. Angela Ploomi and Dr. Luule Metspalu with their advices and valuable comments throughout my studies.

I greatly appreciate Dr. Katrin Jõgar and Dr. Aare Kuusik for kind help for assistance in experiments and with the professional collaboration.

I would thank Dr. Ingrid H. Williams and Dr. Eha Kruus for linguistic help.

I thank all my colleagues in Institute of Agricultural and Environmental Sciences for their support throughout my studies and for supporting at the difficult times.

I am grateful to Dr. Reet Karise and doctoral student Riin Muljar for their review of my thesis and critical and valuable comments on my thesis.

I would like to express my sincerest gratitude to all my teachers for their hard work, without them I wouldn’t be who I am right now.

I am very thankful to my husband Rein, and our sons Reimo and Kristo for their patience and help during these study years. I also thank my wonderful father Hallik Mällo, my sister and her family, and all my rela- tives and friends for their tolerance, support and understanding during the studies and completion of my thesis. This work is dedicated to the memory of my wonderful mother, Ene Mällo.

This study was carried out at the Institute of Agricultural and Environ- mental Sciences of the Estonian University of Life Sciences. I gratefully acknowledge the funding sources that made my Ph.D. work possible. The research and my studies in Estonian University of Life Sciences was funded by the Estonian Science Foundation (grants numbers 8895, 9449), State Forest Management forest protection project number 8-2/ T12115MIMK, Estonian target financing project number SF170057s09, Archimedes foundation ESF DoRa Programme scholarships, Estonian Students Fund in USA, Estonian Word Council Inc., Raefond of Esto- nian University of Life Sciences and Doctoral School of Earth Sciences and Ecology.

PUBLICATIONS 2816

RESEARCH ARTICLE Oxygen convective uptakes in gas exchange cycles in early diapause pupae of Pieris brassicae

Katrin Jõgar*, Aare Kuusik, Angela Ploomi, Luule Metspalu, Ingrid Williams, Külli Hiiesaar, Irja Kivimägi, Marika Mänd, Tea Tasa and Anne Luik Estonian University of Life Sciences, Institute Agricultural and Environmental Sciences, Department of Plant Protection, Kreutzwaldi 1, Tartu 51014, Estonia *Author for correspondence ([email protected])

Accepted 28 May 2011

SUMMARY Oxygen convective uptakes in gas exchange cycles were directly recorded in early diapause pupae of Pieris brassicae L. (Lepidoptera; Pieridae) by means of O2 coulometric respirometry. This method was combined with flow-through CO2 respirometry, the two systems being switchable one to the other. During recording with both systems, measurements were also taken with infrared actography. The pupae displayed short discontinuous gas exchange cycles lasting 40–70�min. No true C phase was found by flow-through measurements; instead, flutter opening of the spiracles with discrete convective O2 uptakes began shortly after the O phase whereas CO2 release was suppressed by the inward directed passive suction ventilation. The F phase was characterized by a series of small CO2 bursts (flutter events). Between these bursts, novel sub-phase ‘miniflutter’ was observed, which consisted of six to 10 miniature inspirations without any CO2 emission. During the flow-through measurements, oxygen convective uptakes were indirectly recorded by the infrared actograph as sudden extensions (lengthening) of the abdominal segments at each spiracular microopening. Key words: oxygen uptake, Pieris brassicae, gas exchange cycle, butterfly, flutter, coulometric respirometry.

INTRODUCTION detectors or diaferometers (Punt et al., 1957). Simultaneous Breathing in many insects is characterised by discontinuous gas measurements of O2 uptake and CO2 release by flow-through exchange cycles (DGCs), during which carbon dioxide is released respirometry in the tok-tok beetle, Psammodes striatus, showed a periodically. To date, the focus of studies of DGCs has been on peak of O2 consumption at the beginning of the O phase, together CO2 release as measured by flow-through CO2 respirometry, with a burst of CO2 release (Lighton, 1988). Oxygen uptake also whereas O2 consumption during the DGC has been little studied. peaked during the release of CO2 in the giant burrowing cockroach, Classically, the DGC consists of three phases (Schneiderman, Macropanesthia rhinoceros (Woodman et al., 2007). 1960; Lighton, 1996; Chown and Nicolson, 2004). During the open The F phase cannot be detected by flow-through O2 respirometry (O) phase, the spiracles are open and CO2 is released in a burst. without a significant diffusive component, because the inward bulk The O phase is followed by the facultative closed (C) phase, when flow of air into the tracheal system is functionally equivalent to a the spiracles are closed and little or no gas exchange occurs. Within minute and probably undetectable reduction in the flow rate of air the C phase, sub-atmospheric pressure is created in the tracheae through the respirometer chamber (Lighton, 1988; Lighton, 1994). because of O2 consumption by the tissues and CO2 buffering by Thus, single microopenings of the spiracles and air convective means of bicarbonates in the tissue and haemolymph (Wobschall uptakes into the tracheae cannot be detected by flow-through and Hetz, 2004). After the C phase, the flutter (F) phase occurs, respirometry. during which the spiracles open and close rapidly in succession Special techniques are required to record PSV during (fluttering). When open, air is sucked through the spiracles into the microopening of the spiracles in the F phase. Schneiderman used tracheae by convection along the negative pressure gradient. This cannulated spiracles to measure partial pressure and thus described is known as passive suction ventilation (PSV); thus PSV occurs in the rhythms of passive air uptake in silkworm pupae (H. cecropia) the absence of muscular movement (see Miller, 1974; Miller, 1981). (Schneiderman, 1960). Sláma recorded a sawtooth pattern of PSV during the F phase has been described as a mechanism for abdominal retractions with contact transducers in lepidopteran restricting water loss in insects (see Kestler, 1978; Kestler, 1982; pupae (including the large cabbage white Pieris brassicae) (Sláma, Kestler, 1991). DGC is regarded as CFO cycles when CO2 bursts 1984; Sláma, 1988). This pattern was caused by the microopening are not actively ventilated by abdominal pumping, but as CFV (V, of the spiracles and passive inspirations. A similar pattern of passive ventilation) cycles when the CO2 bursts are associated with pumping inspirations was recorded by Sláma and Neven in young pupae of movements (Kestler, 1985; Kestler, 2003). the codling moth, Cydia pomonella (Sláma and Neven, 2001). Hetz Discontinuous O2 uptakes and CO2 release during the O phase et al. used miniaturized amperometric sensors to make direct O2 has been demonstrated in some Carabidae beetles and in pupae of measurements within the tracheal system of lepidopteran pupae the Cecropia moth, Hyalophora cecropia, using heat conductivity (Hetz et al., 1994). Wobschall and Hetz recorded O2 uptake directly

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74 Oxygen convective uptakes 2817

in diapausing Atlas moth (Attacus atlas) pupae by simultaneous 3�h. Tests with each individual were made twice. We confirmed, measurements of tracheal pressure and volume changes with preliminary experiments, that the switch from still air to flowing –1 (plethysmometry) in the tracheal system, while combining CO2 air (120�ml�min ) and vice versa did not significantly change the measurements by flow-through respirometry (Wobschall and Hetz, frequency of the DGC. Body movements were visually observed 2004). Coulometric (volumetric-manometric) respirometry has been under a stereomicroscope (SZ-ZTW, Olympus, Japan). used to directly record O2 convective uptakes in diapausing 2–5�month old pupae of the cabbage moth, Mamestra brassicae Coulometric respirometry (Jõgar et al., 2007), and P. brassicae (Jõgar et al., 2004; Jõgar et Coulometric respirometers usually work in an interrupted regime al., 2005; Jõgar et al., 2008). However, there is a lack of information (on–off) of electrolysis (e.g. Heusner et al., 1982). By contrast, our about the gas exchange patterns, including O2 convective uptake coulometric respirometry (a volumetric manometric system) was during the initiation phase of diapause (early diapause) (see Kostál, characterised by a continuously (uninterrupted) O2-compensating 2006; Belozerov, 2009). system (Kuusik, 1977; Kuusik et al., 1996; Tartes et al., 1999; Tartes Coulometric respirometry was combined with flow-through CO2 et al., 2002). This setup has also been described by Lighton (Lighton, respirometry. We suppose that the flutter events observed by 2008). This respirometer ensures continuous and adequate replacement coulometric O2 measurements can usefully be directly compared of consumed O2 with electrolytically produced O2. The insect itself with flow-through CO2 respirometry. plays an active role in this self-regulating system. The rates of O2 The main aim of the present investigation was to describe the production and O2 consumption by the insect are indicated on graphs –1 pattern of O2 convective uptakes and associated body movements as VO2 (ml�h ). The system also records transient changes in the rate in young pupae of P. brassicae. To achieve this, coulometric O2 of release of CO2. In our respirometer, we did not use the switching respirometry was combined with flow-through CO2 respirometry, electrodes of electrolysis; instead, the electrolysis current was directly and discrete O2 discrete uptake was simultaneously recorded connected with a photoelement. High sensitivity of the respirometer indirectly using an infrared (IR) actograph. to pressure changes in the respiration chamber was achieved by replacing the standard photodiode with the photosensitive element of MATERIALS AND METHODS a transistor (KT302A, Semitronics, Freeport, NY, USA), which has Insects a very small photosensitive area (approximately 0.5�mm2). In this way, For laboratory experiments, eggs of Pieris brassicae (Linnaeus the smallest movement in the meniscus of ethanol inside the U-shaped 1758) (second generation) were collected from cabbage fields near capillary was reflected as a signal on the recording trace (Fig.�1). The Tartu, Estonia (58°23�N, 26°41�E), during July and August 2009. electrolysis current depended on the intensity of the light falling on They were reared in a laboratory under short-day conditions the phototransistor. The ethanol meniscus in the glass capillary served (12�h:12�h light:dark) at 21±1°C and ambient air humidity (55–65% as a shutter to screen the photosensitive area from light. The relative humidity). The larvae that hatched from the eggs were fed electrochemical equivalent of O2 generation has been reported as –1 –1 on leaves cut from cabbage plants, which were replaced with a fresh 209.5��l�O2�mA �h (Taylor, 1977). This value was used to convert supply daily. After pupation, each pupa was placed in an Eppendorff tube and kept in laboratory conditions. For the experiments, twenty-five 2�week old (14±2�days) pupae were used. Each pupa was weighed to 0.1�mg with an analytical 11 11 balance before experimentation (Explorer Balances, max. 62�g; Ohaus Corporation, Nänikon, Switzerland). Pupal body mass ranged 5 2 5 + 12 V from 0.383 to 0.411�g (Table�1). During respiratory measurements, temperature and humidity conditions were recorded using a digital HygroClip probe (HygroPalm, Rotronic Company, Basserdorf, 12 10 13 Switzerland). All measurements were made at 21±1°C and ambient 5 1 5 air humidity (50–55% relative humidity). The Eppendorff tube with pupa was used as the insect chamber in the respiratory systems; this Pt avoided handling stress. The respiratory measurements of the first 9 30�min were discarded; recordings in both systems lasted at least 7 8

Table 1. Characteristics (mean ± s.d.) of the discontinuous gas Cu exchange cycle in 2�week old pupae of Pieris brassicae (N�25) in 3 4 the initiation phase of diapause

Body mass (g) 0.397±0.014 6 14 Metabolic rate –1 –1 VCO2 (ml�g �h ) 0.042±0.0057 Fig.�1. Design of the electrolytic continuously O2-compensating differential V (ml g–1 h–1) 0.051±0.0027 O2 � � volumetric-manometric closed-system respirometer. 1, Insect chamber with Discontinuous gas exchange cycle pupa; 2, empty insect chamber; 3, vessel with potassium hydroxide Frequency (mHz) 0.26±0.0002 solution; 4, electrolysis unit, CuSO solution with platinum (Pt), and copper Period (min) 58.25±11.72 4 (Cu) electrodes; 5, taps for switching from volumetric-manometric Small bursts of CO during flutter 2 respirometry to flow-through CO respirometry; 6, glass capillary half-filled Number 25.06±3.69 2 with ethanol; 7, light source; 8, photo transistor; 9, infrared (IR) emitter Frequency (mHz) 12.20±0.32 diode; 10, IR sensor diode; 11, connections to the flow-through Period (s) 90.75±13.19 respirometry system; 12, compensating vessel; 13, current amplifier; 14, Oxygen uptakes between two flutter bursts 7.49±2.63 microsyringe.

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75 2818 K. Jõgar and others

–1 the readings of the event recorder to O2 consumption values (�l�O2�h it records not only heartbeats but also all other abdominal –1 or �mol�O2�h ). contractions, including muscular ventilation. An IR-emitting diode The coulometric respirometer allowed simultaneous recording of was placed on one side of the respirometer chamber near the ventral O2 consumption, sudden O2 (air) uptake (known as PSV) by side of the abdomen, while an IR-sensitive diode (TSA6203, convection into the tracheae at microopenings of the spiracles, Mikrotechna, Prague, Czech Republic) was placed on the opposite discrete CO2 releases by bursts, abdominal pumping movements and side of the chamber (see Metspalu et al., 2001; Metspalu et al., 2002). heartbeat patterns (see Jõgar et al., 2004; Jõgar et al., 2007). Rapid The light from the IR diode (BP104, Mikrotechna) was modulated changes in pressure (lasting seconds) in the insect chamber, caused by contractions of the heart and skeletal muscles. The level of output by active body movements of the insect or other rapid events, were voltage reflected the vigour of the muscular contractions of the insect not compensated and led to corresponding rapid changes in the (Hetz et al., 1999). Sudden extensions (lengthening) of abdominal electrolysis current, reflected as spikes on recordings. Thus, our segments (PSV) are recorded as relatively long upward spikes coulometric respirometer also served as an activity detector. synchronous with microopenings of the spiracles (Figs�3, 4). Weak A rapid O2 convective uptake resulted in adequate air volume regular muscular contractions of the abdomen resulted in two-phased decrease in the insect chamber and the ethanolic meniscus shifted relatively short spikes on the recording traces we refer to as down by a fraction of a millimetre. As a result, more light fell on abdominal pulsations (Fig.�5). Regular, high-frequency, low- the sensitive area of the transistor and an upward signal was recorded amplitude signals were interpreted as heartbeats (Fig.�3). (Fig.�2). Downward signals indicated CO2 release by bursts (Tartes et al., 1999). The volume of air uptake was estimated by extracting Data acquisition and statistics air from the insect chamber with a microsyringe (1��l volume, Computerised data acquisition and analysis were performed using Agilent Technologies, Espoo, Finland). The calibration of convective DAS 1401 A/D hardware and TestPoint software (Keithley, air uptake is shown in Fig.�2. Metrabyte, Cleveland, OH, USA) with a sampling rate of 10�Hz. Four bipolar channels allowed simultaneous recording of four events. Flow-through CO2 respirometry Mean (±s.d.) standard metabolic rate was calculated automatically The infrared gas analyser or flow-through CO2 respirometer (Infralyt- using STATISTICA (version 8, StatSoft, Tulsa, OK, USA). 4, Saxon Junkalor GmbH, Dessau, Germany) was used to confirm Statistical comparisons were made with one-way ANOVA (analysis that the presumed CO2 signals, i.e. the downward spikes on the of variance). Significant ANOVAs were followed with the Fisher’s recording trace of the electrolytic respirometer, were actually due to least significant difference (LSD) test. The significance level was CO2 bursts, and to measure them quantitatively. The respirometer was set at P<0.05. calibrated at different flow rates by means of calibration gases (Trägergase, Saxon Junkalor GmbH) and with gas injection. An air RESULTS flow rate of 120�ml�min–1 was used. The insect chamber could be At the initiation phase of diapause (14±2�days old), P. brassicae switched either to the flow-through CO2 respirometer or to the pupae displayed DGCs lasting 40–70�min (Table�1), whereas the coulometric respirometer without disturbing the insect (Fig.�1). During duration of CO2 release by burst was 2–6�min (3.1±0.1). Recordings the measurements with coulometric respirometry, the empty by flow-through respirometry showed a typical pattern. After the respiration chamber served to determine the baseline of the O-phase CO2 emission had ceased, the C phase began, which was measurements. followed by the F phase with small bursts of CO2 release (Fig.�3). No true C phase was found by flow-through respirometry. Shortly IR actography after the end of the O phase, coulometry revealed convective O2 Both the coulometric (electrolytic) respirometer and the flow- uptake. During this time, CO2 release was suppressed by the inward- through respirometer were combined with an IR insect cardiograph directed PSV. This convective O2 uptake indicates an earlier (opto-cardiography); we refer to this as the IR actograph, because beginning of the F phase than detectable with the flow-through

0.40 1.3 * * 1.0 0.36 * *

) 0.24

–1 0.32 0.8 2 min 0.4 µl 0.28 ) 0.6 –1 0.18 0.24 0.2 µl 0.3 u de (V) u de (V) (ml h

0.20 2 0.12 0.2 0.16 0.1 µl –0.2 CO su mption (ml h V Amplit 0.12 Amplit

con –0.2 2 0.08 –0.7 0.06

O OC F 0.04 0 –1.2 0 –0.6 0 1 2 3 4 5 6 7 8 0 3 6 9 12 15 18 Time (min) Time (min)

Fig.�2. Calibration with different volumes of air extracted from the insect Fig.�3. Discontinuous gas exchange in P. brassicae pupa recorded with chamber by means of coulometric measurements (lower trace, three left CO2 respirometry. Note the discrete small bursts of the F phase (lower spikes). The other four spikes are caused by sudden oxygen uptake by trace). The upper trace shows a series of abdominal extensions or Pieris brassicae pupae, which are synchronous with abdominal extensions lengthening (upper spikes) due to microopening of the spiracles and O2 (asterisks) (upper trace, IR probe actograph). By these measurements, the uptakes (IR probe actograph); the abdominal movements during O phase volume of first oxygen uptake was 0.1��l and the second was 0.2��l. were identified as heartbeats.

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76 Oxygen convective uptakes 2819

Fig.�4. Typical pattern of convective O2 uptake by coulometric 0.12 * 1.00 respirometry recorded in P. brassicae pupa at the time that * * corresponded to the closed phase by the flow-through CO2 ) respirometer. The spikes are gradually shortened due to the pressure –1 0.10 * * 0.95 * * * rises inside the trachea. Asterisks (upper trace) indicate the extensions (lengthening) of abdominal segments. Arrow indicates a 0.08 0.90 CO2 release by miniburst. u de (V) 0.06 0.85 su mption (ml h Amplit 0.04 con 0.80 2 O 0.02 0.75

0 0.70 0 30 60 90 120 150 180 Time (s)

system. Thus the F phase lengthened on account of the C phase. At (Fig.�7B). In a few pupae (never group, N�6), CO2 bursts occurred each microopening of the spiracles and passive convective oxygen without active ventilation (CFO cycles) (Fig.�7C). Ventilating uptake event, signals of abdominal lengthening were recorded movements (amplitude 1–2�V) associated with CO2 bursts were (Figs�3, 4). Recordings of coulometric respirometry showed clear, visible externally as twisting abdominal movements. gradually shortened signals due to convective oxygen uptake Active ventilation during the bursts of CO2 showed individual (Fig.�4). Each of the two to three first microopenings and O2 uptakes variation in the vigour of contractions and their number (from one lasted less than 0.5�s. Oxygen convective uptakes during the to 15). In pupae with only one to five muscular (active) ventilating interburst period increased metabolic rate by 5–6% compared with movements accompanying the burst, as well as in those lacking the metabolic rate when these uptakes were absent. Flow-through active ventilation, a relatively low level of CO2 release was measurements also indicated abdominal lengthening concurrent with observed. In contrast, pupae with vigorously ventilated bursts the small bursts of CO2 during flutter (Fig.�5). showed a significantly higher level of CO2 release (Fig.�7A). Each Between two large CO2 bursts, a series of small bursts of CO2 burst lasted 3–6�min in the always group of pupae, but 2–2.5�min were recorded by coulometric respirometry (Fig.�6A). Each small in the occasionally and never groups. Statistical comparison of CO2 burst started with a brief uptake of air into the tracheae, recorded release frequency (ANOVA, F24.2�41.8, P>0.05) did not show a by the IR actograph as a sudden extension of the abdomen, significant difference. The energy cost of muscular ventilation during indirectly indicating air (O2) uptake (Fig.�5). Between two a burst was not studied. –1 consecutive small CO2 bursts, a series of air uptakes (miniature Abdominal two-phase regular contractions (5–7�min ) of low inspirations) were recorded, which we considered as ‘miniflutter’ amplitude (0.2–0.3�V) (referred to as abdominal pulsations) (Fig.�5, (Fig.�6A,B; Table�1). These uptakes were irregular with respect to Fig. 6B) occurred periodically; these were not visible externally. spike height and interval. During such miniflutters, no emissions In some pupae showing no active ventilation during CO2 release of CO2 were recorded. by bursts, very regular low amplitude (0.1–0.2�V) pulsations Simultaneous recording with the IR actograph during flow- (57–70�min–1) were recorded; these we interpreted as heartbeats through CO2 respirometry indicated that pupae differed in the type (Fig.�3). of body movement associated with the respiratory patterns of CO2 release. In some pupae, CO2 bursts were always concurrent with DISCUSSION abdominal ventilating movements (CFV cycles) (always group, N�9) Our results showed that, in the initiation phase of diapause, P. (Fig.�7A), whereas in others (occasionally group, N�10), only some brassicae pupae display relatively short DGCs (40–70�min), with CO2 bursts were concurrent with abdominal ventilating movements CO2 bursts lasting 2–6�min. This contrasts with earlier studies, using

Fig.�5. Three small discrete bursts of CO2 (lower trace) during flutter 1.05 0.16 * in a P. brassicae pupa, recorded with flow-through respirometry. The upper trace is a simultaneous recording from the IR probe actograph showing abdominal pulsations as two-phase signals (smaller spikes)

) 0.12 * * and abdominal extensions (spikes indicated by asterisks). At the –1 * 0.85 beginning of each small burst, abdominal extension (lengthening) * occurred due to microopening of the spiracles and air convective * u de (V) (ml h

2 uptake. 0.08 CO V 0.65 Amplit 0.04

0 0.45 0 1 2 3 4 Time (min)

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77 2820 K. Jõgar and others

0.08 Fig.�6. (A)�A discontinous gas exchange cycle in a P. brassicae A pupa recorded by coulometric respirometry, showing an interburst period with small O phases (small CO2 bursts). CO2 CO2 (B)�Detail of the shaded area in A. The lower trace (coulometric 0.06 respirometry) shows the convective uptake of air at the microopening (mini-flutter) of the spiracles between two small bursts of CO2, recorded in pupae of P. brassicae. The upper 0.04 trace is a simultaneous recording from the IR probe actograph showing abdominal pulsations (downward spikes) and sudden abdominal extension (lengthening) at each microopening of the spiracles (upward spikes indicated by asterisks). 0.02 ) –1

0 0 10 20 30 40 50 60 Time (min) 0.06 su mption (ml h B ** *

con * * 2 * 0.7 O * * * * * 0.04 u de (V) 0.3

0.02 CO2 Amplit CO2

0 –0.1 0 40 80 120 Time (s)

P. brassicae pupae more than 2�months old, which displayed longer were commonly observed to be irregular with respect to frequency DGCs (8–23�h) (Harak et al., 1999; Jõgar et al., 2004; Jõgar et al., and amplitude, if inferred from the CO2 release pattern (e.g. 2005) with CO2 bursts lasting 13–18�min (Harak et al., 1999; Kuusik Wobshall and Hetz, 2004). et al., 1980; Tartes et al., 1999). The young pupae we used with Our flow-through CO2 measurements showed no CO2 release for their short DGCs were convenient for studying flutter events. They a short time after the O phase. The coulometric respirometry and had a relatively high metabolic rate; in 2–3�month old pupae IR actographic recordings showed rapid and clear uptakes of air –1 –1 metabolic rate is at least two times lower (12–28�ml�O2�g �h ) shortly after the O phase, indicating the beginning of the F phase. (Kuusik 1977; Jõgar et al., 2004; Jõgar et al., 2005). An earlier study by Tartes et al. revealed that air convective uptakes Commonly, after the O phase, a period with no CO2 release occurs began immediately after the O phase (Tartes et al., 2002). Air (C phase); later, the CO2 level was marginally elevated (F phase) convective uptakes, shortly after large CO2 bursts, also occurred in (Chown et al., 2006). The present study revealed that in young P. old diapausing M. brassicae pupae (Jõgar et al., 2007). We suggest brassicae pupae, the DGC measured with flow-through respirometry that, at the beginning of the flutter, air uptakes were convective but was characterised by a C phase, at the end of which a series of O2 later were diffusive-convective. These results concur with the convective uptakes was found. Thus, the C phase in those pupae plethysmometry flow-through measurements of Wobschall and Hetz was not as closed as previously thought. Between two large CO2 (Wobschall and Hetz, 2004), revealing that the convective uptakes bursts, almost regular small CO2 bursts were recorded. Each small of O2 dominate at the beginning of the flutter phase but that, in the burst started with sudden uptake of air into the trachea (PSV). The later F phase, diffusion takes over from convection as the chief main finding in the present study was a series of irregular mechanism of O2 uptake. Wobschall and Hetz showed that, in microopenings of the spiracle(s) with convective O2 uptakes (mini- diapausing moth pupae (A. atlas), uptake of air into the tracheal flutter) found between small bursts. During the mini-flutter, no system at the beginning of the F phase along the negative hydrostatic recordable CO2 emission occurred. pressure gradient may initially inhibit CO2 release from the tracheae There are several examples where the interburst period consists (Wobschall and Hetz, 2004). We suppose that, at the beginning of of discrete small CO2 bursts. Such bursts were described by Lighton the F phase of P. brassicae pupae, CO2 emission was also inhibited. (Lighton, 1988) in the tok-tok beetle, P. striatus; in this beetle, each One may suppose that water is conserved only at the beginning of burst was accompanied by active abdominal movement. Discrete the F phase when clear convective O2 uptakes (PSV) occur, but not CO2 emissions during the F phase have also been reported by later when diffusion is the dominating mechanism of the fluttering Duncan et al. in the tenebrionid beetle, Pimelia grandis (Duncan et period. al., 2002), and by Kovac et al. in resting honeybees Apis mellifera The duration of the F phase may be underestimated, as the F (Kovac et al., 2007). However, spiracle openings within the F phase phase may start before the CO2 measurements can detect it (Hetz

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78 Oxygen convective uptakes 2821

0.9 3 1985). In our measurements in P. brassicae pupae, each miniature A inspiration was synchronised with rapid extension of the abdomen, 0.8 2 confirming that a convective component was always present in O2 0.7 1 uptakes. We observed in P. brassicae a relatively longer flutter phase 0.6 compared with that in some other insects, such as the carabid 0 0.5 Pterostichus niger (Kivimägi et al., 2011) and the bumblebee –1 Bombus terrestris (Karise et al., 2010). 0.4 Gas exchange patterns are known to vary between and within 0.3 –2 individuals (see Chown, 2001; Chown et al., 2002; Marais and

0.2 –3 Chown, 2003). In the present study, variation was found between individuals in the duration of CO2 release and metabolic rates, but 0.1 –4 not in DGC frequency. Individuals displayed different gas exchange 0 cycles. Most showed DGCs with all CO2 bursts actively ventilated, a few with no bursts actively ventilated, and others with only some 0.9 3 bursts actively ventilated. B 0.8 2 Beside active ventilation, some other types of body movements, differing in frequency and amplitude, were observed in the present 0.7 1 study. The regular but periodically occurring abdominal pulsations 0.6 ) in P. brassicae pupae correspond, in our opinion, to the extracardiac

–1 0 0.5 hemocoelic pulsations described in lepidopteran pupae (Sláma,

u de (V) 1984; Sláma, 1999). These pulsations and other abdominal (ml h –1 2 0.4

CO movements play an important role in the regulation of pupal V 0.3 –2 Amplit respiration and haemolymph circulation (Sláma and Neven, 2001). In P. brassicae pupae, we interpreted high-frequency but low- 0.2 –3 amplitude signals, accompanied by CO2 release in bursts, as 0.1 –4 heartbeats. In a previous study using thermographic measurements, 0 we demonstrated heartbeat reversal, correlated with gas exchange cycles and twisting abdominal movements in diapausing P. 3 0.9 brassicae pupae (Jõgar et al., 2005). Heartbeat reversal correlated C with gas exchange cycles has also been reported in saturnid moth 0.8 2 pupae (Wasserthal, 1996; Hetz et al., 1999; Sláma, 2003). 0.7 1 Manometric O2 respirometry methods have been criticized and 0.6 their readings mistrusted because these methods usually do not allow 0 separation of active and resting metabolism (see Van Voorhies et 0.5 –1 al., 2008). Nevertheless, some volumetric manometric methods, 0.4 including our coulometric respirometry, are regarded as useful (Klok –2 0.3 and Chown, 2005). We are convinced that in gas exchange studies –3 of insects, coulometric respirometry supplemented by the flow- 0.2 through method has clear advantages. Lighton pointed out that 0.1 –4 coulometric continuously recording respirometry deserves to be 0 more widely used (Lighton, 2008). 0 1 2 3 In summary, in this study we have shown that the pattern of gas Time (h) exchange in P. brassicae pupae may be effectively investigated by Fig.�7. Examples of individual variation in early diapause pupae of P. the combined use of coulometric respirometry and flow-through CO2 brassicae in the types of gas exchange patterns taking in account visible systems by switching the same respiration chamber from one system body movements: (A) CO2 release by bursts accompanied by active to the other without disturbing the insect. By combining IR ventilation (twisting abdominal movements) (CFV cycles); (B) CO2 release actography in parallel with both types of respirometry, it was by bursts with only some accompanied by active ventilation; (C) CO2 possible to record rapid air uptakes. Thus, on our recording traces, release by bursts with no active ventilation (CFO cycles). The upper trace shows reccordings of IR probe actograph and the lower trace recordings of the patterns of microopenings of the spiracles were clearly indicated. flow-through respirometry. ACKNOWLEDGEMENTS The research was supported by the Estonian Science Foundation (grant nos 7130 and 6722) and Estonian target financing project no. SF170057s09. We thank the et al., 1994; Wobschall and Hetz, 2004). In pupae of P. brassicae, editor and two anonymous reviewers, who provided helpful and much-appreciated comments on earlier drafts of the manuscript. the duration of the F phase may also be longer than estimated by the flow-through system, as far as CO release was prevented at the 2 REFERENCES beginning of the F phase by convective air uptakes. Wobschall and Belozerov, V. N. (2009). New aspects in investigations of diapause and non-diapause Hetz showed that small volume and pressure decreases occurred dormancy types in insects and other arthropods. Entomol. Rev. 89, 127-136. between the microopenings in the F phase (Wobschall and Hetz, Chown, S. L. (2001). Physiological variation in insects: hierarchical levels and implications. J. Insect Physiol. 47, 649-660. 2004). This confirmed a small but significant contribution of Chown, S. L. and Nicolson, S. W. (2004). Insect Physiological Ecology: Mechanisms suction ventilation during each microopening (see also Kestler, and Patterns. Oxford: Oxford University Press.

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79 2822 K. Jõgar and others

Chown, S. L., Addo-Bediako, A. and Gaston, K. J. (2002). Physiological variation in indirect effects revealed in the respiratory metabolism in diapausing pupae of Pieris insects: large-scale patterns and their implications. Comp. Biochem. Physiol. 131B, brassicae (Lepidoptera, Pieridae). Acad. Sci. Estonian SSR 29, 198-211 (in Russian 587-602. with English summary). Chown, S. L., Gibbs, A. G., Hetz, S. K., Klok, C. J., Lighton, J. R. B. and Marais, Kuusik, A., Harak, M., Hiiesaar, K., Metspalu, L. and Tartes, U. (1996). Different E. (2006). Discontinuous gas exchange in insects: a clarification of hypotheses and types of external gas exchange found in pupae of greater wax moth Galleria approaches. Physiol. Biochem. Zool. 79, 333-343. mellonella (Lepidoptera: Pyralidae). Eur. J. Entomol. 93, 23-35. Duncan, F. D., Krasnov, B. and McMaster, M. (2002). Novel case of a tenebrionid Lighton, J. R. B. (1988). Simultaneous measurement of oxygen uptake and carbon beetle using discontinuous gas exchange cycle when dehydrated. Physiol. Entomol. dioxide emission during discontinuous ventilation in the tok-tok beetle, Psammodes 27, 79-83. striatus. J. Insect Physiol. 34, 361-367. Harak, M., Lamprecht, I., Kuusik, A., Hiiesaar, K., Metspalu, L. and Tartes, U. Lighton, J. R. B. (1994). Discontinuous ventilation in terrestrial insects. Physiol. Zool. (1999). Calorimetric investigations of insect metabolism and development under the 67, 142-162. influence of a toxic plant extract. Thermochim. Acta 333, 39-48. Lighton, J. R. B. (1996). Discontinuous gas exchange in insects. Annu. Rev. Entomol. Hetz, S. K., Wasserthal, L. T., Hermann, S., Kaden, H. and Oelssner, W. (1994). 41, 309-324. Direct oxygen measurements in the tracheal system of lepidopterous pupae using Lighton, J. R. B. (2008). Measuring Metabolic Rates. A Manual for Scientists, 201 pp. miniaturized amperometric sensors. Bioelectrochem. 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80 II Physiological Entomology (2011) 36, 62–67 DOI: 10.1111/j.1365-3032.2010.00768.x

Gas exchange patterns of Pterostichus niger (Carabidae) in dry and moist air

IRJAKIVIM AGI¨ 1,AAREKUUSIK 1,KATRINJ OGAR˜ 1, ANGELAPLOOMI 1,INGRIDH.WILLIAMS 1,LUULEMETSPALU 1, K ULLIHIIESAAR¨ 1,IVARSIBUL 2,MARIKAM AND¨ 1 and ANNELUIK 1 1Department of Plant Protection, Estonian University of Life Sciences, Tartu, Estonia and 2Department of Silviculture, Extonian University of Life Sciences, Tartu, Estonia

Abstract. Gas exchange patterns of adult male Pterostichus niger Schaller after hydration (i.e. given access to food and water) are compared in dry air [5–7% relative humidity (RH)] and moist air (90–97% RH) by means of ﬂow-through CO2 respirometry combined with infrared probe actography. Of thirty beetles examined, slightly more than 50% showed continuous gas exchange and are not considered further. Of the remaining beetles, the majority (approximately 71%) display a pattern of cyclic gas exchange in both dry and moist air (i.e. CO2 gas is released in bursts, with a low level of CO2 release during the interburst periods). A minority of the beetles (four out of 30) are found to exhibit discontinuous gas exchange in both dry and moist air; this is characterized by three clearly separated states of the spiracles: closed (C), ﬂutter (F) and open (O) phases. The pattern of cyclic gas exchange is associated with weak abdominal pulsations. After switching from moist to dry air, a small modulation of the discontinuous gas exchange cycles (maximum mean CO2 production rate) occurs, providing no clear support for the hygric theory of discontinuous gas exchange in this species (i.e. that it serves to restrict respiratory water loss). Key words. Carabidae, continuous respiration, cyclic gas exchange, discontinuous gas exchange, metabolic rate, Pterostichus niger.

Introduction F phase, without the help of muscular contraction; this is referred to as passive suction ventilation (Jogar˜ et al., 2004, At rest, many insects display cycles of discontinuous gas 2008). During the third or open (O) phase, CO2 is released exchange during which gaseous CO2 is released in bursts, in bursts, often supported by active ventilation (Slama,´ 1999; and uptake of O2 is often also cyclic (Kestler, 1985; Lighton, Sibul et al., 2004). The period between two bursts is known 1994, 1996; Chown & Nicolson, 2004; Hetz & Bradley, 2005). as the interburst period. Discontinuous gas exchange has three classically described Gibbs & Johnson (2004) describe cyclic gas exchange as a phases according to the state of the spiracles. During the pattern in insect gas exchange during which there are rhythmic constriction or closed (C) phase, no gas exchange occurs increases and decreases in CO2 release that go to zero. Accord- through the spiracles. The next, or flutter (F) phase, is ing to Marais et al. (2005), cyclic gas exchange is likely to be characterized by rapid opening and closing of the spiracles, or the ancestral pattern of respiratory gas exchange in insects at ‘fluttering’. During this phase, O2 enters the tracheal system of rest. the insect by bulk flow as a result of convection, by diffusion or Several hypotheses exist concerning the origins and function by a combination of convection and diffusion. In lepidopteran of discontinuous gas exchange (Chown et al., 2006). The pupae, air is sucked passively into the tracheae during the more widely discussed adaptative hypotheses are the hygric and oxidative damage theories, whereby modification of the Correspondence: Irja Kivimagi,¨ Department of Plant Protection, strongly periodic patterns of CO2 emission are considered to be Estonian University of Life Sciences, Kreutzwaldi 1, 51014, Estonia. adaptative changes to avoid excess water loss and/or oxidative Tel.: 372 7 313351; e-mail: [email protected] + damage (Hetz & Bradley, 2005; White et al., 2007; Terblanche © 2010 The Authors 62 Physiological Entomology © 2010 The Royal Entomological Society

82 Gas exchange of P. niger in dry and moist air 63 et al., 2008; Terblanche & Chown, 2010). None of the theories, relative humidity (RH)], and supplied with water and cat food however, can exclude the non-adaptative hypothesis (Chown (Friskies Junior 1; Purina, Nestle´ S.A., Switzerland) ad libi- & Holter, 2000). However, discontinuous gas exchange is tum. Under these laboratory conditions, the beetles survived reported for many adult insects, and may have originated well for several months, but were tested within 2 weeks of independently at least five times within the class Insecta collection. (Lighton, 1996; Marais et al., 2005; Chown et al., 2006; Thirty P. niger beetles were used in the experiments. Before Terblanche & Chown, 2010). Nevertheless, discontinuous gas testing, they were starved for 24 h to reduce variability associ- exchange may have been primarily a characteristic of the ated with specific dynamic action (Salvucci & Crafts-Brandner, respiratory system at rest (Chown & Holter, 2000) and then 2000; Bradley et al., 2003; Terblanche et al., 2005). During subsequently been selected for to reduce water loss or oxidative this starvation period, they were held in moist conditions (over damage (Chown & Nicolson, 2004; Terblanche et al., 2008; 80% RH) and given access to water. Preliminary experiments Terblanche & Chown, 2010). showed that the species is very susceptible to dry conditions Gas exchange patterns vary with metabolic rate (Contreras and cannot be dehydrated for longer than 15–20 h. & Bradley, 2009) and with the state of hydration of the Each test individual was weighed to the nearest 0.01 mg insect. To date, there are only a few studies concerning (OHAUS Digital Explorer Balances, Pine Brook, New Jer- a direct effect of environmental humidity on gas exchange sey). Only male individuals with a body mass of 0.20–0.22 g of insects. Slama´ et al. (2007) show a clear pattern of (mean SD, 0.21 0.03 g) were used in tests. Observations ± ± discontinuous gas exchange in the termite Prorhinotermes of the abdominal movements were made using a stereomicro- simplex in a dry respirometer chamber but, in a moist scope (SZ-ZTW; Olympus, Japan) after removal of the elytra chamber, the same species displays continuous gas exchange. from the beetles. Terblanche et al. (2008) show that, in pupae of the moth Test insects (30 individuals) were divided into two groups by random selection. In one group (n 15), environmental con- Samia cynthia, discontinuous gas exchange is not abandoned = in conditions of high atmospheric O2, or in high or low ditions were changed from dry to moist air, and, in the other group (n 15), from moist to dry air. Each test beetle was gas moisture levels, thus supporting the oxidative damage = hypothesis. placed in the respiratory chamber and left undisturbed for 1 h In this present study, respiratory gas exchange is examined before the start of the measurement period, in the humidity con- in adults of the carabid beetle Pterostichus niger Schaller under ditions that would be used in the test. Tests with each individual different moisture conditions. The species is eurytopic (i.e. able were made on the same day. Each recording lasted at least 3 h to withstand a wide range of environmental conditions) and in dry or moist conditions. Of the 30 beetles, 16 showed con- is common in a variety of open habitats, occurring also in tinuous respiration and were excluded from further analysis. almost every type of forest community (Witzke, 1976; Sustek,ˇ 1994). It shows a preference for deciduous and mixed stands Flow-through CO respirometry of trees on humus-rich, rather moist soil, although it is found 2 in hedgerows and on upland heaths. The adult beetles are An infrared differential gas analyzer, comprising a flow- moderately hygrophilous, and are usually active at night, hiding through respirometer (Imfralyt-4; Saxon Junkalor GmbH, under stones, loose bark and under the soil surface during the Germany) that is adapted for entomological research, was day (Sustek,ˇ 1994). used to measure rates of CO production (Lighton, 2008). Some predictions are made with respect to the relative 2 The gas analyzer was calibrated at different flow rates using importance to P. niger of the different adaptive theories of calibration gases (Tragergase;¨ Saxon Junkalor GmbH), with respiratory gas exchange. If discontinuous gas exchange has gas injection (Kuusik et al., 2002). When the respirometry evolved for water conservation (the hygric hypothesis), then was carried out in dry air, the insect chamber (volume 3 mL) it is expected that it would be used in conditions of low was perfused with dry (5–7% RH), CO2-free air [produced humidity to restrict water loss and would cease when humidity by passing air over Drierite (W. A. Hammond Drierite Co. is high. By contrast, the oxidative damage hypothesis predicts Ltd, Xenia, Ohio) and soda-lime granules] at a flow rate of that humidity conditions should have little or no effect on the 1 3 mL s− . Moist air (90–97% RH) was produced by bubbling prevalence or extent of discontinuous gas exchange. the air flow through a wash bottle of distilled water. Baseline drift of the analyzer was corrected during analysis from the measurements at the beginning and end of each trial with the Materials and methods respirometer chamber empty (Gray & Bradley, 1996; Duncan, 2003; Duncan & Byrne, 2005). Standard metabolic rates were Insects calculated as the mean CO2 production rates (VCO2) over three to ten complete cycles of continuous or discontinuous Experiments were carried out with adults of the carabid bee- gas exchange. tle P. niger (Coleoptera: Chrysomelidae) that were collected The temperature and humidity in the insect chamber were from their natural hibernation sites, comprising tree stumps measured continuously with a digital thermometer-hygrometer (humid environment) in the vicinity of Tartu, Estonia, in 2008 (HygroPalm Humidity temperature Indicator; Rotronic, U.K.). and 2009. The beetles were housed in 5-L bins half-filled with The respiratory measurements were made at 20 0.5 ◦C in ± soil, maintained at room temperature [20–23 ◦C over 80% a heating–cooling thermostat.

83 64 I. Kivim¨agi et al.

Infrared probe actography

The respirometry was combined with an infrared-optical device commonly known as an insect infrared cardiograph or optocardiograph (Hetz, 1994; Hetz et al., 1999). Two emitting diodes (TSA6203) were placed on one side of the insect chamber and two infrared sensor diodes (BP104) were placed on the opposite side. The light infrared-diode was modulated by the abdominal contractions. The level of output voltage reﬂected the vigour of the muscular contractions of the insect.

Data acquisition and statistical analysis

Computerized data acquisition and analysis were performed using an analog-to-digital converter and testpoint software with a sampling rate of 10 Hz (DAS 1401; Keithley- Metrabyte, Keithley Instruments Inc., Cleveland, Ohio). The mean metabolic rate was calculated automatically, by averaging data over a period involving at least eight cycles of gas exchange. Tests were performed using the statistics package statsoft, version 5.6, (StatSoft, Inc., Tulsa, Oklahoma). Data are shown as the mean SD with sample sizes and were compared using analysis of± variance (anova), Wilcoxon matched pairs test, Student’s t-test and chi-squared test. Means of gas exchange patterns for each beetle in dry and moist conditions were compared using a Wilcoxon matched pairs test. One-way anova was used to analyze different gas exchange patterns Fig. 1. An example of a pattern of discontinuous gas exchange in of test beetles in dry and moist conditions. Significant anovas adult Pterostichus niger (weighing 0.21 g) in moist air (A) and dry were analyzed further using Fisher’s least significant difference air (B) recorded by flow through respirometry (lower trace, VCO2), test. P < 0.05 was considered statistically significant. simultaneously by infrared probe actography (upper trace).

Results

In the current experiment, ten beetles (four moist to dry, six dry to moist) displayed cyclic gas exchange and four beetles (three moist to dry, one dry to moist) showed discontinuous gas exchange.

Discontinuous gas exchange

A minority (n 4) of the P. niger that were tested showed a clear pattern of= discontinuous gas exchange in dry air, as well as in moist air. The pattern of gas exchange in these four individuals was characterized by a closed (C) phase, when CO2 emission went to zero or close to zero, and a flutter phase Fig. 2. Pattern of discontinuous gas exchange of Pterostichus niger (F) (Figs 1A, B and 2). The open (O) phase of the discontin- (0.22 g) recorded in moist air at higher resolution. Closed (C), flutter uous gas exchange was associated with vigorous and regular (F) and open (O V) phases are clearly separated. Note that the bursts = pumping movements of the abdomen i.e. with active venti- of CO2 release are accompanied by ventilation movements (horizontal lation (Figs 1A, B and 2). This pattern of discontinuous gas bar; upper trace, measured by infrared actography, V). exchange was recognized as constriction–flutter–ventilation cycles. The duration of a ventilation period contributed approximately 60–80% of the total length of a cycle. The peak of Cyclic gas exchange the CO2 burst lasted only 1–2 min, after which the level of A cyclic gas exchange pattern was observed in ten of CO2 emission remained high for a longer time. The maximum 1 the 14 beetles at rest. The bursts of CO release during VCO2 (mean SD) was 0.58 0.039 mL h− and 0.48 2 ± 1 ± cyclic gas exchange were associated with weak abdominal 0.028 mL h− in moist and dry conditions, respectively ±(t 6.82, d.f. 3, P < 0.05; Student’s t-test). movements (Fig. 3). Very weak movements of the abdominal = = © 2010 The Authors Physiological Entomology © 2010 The Royal Entomological Society, Physiological Entomology, 36, 62–67

84 Gas exchange of P. niger in dry and moist air 65

Discussion

Under the experimental conditions examined, the respiration pattern, as well the type of body movement, varies between individuals of P. niger. Only a few P. niger show a true pattern of discontinuous gas exchange, with distinct closed (C) and flutter (F) phases. In individuals with cyclic gas exchange, no clear C and F phases are found. Bursts of CO2 release during cyclic gas exchange are associated with weak abdominal contractions (i.e. pulsations). In individuals showing discontinuous gas exchange, vigorous abdominal contractions (i.e. pumping movements) accompany each burst of CO2 expulsion. The metabolic rate of individuals with cyclic gas exchange is higher than in individuals with discontinuous gas exchange. Gibbs Fig. 3. An example of cyclic gas exchange in Pterostichus niger & Johnson (2004) show that metabolic rate varies with gas (0.21 g). Release of CO2 by bursts are accompanied by abdominal pulsations recorded with flow-through respirometry (lower trace) and exchange patterns, being lowest in individuals using discon- in parallel with an infrared probe actograph (upper trace, V). Two tinuous gas exchange, intermediate for individuals using cyclic activity periods are indicated by asterisks. The arrow separates the gas exchange and highest for individuals using continuous gas measurements in dry air (left) and moist air (right). exchange. Long burst periods (B phase) are recorded from individuals exhibiting both discontinuous and cyclic gas exchange patterns. tergites were designated as abdominal pulsations. These very The long burst phase is also described in dung beetles (Davis regular movements were recorded by the infrared probe et al., 1999; Duncan & Byrne, 2002; Duncan et al., 2009). actograph, although they were barely visible by eye, even A common pattern of gas exchange in P. niger is cyclic gas under a stereomicroscope. A characteristic feature of this exchange associated with very weak and uniform body move- cyclic gas exchange was a ‘long burst’ period, which started ments or abdominal pulsations, and these pulsations stop for with a short peak of CO2 release, lasting 3–5 min (4.2 just a short time before the next burst. This pattern without 0.5 min). After the CO2 emission peak, a very even clear C and F phases does not differ between dry and moist ± level of CO2 emission was recorded. The long burst period air. The overall pattern differs from the intracyclic activity in was followed by a short interburst period, during which the study by Kestler (1991), and also differs from the inter- the pulsations stopped for a short time (3–5 min), until the burst activity in the cockroach Perispaeria spp. (Marais & next burst of CO2 release (Fig. 3). When the pulsations Chown, 2003) and the bumble bee Bombus terrestris (Karise stopped, CO2 release decreased abruptly but never reached et al., 2009). baseline. After switching from dry to moist air flow in the Pterostichus niger has a mode of breathing that is different insect chamber, the general pattern of cyclic gas exchange from another member of the same genus, Pterostichus stygicus. did not alter, although short periods of activity appeared Pterostichus stygicus shows rapid cycles of collapse and then (1–2 per h), in which abdominal pulsations were coincident reinflation of the tracheal tubes that are synchronous with with the CO2 bursts (Fig. 3). The standard metabolic rate convective expulsion of CO2, although larger bursts of CO2 was higher in the beetles displaying cyclic gas exchange than release, which are characteristic of discontinuous and cyclic in individuals showing discontinuous gas exchange (anova: gas exchange, are absent (Socha et al., 2008). F1.13 82.28 in dry air; F1.13 58.6 in moist air, P < Because bursts of CO2 release are observed for P. niger 0.001;= Fisher’s least significant difference= test P < 0.0001; in both dry and humid air, the hygric hypothesis for the Table 1). However, statistical comparison using merely a origin of discontinuous gas exchange is not supported, at frequency test of ratios (χ2 test) showed no significant least in this species. Multiple gas conditions are not tested difference between tested groups (χ2 1.40, P 0.2367, simultaneously in the current experiments; therefore, there is d.f. 1). = = no evidence to support or challenge the alternative oxidative =

Table 1. Summary statistics and results from paired tests of gas exchange in Pterostichus niger in dry and moist air (mean SD) and metabolic rate ± between different gas exchange patterns: discontinuous gas exchange (DGE) and cyclic gas exchange (CGE).

n Dry air Moist air t P

1 b b DGE VCO2 (mL h− ) (SMR) 4 0.07 0.0056 0.07 0.0063 0.0344 0.970 1 ± ± DGE burst (h− ) 4.00 0.17 4.00 0.15 0.0229 0.983 1 ± a ± a − CGE VCO2 (mL h ) (SMR) 10 0.11 0.012 0.11 0.017 0.3455 0.752 − ± ± − CGE burst (h 1) 7.50 0.64 7.50 0.72 0.1031 0.920 − ± ± Vertical comparison was performed by one-way analysis of variance (different superscript letters indicate statistically signiﬁcant differences, P < 0.05) and horizontal comparison by Wilcoxon matched pairs test. SMR, standard metabolic rate.

85 66 I. Kivimägi et al. damage theory. Until recently, discontinuous gas exchange Duncan, F.D., Forster,¨ T.D. & Hetz, S.K. (2009) Pump out the has been considered as the main mechanism for conserving volume – the effect of tracheal and subelytral pressure pulses on water in several species of insect (White et al., 2007; Lighton, convective gas exchange in a dung beetle, Circellium bacchus 2007; Nespolo et al., 2007). However, cyclic gas exchange (Fabricus). Journal of Insect Physiology, 56, 551–558. Gibbs, A.G. & Johnson, R.A. (2004) The role of discontinuous gas may also help to conserve water (Gibbs et al., 2003). Insects exchange in insects: the chthonic hypothesis does not hold water. may alter their gas exchange patterns to cope with changes in Journal of Experimental Biology, 207, 3477–3482. atmospheric moisture over longer time scales. For example, Gibbs, A.G., Fukuzato, F. & Matzkin, L.M. (2003) Evolution of water Drosophila sp. selected for desiccation resistance show an conservation mechanisms in Drosophila. Journal of Experimental increased presence of cyclic gas exchange (Gibbs et al., 2003). Biology, 206, 1183–1192. However, it needs to be considered that discontinuous gas Gray, E.M. & Bradley, T.J. (1996) Evidence from mosquitoes suggests exchange may have originated in insects living in hypercapnic that cyclic gas exchange and discontinuous gas exchange are two manifestations of a single respiratory pattern. Journal of (high CO2) and hypoxic conditions to aid in the release of CO2 (Chthonic hypothesis; Lighton, 1996). Experimental Biology, 209, 1603–1611. In summary, despite showing that P. niger may exhibit Hetz, S.K. (1994) Untersuchungen zu Atmungs, Kreislauf und Säure- Basen-Regulation an Puppen der tropischen Schmetterlingsgattun- either cyclic or discontinuous gas exchange in both moist and gen Ornithoptera, Troides und Attacus. Dissertation, Friedrich- dry air, there is insufficient support for the oxidative damage Alexander-Universitat,¨ Erlangen-Nurnberg.¨ theory based on the data obtained in the present study because Hetz, S.K. & Bradley, T.J. (2005) Insects breathe discontinuously to only a limited number of individuals are examined, and no avoid oxygen toxicity. Nature, 433, 516–519. testing is made under multiple gas conditions. Hetz, S.K., Psota, E. & Wasserthal, L.T. (1999) Roles of aorta, ostia and tracheae in heartbeat and respiratory gas exchange in pupae of Troides rhadamantus Staudinger 1888 and Ornithoptera priamus L. 1758 (Lepidoptera, Papilionidae). International Journal of Insect Acknowledgements Morphology, 28, 131–144. Jogar,˜ K., Kuusik, A., Metspalu, L. et al. (2004) The relations between The authors are grateful to Robert Weaver and the anonymous the patterns of gas exchange and water loss in diapausing pupae referees for their critical and valuable comments on earlier of large white butterfly Pieris brassicae (Lepidoptera: Pieridae). drafts of this manuscript. The research was supported by European Journal of Entomology, 101, 467–472. the Estonian Science Foundation (grant numbers 7130, 6722, Jogar,˜ K., Kuusik, A., Metspalu, L. et al. (2008) Effects of Neem EC 6781 and 7391) and Estonian target financing project number on gas exchange, Tracheal ventilation, and water loss in diapausing SF170057s09. pupae of Pieris brassicae. Entomologia Experimentalis et Applicata, 2, 165–173. Karise, R., Kuusik, A., Mand,¨ M. et al. (2009) Gas exchange patterns of bumble bee foragers before and after exposing to lowered References temperature. Journal of Insect Physiology, 56, 529–535. Kestler, P. (1985) Respiration and respiratory water loss. Environmen- Bradley, T., Brethorst, L., Robinson, S. & Hetz, S.K. (2003) Changes tal Physiology and Biochemistry of Insects (ed. by K.H. Hoffman), in the rate of CO2 release following feeding in the insect Rhodnius pp. 137–281. Springer, Germany. prolixus. Physiological and Biochemical Zoology, 76, 302–309. Kestler, P. (1991) Cyclic CO2 release as a physiological stress Chown, S.L. & Holter, P. (2000) Discontinuous gas exchange cycles indicator in insects. Comparative Biochemical Physiology, 100C, in Aphodius fossor (Scarabaeidae) a test of hypotheses concerning 207–211. origins and mechanisms. Journal of Experimental Biology, 203, Kuusik, A., Martin, A.-J., Mand,¨ M. et al. (2002) Interrelations of gas 397–403. exchange cycles, body movements and heartbeats in the foragers Chown, S.L. & Nicolson, S.W. (2004) Insect Physiological Ecology: of bumblebee Bombus terrestris (Hymenoptera: Apidae) at low Mechanisms and Patterns. Oxford University Press, U.K. temperatures. European Journal of Entomology, 99, 209–214. Chown, S.L., Gibbs, A.G., Hetz, S.K. et al. (2006) Discontinuous gas Lighton, J.R.B. (1994) Discontinuous ventilation in terrestrial insects. exchange in insects: a clarification of hypotheses and approaches. Physiological Zoology, 67, 142–162. Physiological and Biochemical Zoology, 79, 333–343. Lighton, J.R.B. (1996) Discontinuous gas exchange in insects. Annual Contreras, H.L. & Bradley, T.J. (2009) Metabolic rate controls res- Review of Entomology, 41, 309–324. piratory pattern in insects. Journal of Experimental Biology, 212, Lighton, J.R.B. (2007) Why insects evolved discontinuous gas 424–428. exchange. Current Biology, 17, 645–647. Davis, A.L.V., Chown, S.L. & Scholtz, C.H. (1999) Discontinuous gas Lighton, J.R.B. (2008) Measuring Metabolic Rates: A Manual for exchange cycles in Scarabaeus dung beetles (Coleoptera: Scarabaei- Scientists. Oxford University Press, New York, New York. dae): mass scaling and temperature dependence. Physiological and Marais, E. & Chown, S.L. (2003) Repeatability of standard metabolic Biochemical Zoology, 72, 555–565. rate and gas exchange characteristics in a highly variable cockroach, Duncan, F.D. (2003) The role of the subelytral cavity in respiration Perisphaeria sp. Journal of Experimental Biology, 206, 4565–4574. in a tenebrionid beetle, Onymacris multistriata (Tenebrionidae: Marais, E., Klock, C.J., Terblanche, J.S. & Chown, L. (2005) Insect Adesmiini). Journal of Insect Physiology, 49, 339–346. gas exchange patterns: a phylogenetic perspective. Journal of Duncan, F.D. & Byrne, M.J. (2002) Respiratory airflow in a wingless Experimental Biology, 208, 4495–4507. dung beetle. Journal of Experimental Biology, 205, 2489–2497. Nespolo, R.F., Artacho, P. & Castaneda,˜ L.E. (2007) Cyclic gas- Duncan, F.D. & Byrne, M.J. (2005) The role of the mesothoracic exchange in the Chilean red cricket: inter-individual variation spiracles in respiration in flighted and flightless dung beetles. and thermal dependence. Journal of Experimental Biology, 210, Journal of Experimental Biology, 208, 907–914. 668–675.

86 Gas exchange of P. niger in dry and moist air 67

Salvucci, M.E. & Crafts-Brandner, S.J. (2000) Effects of temperature Terblanche, J.S. & Chown, S.L. (2010) Effects of flow rate and and dietary sucrose concentration on respiration in the silverleaf temperature on cyclic gas exchange in tsetse flies (Diptera, whitefly, Bemisia argentifolii. Journal of Insect Physiology, 46, Glossinidae). Journal of Insect Physiology, 56, 513–521. 1461–1467. Terblanche, J.S., Klok, C.J. & Chown, S.L. (2005) Temperature- Sibul, I., Kuusik, A. & Voolma, K. (2004) Patterns in abdominal dependence of metabolic rate in Glossina morsitans morsitans pumping, miniature inspirations and heartbeats simultaneously (Diptera, Glossinidae) does not vary with gender, age, feeding, preg- recorded during cyclical gas exchange in adult Hylobius abietis nancy or acclimation. Journal of Insect Physiology, 51, 861–870. (Coleoptera: Curculionidae) using a respirometer and IR actographs. Terblanche, J.S., Marais, E., Hetz, S.K. & Chown, S.L. (2008) Control European Journal of Entomology, 101, 219–225. of discontinuous gas exchange in Samia cynthia: effects of Slama,´ K. (1999) Active regulation of insect respiration. Annals of the atmospheric oxygen, carbon dioxide and moisture. The Journal of Entomological Society of America, 92, 916–929. Experimental Biology, 211, 3272–3280. Slama,´ K., Sobotnik, J. & Hanus, R. (2007) Respiratory concerts White, C.R., Blackburn, T.M., Terblanche, J.S. et al. (2007) Evolu- revealed by scanning microrespirography in a termite Prorhinoter- tionary responses of discontinuous gas exchange in insects. Pro- mes simplex (Isoptera: Rhinotermitidae). Journal of Insect Physiol- ceedings of the National Academy of Sciences of the United States ogy, 53, 295–311. of America, 104, 8357–8361. Socha, J.J., Lee, W.-K., Harrison, J.F. et al. (2008) Correlated patterns Witzke, G. (1976) Beitrag zur Kenntnis der Biologie und Okologie¨ of tracheal compression and convective gas exchange in a carabid des Laufkafers¨ Pterostichus (Platysma) niger, Schaller 1783 beetle. Journal of Experimental Biology, 211, 3409–3420. (Coleoptera, Carabidae). Zeitschrift für Angewandte Zoologie, 2, Sustek,ˇ Z. (1994) Classification of the carabid assemblages in the 145–162. floodplain forests in Moravia and Slovakia. Carabid Beetles: Ecology and Evolution (ed. by K. Desender et al.), pp. 371–376. Accepted 21 October 2010 Kluwer, The Netherlands. First published online 7 December 2010

III Agronomy Research 7(Special issue I), 328–334, 2009

Physiology of a carabid beetle Platynus assimilis

I. Kivimägi1, A. Ploomi1, L. Metspalu1, E. Svilponis1, K. Jõgar1, K. Hiiesaar1, A. Luik1, I. Sibul2 and A. Kuusik1

1Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia; e-mail: [email protected] 2Institute of Forestry and Rural Engineering, Estonian University of Life Sciences, Kreutzwaldi 5, Tartu 51014, Estonia

Abstract. Predacious carabid beetle Platynus assimilis Paykull (Coleoptera: Carabidae) is a fast-moving insect which should be considered as an important component of biological control in organic farming. In this study we tested some factors of potentially dangerous influence of pesticide Fastac (synthetic pyrethroid) on overwintering physiology of adult ground beetle under laboratory conditions. Cold-hardiness (measured by supercooling point SCP) was determined 2 weeks after exposure to pesticide treated-food. Pesticide had decreased cold- hardiness of the ground beetles. Weak supercooling capacity could be harmful to overwintering insects in cold winters without snow cover.

Key words: Platynus assimilis, overwintering, respiration, alpha-cypermethrin

INTRODUCTION

Carabid beetles (Coleoptera: Carabidae) are species rich and abundant in arable habitats. Polyphagous carabid adults and larvae are important natural pest-control agents known to feed also on agricultural pests. Their food list contains a wide range of aphids, dipteran eggs, larvae and pupae, eggs and larval stages of the Colorado potato beetle Leptinotarsa decemlineata, slugs (Kromp, 1999). Carabids react sensitively to anthropogenic changes in habitat quality and are also affected by intensive agricultural cultivation. They can be affected by deep ploughing as well as by crop treatment with pesticides (Kromp, 1999). In organic farms it isn’t allowed to use pesticides, but land- use in neighbouring conventional farms presents a probable risk. Carabid beetles may feed on chemically treated plants or pests after which they can move relatively fast to the organic fields because beetles long legs allow them to walk or run quickly on the soil surface (Kromp, 1999). So, the pesticide applications in conventional farms may cause ecological damage also in organic farms by killing beneficial organisms. Pyretroids are contact insecticides that have a broad and a long-lasting effect and are poisonous for almost all pollinating insects (Barten at al., 2006). Pyrethroids may bioconcentrate through the food web (Solomon et al., 2001). Pyrethroid Fastac (active substance alpha-cypermethrin) is a widely used insecticide in Estonian agriculture. The chemical compound is a structural analog of chrysanthemum plant pyrethrins, which are permitted for use in organic farming (Coats et al., 1989). This pesticide is highly toxic to insects and aquatic organisms (Muller-Beilschmith, 1990; Solomon et al.,

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90 2001; Karise, 2007) but have relatively low toxicity to terrestrial vertebrates (Solomon et al., 2001; Yarkov et al., 2003). The influence of pyrethroids on carabids physiology (inclusively overwintering physiology) has not been studied yet. Adults of the ground beetle Platynus assimilis (Paykull), is a night-active on soil surface dwelling beetle that is a well known common predator in agricultural fields (Thiele, 1977; Lövei & Sunderland, 1996), were chosen for test object in our experiment. The aim of current investigation was to assess the influence of Fastac on adult of P. assimilis under laboratory conditions. The poisoning effect assessment criteria were cold-hardiness evaluated by supercooling point (SCP), weight and respiration.

MATERIALS AND METHODS

Insects. Adults of P. assimilis were collected from the Tartu County, Estonia, in their hibernating sites – tree stumps. The tests were carried out in January 2009. Chemical substance. Fastac 50 is a commercial formulation of alpha- cypermethrin (a.i. 50g l-1). In the study, 0.15%, dose was used according to the recommendations for field spraying. Laboratory tests. Test material collected from hibernating sites (52 individuals in total) was separated into two parts: treatment (24 individuals: 13 female and 11 male beetles) and control (28 individuals: 13 female and 15 male beetles). Beetles were weighed twice: before the treatment and at the end of study (14th day). Alpha- cypermethrin was diluted in distilled water by dose recommendations. The beetles were fed with cat food (Friskies Junior-1 Purina) every fourth day. Feeding the laboratory-reared ground beetles by cat food is suggested also by Tréfás et al. (2001) and Ploomi (2004). For the insecticide exposure, food pieces were dipped in the emulsion of alpha-cypermethrin (treated) or in the distilled water (control group) for 10 seconds. The experiment lasted 14 days, while individual beetles were kept in plastic boxes (0.5 l) on wet tissue at room temperature (221°C). Respiration rate of P. assimilis was measured on the first day before treatment and 2 days after the last feeding (15th day after the onset of the experiment). An infrared gas analyser or IRGA (Infralyt-4. VEB, Junkalor, Dessau) adapted for entomological research (Kuusik et al. 2001, 2004; Metspalu et al. 2001) was used to record the bursts of carbon dioxide release. By means of IRGA it has been proven, that the presumed CO2 signals, i.e. the downward peaks on the recording of the electrolytic respirometer, were actually due to CO2 bursts, and the instrument was used to measure the respiration level quantitatively. This flow-through respirometer was calibrated at different flow rates by means of calibration gases (Trägergase, VEB, Junkalor, Dessau), with gas injection. Air flow rate was commonly 3.6 l per h, by which the rate -1 of carbon dioxide release was measured (VCO2 ml h ). The flow-through respirometry combined with an infrared optical device referred to as infrared actograph has commonly been used’ as an insect IR cardiograph (Hetz et al. 1999, Kuusik et al. 2001) or optocardiographic method (Slama 2001). Two IR-emitting diodes (TSA6203) were placed on one side of the insect chamber, and two IR sensor diodes (BP104) were placed on the opposite side. The light from the IR-diode was modulated by the abdominal contractions. The level of output voltage reflected the vigour of the

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91 muscular contractions of the insect. CO2 releases and abdominal movements were measured on P. assimilis before and after the treatment. For measuring supercooling points (SCP) ground beetles were anesthezised by ether. The beetles were positioned so that its integument (thoracic tergit) was contacted with the copper-constantan thermocouple, placed in a glass vial, and then transferred to the circulator bath (Ministat 230w-2, Huber, –33°C to +200°C). SCP was determined using a 0.5 C min−1 cooling rate. The temperature was registered and saved by a data logger (Almemo 2890-9, Ahlborn). Data acquisition and statistics. Computerised data acquisition and analysis were performed using an analog-to-digital converter and TestPoint software with 10 Hz sampling rate (DAS 1401, Keithley-Metrabyte). The CO2 rate was automatically calculated by averaging data over a period involving at least 12 cycles of gas exchange. Means, standard error and standard deviations are reported. Tests were performed using a statistic package StatSoft ver.8, Inc/USA. Means were compared by Student’s t-test and Wilcoxon Matched Pairs Test.

RESULTS AND DISCUSSION

The results of the current study demonstrated the influence of the treatment by insecticide Fastac on the physiology of P. assimilis. Body mass. The measured body masses showed the feeding activity during the study period. If the mean of the body mass of the control group in the beginning of study was 0.038 ± 0.002 g, then in the end of the study it was 0.042 ± 0.003 g (Fig. 1). The body mass of the control group of the beetles increased significantly (P < 0.05). On the other hand, the average body mass of carabid beetles of the treated group in the end of the test remained on the same rate like it was in the beginning of the experiment (0.040 ± 0.002 g). 0,050

* n.s. 0,048

0,046

0,044

0,042

0,040 BODYMASS (g) 0,038

0,036 Mean 0,034 Mean±SE Mean±SD 0,032 A B C D Fig. 1. The influence of treatment on body mass of the ground beetles P. assimilis. A - the body mass of control group before feeding; B- the body mass of control group after feeding; C- the body mass of treatment before feeding; D- the body mass of treatment after feeding. Asterisk indicates the significant difference (Wilcoxon Matched Pairs Test, P < 0.05, n = 28 (on control group); n = 24 (on treated group).

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92 Although the voltage-sensitive sodium channel is likely to be the principal site of pyrethroid action, it’s probably not the only target for insect-selective neurotoxins. Insect neurosecretory neurons are sensitive to very low concentrations of pyrethroids, and disruption of the neuroendocrine system has been involved as a factor contributing to the irreversible effects of pyrethroids in insects (Soderlund & Bloomquist, 1989). All organisms generate energy from the food they eat. Alfa-cypermetrin probably inhibits or disrupts energy production: while the beetle can eat and digest food after being poisoned, it cannot produce energy from food. Eventually, the insect stops eating and sometimes even moving (Brown, 2006). Lack of the increase of body mass on treated group is also explained with transpiration water loss on toxicated insects (Kuusik et al., 1995). Respiration. P. assimilis respiration rate measured before (Fig. 2) and after the treatment (Fig. 3) demonstrate clearly the differences between treatments by alpha- cypermethrin. The simultaneous recording of the flow-through CO2 respirometer and infrared actograph showed in insects (Fig. 2) regular muscular contractions due to active continuous ventilation (upper trace) and the sinusoidal weak cyclic release of CO2 (lower trace) (lower trace) in the resting state before the treatment (Fig. 2). After the treatment by alpha-cypermethrin insects had greater mean rate of carbon dioxide outbursts (Fig. 3) and constant cyclic CO2.release was disappeared. Treatment with alpha-cypermethrin caused irregular muscular contractions of P. assimilis.The respirometry and IR actography revelated that adult P. assimilis has relatively high expenditure of metabolic energy during the the irregular muscular contractions. After the treatment with alfa-cypermetrin, a typical symptom of influence of this pyrethroid on carabide beetles was paralyse after 2–3 days. Pyrethroids are nerve poisons that effect nerve axon. The main target site is neuronal sodium channels and it increases sodium entry into the nerve cell and induces depolarization of the nerve membrane and blocks of nerve conduction. The normal function of the nervous system is affected, stimulating repetitive nerve discharges leading to paralysis. The paralysis is often preceded by spastic activity of the organism due to the hyper-activity of nerve endings. The spastic activity is caused by sodium channels repeatedly polarizing and depolarizing, mimicking neuro-transmission where none is actually taking place (Vijverberg & van den Bercken, 1990). Supercooling point (SCP). The study demonstrated that the mean SCP on treated group was higher than control group (see Fig. 4). Control group mean SCP was –5.5°C and treated group mean SCP was –5°C, which is statistically significant (Student t-test; t = -2.63253, df = 50; P < 0.05). The first toxic effect on the treatment group by neurotoxicants is the dehydration on their bodies. The neurotoxicant caused faster excretion of body fluids from digestive system or water transpiration which increases SCP (Kuusik et al., 1995). By comparing treated group versus control, insects in treated group displayed larger variance in SCP. Its shows that some insects from treated group are more vulnerable than others, for some reasons (illness, certain winter damages etc.). On the other hand, pyrethroids cause extensive damage to haemolymph (Saleem et al., 1998), which may increase the SCP. Pyrethroids can affect other biochemical changes, for instance level of glucose and trehalose (M’diaye & Bounias, 1993).

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0.5 3

2 0.375 )

-1 1

(ml h (ml 2 Volts

CO 0.25 0 V

-1 0.125

-2 0 1 2 3 4 5 Time (min)

Fig. 2. A simultaneous recording of infrared actograph (upper trace, Volts) and infrared gas analyser (lower trace) representing active and regular tracheal ventilation (upper trace) and weak sinusoidal curve due to cyclic release of CO2 in adults P. assimilis before treatment with alpha-cypermethrin.

0.5 4

0.375 2 ) -1

(ml h (ml 1 2 Volts

CO 0.25 V 0

-1 0.125

-2 1 2 5 0 3 4 Time (min)

Fig. 3. A simultaneous recording of infrared actograph (upper trace, Volts) and infrared gas analyser representing a continuous release of CO2 without cyclicity (lower trace) in adults P. assimilis after treatment with alpha-cypermethrin. Note that the irregular pattern of muscular contractions due to locomotor activity (upper trace).

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94 -4,0

-4,2 Mean Mean±SE -4,4 Mean±SD

-4,6

-4,8

-5,0 *

-5,2 Supercooling point -5,4

-5,6

-5,8

-6,0 Control Treatment Fig. 4. The influence of the treatment on supercooling points of P. assimilis. Asterisk indicates the significant difference (Student t-test; P < 0.05, n = 28 (on control group); n = 24 (on treated group).

CONCLUSIONS

Alpha-cypermethrin affected normal function of the nervous system and caused increasing metabolic rate. The carabid beetle respond to the treatment with alpha- cypermethrin, showing paralyze on the second and third day after the treatment. Reduced mobility means that the beetles are less able to migrate from pesticide-treated fields and thus are exposed to potentially lethal effects for longer periods. The respiration and transpiration systems are the vulnerable targets of the alpha- cypermethrin used on the exposed carabids.

ACKNOWLEDGEMENTS. This research was supported by the Estonian Science Foundation grants No. 7130, 6722, 6781 and Estonian Ministry of Education and Research targeted financing projects No. SF170057s09, SF0170014s08 and SF0170021s08.

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96 IV Eur. J. Entomol. 110(1): 000–000, 2013 http://www.eje.cz/pdfs/110/1/@ ISSN 1210-5759 (print), 1802-8829 (online)

Gas exchange patterns in Platynus assimilis (Coleoptera: Carabidae): Respiratory failure induced by a pyrethroid

1 1 1 1 1 1 IRJA KIVIMÄGI , AARE KUUSIK , ANGELA PLOOMI , LUULE METSPALU , KATRIN JÕGAR , INGRID H. WILLIAMS , 2 1 1 1 IVAR SIBUL , KÜLLI HIIESAAR , ANNE LUIK and MARIKA MÄND

1Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014, Estonia; e-mails: [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; [email protected]; marika.mä[email protected] 2Institute of Forestry and Rural Engineering, Estonian University of Life Sciences, Kreutzwaldi 5, Tartu 51014, Estonia; e-mail: [email protected]

Key words. Carabidae, Platynus assimilis, alpha-cypermethrin, resting metabolic rate, non-target, respiration

Abstract. Discontinuous gas exchange (DGE) is the main (23 individuals) breathing mode in resting adult Platynus assimilis. Few of the beetles tested (13 individuals) displayed a pattern of cyclic gas exchange or CGE. The burst of CO2 release in DGE and CGE was always accompanied by abdominal pumping (active ventilation or V). Seven individuals displayed a pattern of continuous respiration, characterized by regular abdominal pumping. Resting metabolic rate (RMR) in continuously breathing beetles was higher than in those using DGE and CGE. After treatment with sub-lethal doses of alpha-cypermethrin DGE ceased. Treated beetles were characterized by continuous pumping and almost regular periods of activity. RMR increased significantly after treatment with a pyrethroid.

INTRODUCTION the secondary consequences of this, rather than to any Carabid beetles (Coleoptera: Carabidae) are important direct cytotoxicity (Ray & Fry, 2006). Pyrethroids may polyphagous natural pest control agents in agricultural become more concentrated in organisms in the upper fields. They are sensitive to anthropogenic changes in parts of food webs (Solomon et al., 2001). habitat quality and affected by intensive agriculture. They Treatments with chemical pesticides never kill all the can be influenced by tillage as well as by the treatment of insects in a population and may induce poorly studied crops with pesticides (Kromp, 1990). On conventional sub-lethal and delayed effects in the survivors. Several farms, pesticides threaten the survival of non-target methods have been used to assess the effects of insecti- carabid beetles living in both treated and untreated areas, cides on the physiology of beetles. Sláma & Miller (1987) because carabids move relatively fast. They may contact used a hydraulic transducer to record the neurotoxic pesticides directly or feed on chemically-treated seeds effects of a pyrethroid on pupae of Tenebrio molitor. and pests (Kromp, 1999). Basedow (1987) considers pes- Zafeiridou & Theophilidis (2006) used a force displace- ticide application to be the main reason for the reduced ment transducer attached to the dorsal surface of the numbers of carabids in conventionally-farmed wheat second abdominal segment of T. molitor and to monitor fields. Pesticide use on conventional farms may also the respiratory contractions before and after sublethal poi- cause ecological damage to neighbouring organic farms soning with a pyrethroid. by killing carabid beetles. It is very important to determine the normal physio- Pyrethroids are currently the most commonly used logical state of an insect before studying pathological insecticides in the world (Horton, 2011) and alpha- effects. The physiological state of an insect is most often cypermethrin, the most widely used active ingredient. measured in terms of standard metabolic rate (SMR) or These pesticides are highly toxic to insects and aquatic resting metabolic rate (RMR). The effects of toxicants on organisms (Mueller-Beilschmitdh, 1990; Solomon et al., the SMR are well recorded (Keister & Buck, 1974). Gas 2001; Karise, 2007) but of relatively low toxicity to ter- exchange patterns are also used to characterize the restrial vertebrates (Solomon et al., 2001; Yarkov et al., physiological state of insects, but there are few studies on 2003). Pyrethroids are similarly toxic to both pests and the sublethal and delayed effects of insecticides. In dis- non-target organisms with the molecular targets in insects continuous gas exchange (DGE) CO2 is released periodi- analogous to those in mammals (Marrs & Ballantyne, cally in bursts. The classic DGE consists of three periods 2004). Pyrethroids are primarily toxins targeting the func- or phases, originally termed CFO based on the spiracular tioning of the nervous system (Narahashi et al., 1998). activity (Schneiderman, 1960): constriction (C), fluttering Thus, they owe their insecticidal potency to a rapid func- (F) and open (O) phases. During the C-phase, the spira- tional disruption of an insect’s neuromuscular system and cles are tightly closed and no gas exchange occurs, during

98 the F-phase the spiracular valves open and close rapidly The present study focused on the carabid beetle and finally, in the O-phase, CO2 is released in a burst Platynus assimilis, a mainly night-active, soil-surface- (Chown & Nicolson, 2004; Chown et al., 2006; Lighton, dwelling beetle, which is a common predator in agricul- 1996; Gibbs & Johnson, 2004; Marais et al., 2005). tural fields and forests. One aim of the present study was Oxygen uptake occurs in the F phase by the principle of to measure the resting metabolic rate (RMR) and to char- Passive Suction Ventilation (PSV), which reduces water acterize the gas exchange patterns in adult P. assimilis at loss (Kestler, 1980, 1982). The DGE is commonly termed rest. Another aim was to investigate the effect of suble- the CFO cycle when there is no evidence of active venti- thal doses of alpha-cypermethrin on the levels and pat- lation during the release of CO2. The term, CFV empha- terns of gas exchange in this beetle. sises muscular ventilation (V) during CO2 emission MATERIAL AND METHODS (Kestler, 1971, 1985). A specific strategy to reduce loss of water is the replacement of diffusive O-phases by con- Insects and treatments vective V-phases, wherever possible (Kestler, 2003). Adult beetles of P. assimilis were collected from hibernation In cyclic gas exchange (CGE) there are distinct bursts sites (tree stumps), in Tartu County, Estonia, in January 2010. and inter-burst periods and at least some CO2 production They were kept for one day in the laboratory prior to the experi- is detectable even during the inter-burst periods (Marais ment. Based on preliminary experiments temperature compensa- & Chown, 2003; Nespolo et al., 2007). Thus, during tion of respiration occurs each day at room temperature. These results were similar to earlier studies on temperature compensa- CGE, CO2 release does not decrease to zero or even close tion in several insects (Kuusik et al., 1995). For the experiments, to zero (Gray & Bradley, 2006). 43 beetles (40–42 mg) were selected and placed individually in There is no evidence of cyclic or discontinuous release Petri dishes. Experiments were performed at 22 ± 1°C in a ther- of CO2 during continuous respiration in which the spira- mostat. cles open and close continuously and asynchronously The respiration of the beetles was measured for at least 3 (Gibbs & Johnson, 2004; Terblanche & Chown, 2010). hours before and after treatment. During these measurements the Active individuals typically show continuous respiration temperature and humidity conditions were recorded by means of (Marais et al., 2005). HygroClip probes (HygroPalm, Rotronic Company). There are several hypotheses of the evolutionary origin Fastac 50 EC, which is a commercial formulation of alpha- cypermethrin (a.i. 50g l–1), was used in the experiments on bee- of discontinuous or cyclic release of CO2 (Chown et al., tles showing only DGE. One ml of the formulation (a 5% emul- 2006; Woods, 2011). According to older hypotheses the sion) was diluted in 100 ml distilled water (0.05%, field solution DGE originated as a water-saving mechanism (Schneider- of formulation) and different concentrations (0.01%, 0.001% man, 1960). However, DGE may have other useful func- and 0.005%) were prepared. Preliminary tests showed that con- tions. centrations of 0.05% and 0.01% were lethal for carabid beetles, The theory of a non-adaptive or mechanistic origin of so these concentrations were not used in this study. Thus, bee- DGE (Chown et al., 2006, 2011; Chown, 2011) must also tles were treated with concentrations of either 0.001% or be considered. The model proposed by Förster & Hetz 0.005%. Also, our preliminary studies revealed that topical applications to the thorax or abdomen using acetone as the sol- (2010) assumes that both CO2 and O2 are involved in the vent resulted strong toxic and metabolic delayed effects. For this regulation of spiracle behaviour. The CO2 partial pressure reason we the dipped the beetles into an aqueous emulsion of threshold for spiracle opening is sensed by the spiracle the pyrethoid for 10 s. Dipping is used as an alternative contact muscle and the O2 partial pressure threshold by the seg- method for bioassays of substances on several insect species mental ganglia of the central nervous system (Förster & (Van der Stern, 2001; Cetin et al., 2006; Wanyika et al., 2009). Hetz, 2010; Chown et al., 2011). Separate CO2 and O2 The beetles in the control were dipped in distilled water. systems as a non-adaptive explanation for DGE behaviour Respirometry does not contradict several adaptive explanations (Hetz & To achieve high resolution recordings of active ventilation we Bradley, 2005; Förster & Hetz, 2010; Chown et al., used coulometric respirometry. The coulometric respirometry 2011). The lengthening of the C-phase and shortening of was used to record pumping movements as the volumetric the O-phase restrict water loss (Schimpf et al., 2012). A method only records rhythmic changes in body external volume. very long C-phase, lasting nearly one day, is character- Coulometric respirometry istic of diapausing pupae of the cabbage white butterfly, Pieris brassicae (Jõgar et al., 2011). Coulometric respirometers (a volumetric-manometric system) are characterized by a continuously O2-compensating system Kestler (1991) reports sublethal effects of toxicants on (Kuusik, 1977; Kuusik et al., 1996; Vanatoa et al., 2006; Jõgar CFV cycles (DGE), which are transformed into a pattern et al., 2011). The principles of the coulometric respirometry of continuous respiration. Some early studies also report system we used are described by Lighton (2008). that the Colorado potato beetles, Leptinotarsa decem- In the present study, we used a modified and compact coulo- lineata (Kuusik et al., 2001a), Hylobius abietis (Sibul et metric respirometer (Fig. 1) suitable for small insects, including al., 2004) and pupae of P. brassicae (Harak et al., 1999; P. assimilis. In the insect chamber of the coulometric respiro- Jõgar et al., 2006, 2008) treated with toxic substances do meter the beetle could walk in a restricted area and typically not respire using DGE. Appel et al. (1997) report that remained stationary (resting) occasionally interrupted by short insects do not respire using DGE following treatment periods of walking (activity). with contact insecticides, which makes them more sus- This respirometer ensures continuous and adequate replacement of consumed O2 with electrolytically-produced O2. The ceptible to desiccation and possibly easier to control. insect itself plays an active role in this self-regulating system.

99 Fig. 1. Design of the continuous recording coulometric respirometer used in the experiments. 1 – plexiglass block; 2 – Fig. 3. The pattern in gas exchange of beetles after treatment removable cover; 3 – insect chamber; 4 – current amplifier; 5 – with pyrethroid (Fastac 50 EC) recorded by the coulometric res- light source; 6 – photo-transistor; 7 – compensating vessel. pirometer (lower trace). Middle trace is a more detailed record of the pumping movements recorded during the fraction of the

Simultaneously, the rates of O2 production and O2 consumption lower trace indicated by the arrow. The same pumping move- -1 by the insect (VO2 ml h ) are measured. The system also records ments were simultaneously recorded by the infrared actograph transient changes in the rate of release of CO2. In our respirome- (upper trace); the frequent peaks between pumping movements ter, the electrolysis current was directly connected with a pho- we interpret as heartbeats. Note that the gas exchange is con- toelement instead of switching the electrodes. High sensitivity tinuous. The horizontal line above the lower trace indicates a of the respirometer to pressure changes in the respiration period when the beetle was active. chamber was achieved by replacing the standard photodiode with the photosensitive element of a transistor (KT302A, meniscus in the glass capillary, which served as a shutter to Semitronics, Freeport, NY, USA), which has a very small pho- screen the photosensitive area from light. Every burst of CO2 tosensitive area (approximately 0.5 mm2). In this way, the produced by the beetle caused a small movement in the etha- smallest movement in the meniscus of ethanol inside the nolic meniscus, which shuts off the photosensitive area from the U-shaped capillary was reflected as a signal on the recording light source, resulting in a downward movement in the trace. The electrolysis current depended on the ethanolic recording trace (Fig. 2). The coulometric respirometer allowed simultaneous recording of O2 consumption and abdominal pumping movements (Figs 2, 3). Every pumping stroke resulted in a rapid decrease (contraction) and increase (relaxation) in the volume of the body. These body movements caused pressure-volume pulses in the insect

Fig. 2. An example of the gas exchange cycles of adult Platynus assimilis recorded by the coulometric respirometer (lower trace). The lower trace indicates in great detail the actographic recording of the pumping movements. Each burst of CO2 release is actively ventilated by pumping movements of the Fig. 4. Carbon dioxide emission of an adult of P. assimilis abdomen. Upper trace is a detailed high resolution record of the exhibiting cyclic gas exchange recorded using flow-through res- pumping movements during a burst of CO2 release. The down- pirometry (lower trace). Upper trace is a simultaneous recording ward peak on this recording indicates the bursts of CO2 release of the pumping movements during the bursts of CO2 release horizontal bar. recorded using an infrared opto-cardiograph.

100 –1 –1 TABLE 1. The metabolic rates (VCO2 ml h g ) of individuals of P. assimilis recorded before and after treatment with two concentrations of Fastac 50 EC and distilled water as the control. Metabolic rate –1 –1 Treatment Indiv. (VCO2 ml h g ) Before After 0.005% 1 0.854 1.171 Fastac 50 EC 2 0.976 1.244 3 0.951 1.121 4 0.878 1.098 5 0.976 1.145 6 0.927 1.122 7 0.878 1.120

Fig. 5. A flow-through CO2 respirometer recording of the 8 1.122 1.146 DGE of an adult P. assimilis with a relatively long O-phase and 9 1.002 1.122 short CF-phase (between vertical lines) (lower trace). Upper trace is a synchronous infrared actograph recording of pumping t = 0.000 d.f. = 8 p = 0.007 movements recorded during the bursts of CO2 release. 0.001% 1 0.927 1.098 Fastac 50 EC 2 0.976 1.073 chamber, which was connected to the glass U-capillary. The 3 0.878 1.070 ethanol meniscus in the capillary oscillated in synchrony with 4 1.002 1.171 the abdominal pumping, which was translated into electrical signals that were recorded as discrete spikes on the recording trace. 5 0.927 1.121 In this way, the coulometric respirometer served also as an 6 0.976 1.122 insect activity detector (Fig. 3) and actograph for recording dis- 7 0.925 1.049 crete body movements. 8 0.951 1.120 Flow-through respirometry and IR-actography t = 0.000 d.f. = 7 p = 0.012 –1 Metabolic rates (VCO2 ml h ) and gas exchange patterns were Dist. water 1 0.878 0.902 measured also by a flow-through system, using a differential gas analyzer (DIRGA) and a pressure compensated URAS 26 (ABB 2 0.976 0.927 3 0.927 0.925 4 0.879 0.976 5 0.979 1.002 6 0.925 0.952 t = 6.00 d.f. = 5 p = 0.345

Analytical, Frankfurt, Germany), covering a measuring range of 0 to 500 ppm. The data from the analyzer were sampled at a rate of 10 Hz to PC via the analog output. Ambient air from outside the laboratory was scrubbed of carbon dioxide and water by passing it through columns containing Soda Lime and Drierite. –1 A flow rate of 200 ml min was used. The CO2 channel was calibrated with commercially available span gas (Eesti AGA AS, Estonia; Linde AG, Höllriegelskreuth, Germany). The flow-through respirometry was combined with infrared opto-cardiographic measurements (see Metspalu et al., 2001). An IR-emitting diode was placed on the side of the insect chamber near the ventral side of the insect’s abdomen, while the IR-sensitive diode (TSA6203) was placed on the opposite side Fig. 6. Typical flow-through respirometry recording of the of the chamber. The light from the IR-diode was modulated by pattern in gas exchange of P. assimilis recorded after treatment abdominal contractions. The level of the output voltage with sublethal doses of Fastac 50 EC (lower trace). Note the reflected the vigour of the muscular contractions of the insect beetles ceased breathing by DGE and switched to continuous (Hetz, 1994; Hetz et al., 1999; Mänd et al., 2005; Karise et al., respiration. There are two higher peaks of activity recorded in 2010; Kivimägi et al., 2011). the upper trace that were synchronously recorded by the infrared actograph; the insert is a more detailed record of the Data acquisition and statistics continuous pumping movements. The peaks in CO2 emission Computerised data acquisition and analysis were performed recorded in the lower trace are due to the activity of the beetle using the DAS 1401 A/D analogue-digital converter as hard- and not to cyclic gas exchange. ware and the TestPoint as software (Keithley, Metrabyte, Cleve-

101 land, OH, USA) with a sampling rate of 10 Hz. Mean (± SD) 2003). Essential between-individual variation was found resting metabolic rates were calculated automatically using a in the patterns of gas exchange of beetles that show DGE, statistical program (StatSoft ver. 8, Inc./USA). Statistical com- CGE and continuous gas exchange. parisons were made using the Wilcoxon Matched Pairs Test and Our results indicate that, in P. assimilis, the metabolic one-way ANOVA (analysis of variance). The significance level rate of beetles that show DGE and CGE did not differ, was set at P < 0.05. which contrasts with the literature in which the metabolic RESULTS rate of DGE beetles is commonly recorded as lower than Our results revealed that 23 of the 43 beetles tested that in CGE beetles. However, P. assimilis exhibited an used only the DGE mode of respiration, 13 beetles CGE uncommon pattern of DGE: the O-phase (burst) was and seven beetles continuous respiration. extraordinarily long compared with the whole cycle. This The O-phase of the beetles that used CGE made up pattern of gas exchange is similar to that described by 40–50% of the whole cycle (Fig. 4). The O-phase was Duncan & Dickman (2001) for the carabids Cerotalis sp. followed by a short C-phase and the F-phase was absent and Carenum sp. and Kivimägi et al. (2011) for Pterosti- or at least not separated from the C-phase. chus niger. The O-phase (burst) in all beetles coincided The DGE was characterized by a relatively long with muscular ventilation. There were few resting P. assimilis that exhibited con- O-phase (burst of CO2) making up 80–90% of the whole cycle while the C- and F-phases were not separated from tinuous respiration in which ventilation by muscular each other (Fig. 5). abdominal pumping occurred continually. Abdominal pulsations may be easily confused with heartbeats (Sláma, In both DGE and CGE the burst of CO2 release was always accompanied by active (muscular) ventilation (V) 2000). In P. assimilis we recorded continuous heartbeats or pumping. The mean duration of DGE was shorter than not coordinated with either bursts of CO2 release or the CGE 444.8 ± 8.1 s and 491.9 ± 5.7 s, respectively periods of abdominal pulsation, but Wasserthal (1996) reports that in Thermophilum hexmaculatum the heartbeat (F1,34 = 0.17; p = 0.05). No differences were found in resting metabolic rates between DGE and CGE: 0.94 ± period is synchronised with ventilatory movements of the –1 –1 –1 –1 abdominal tergites. Breathing by muscular abdominal 0.01 (VCO2 ml h g ) and 0.93 ± 0.01 (VCO2 ml h g ), pumping is well known in insects (Miller, 1974, 1981). respectively (F1,34 = 0.17, p = 0.68). Continuous respiration in P. assimilis was characterized The continuous respiration in adult Tenebrio molitor is by the continuous pumping movements recorded by cou- also characterized by abdominal pumping or respiratory lometric respirometry (Fig. 3). Between the pumping contractions occurring continually or periodically movements there were frequent pulsations, which we (Zafeiridou & Theophilidis, 2004, 2006). Sláma (2008, interpreted as heartbeats (see Fig. 3). Individuals using 2010) also describes weak abdominal movements, such as continuous respiration had higher resting metabolic rates extracardiac haemocoelic pulsations. –1 –1 Resting metabolic rate in beetles with continuous active (1.03 ± 0.02 VCO2 ml h g ) than those using DGE (0.94 –1 –1 ventilation was significantly higher than in individuals ± 0.01 (VCO2 ml h g ) (F1,28 = 11.46; p = 0.029). After treatment with sublethal doses (0.005%, 0.001%) using DGE. That metabolic rate varies with the gas of the pyrethroid alpha-cypermethrin the beetles ceased exchange pattern and is lowest in individuals that use DGE and switched to a form of continuous gas exchange DGE and highest in individuals using continuous gas (Fig. 6). The treated beetles became active for short exchange is also reported by Gibbs & Johnson (2004) and periods with a frequency of 1–2 per hour. An increase in Contreras & Bradley (2009, 2011). Treatments of adult P. assimilis with 0.005% and CO2 emission was recorded during each activity period. The activity periods may be easily confused with bursts 0.001% emulsions of alpha-cypermethrin resulted in the beetles switching from DGE to continuous respiration via of CO2 release. These activity periods (struggling) were also confirmed by visual observations under a stereomi- pumping. However, the pumping in treated beetles croscope. The metabolic rate between active periods was occurred at higher frequencies and amplitudes than in measured. The mean resting metabolic rate of beetles untreated beetles characterized by continuous gas treated with 0.005% and 0.001% Fastac 50 EC were exchange. Mild desiccation, sublethal doses of toxicant and handling stress also result in a similar pattern of 1.140 ± 0.015 (n = 9) and 1.104 ± 0.016 (n = 8) VCO2 ml h–1g–1, respectively. Thus the mean resting metabolic rate release of CO2 (Kestler, 1991). significantly increased after treatment in both groups Cessation of DGE may be regarded as the earliest 0.948 ± 0.016 (0.005%) and 1.140 ± 0.016 (0.001%) symptom of poisoning in insects. It may be suggested that –1 –1 disturbances in DGE are due to the paralysis of the VCO2 ml h g , respectively (see also Table 1). opening-closing mechanisms of spiracles. However, it is DISCUSSION also possible that the pesticide increases metabolic rate There was a significant difference in the between- due to uncontrolled muscle activity and it is this that individual variability in gas exchange patterns in adult P. causes them to switch from DGE to a continuous mode of assimilis. Variability within and between individuals in gas exchange. However, there are also other factors that physiological characteristics, including gas exchange pat- might result in the cessation of normal DGE (Kuusik et terns, is regarded as a normal phenomenon in insects al., 2001b). The mechanism in insects may be either auto- (Chown, 2001; Chown et al., 2002; Marais & Chown, intoxication and/or the release of neurohormones, or

102 changes in O2/CO2 thresholds, or in the CO2 capacitance clarification of hypotheses and approaches. — Physiol. Bio- due to changes in the acid-base status (Kestler, 1991). chem. Zool. 79: 333–343. Resting metabolic rate of treated individuals of P. assi- CHOWN S.L., SØRENSEN J.G. & TERBLANCHE J.S. 2011: Water loss milis was significantly higher than that of untreated indi- in insects: An environmental change perspective. — J. Insect Physiol. 57: 1070–1084. viduals. The increase in the metabolic rate of treated bee- CONTRERAS H.L. & BRADLEY T.J. 2009: Metabolic rate controls tles may be a consequence of the metabolic cost of vig- respiratory pattern in insects. — J. Exp. Biol. 212: 424–428. orous pumping. According to Chown & Holter (2000) CONTRERAS H.L. & BRADLEY T.J. 2011: The effect of ambient and Sibul et al. (2008) an increase in metabolic rate may humidity and metabolic rate on the gas-exchange pattern of be due to a small increase in the metabolic cost of con- the semi-aquatic insect Aquarius remigis. — J. Exp. Biol. vective ventilation by muscular contractions. 214: 1086–1091. DGE is advantageous for insect species of different DUNCAN F.D. & DICKMAN C.R. 2001: Respiratory patterns and lifestyles living in a wide range of environments. Thus, metabolism in tenebrionid and carabid beetles from the ceasing to breath by means of DGE is harmful for insects. Simpson Desert, Australia. — Oecologia 129: 509–517. According to Schimpf et al. (2012) insects breathing by FÖRSTER T.F. & HETZ S.K. 2010: Spiracle activity in moth pupae-the role of oxygen and carbon dioxide. — J. Insect means of DGE survive for longer when deprived of food Physiol. 56: 492–501. and water, which indicates that DGE confers a fitness GIBBS A.G. & JOHNSON R.A. 2004: The role of discontinuous gas benefit by reducing water loss. exchange in insects: the chthonic hypothesis does not hold Carabid beetles are important agents of biological con- water. — J. Exp. Biol. 207: 3477–3482. trol in organic farming programs (Kromp, 1990). Our GRAY E.M. & BRADLEY T.J. 2006: Evidence from mosquitoes data indicate that the respiratory system of P. assimilis is suggests that cyclic gas exchange and discontinuous gas vulnerable to pyrethroids, which induce a respiratory exchange are two manifestations of a single respiratory pat- failure in this beetle. When pyrethroids are applied to tern. — J. Exp. Biol. 209: 1603–1611. agricultural land their physiological effect on predaceous HARAK M., LAMPRECHT I., KUUSIK A., HIIESAAR K., METSPALU L. beetles should not be ignored. & TARTES U. 1999: Calorimetric investigations of insect metabolism and development under the influence of a toxic ACKNOWLEDGEMENTS. The authors are grateful to editores plant extract. — Thermochim. Acta 333: 39–48. and the anonymous referees for their critical and valuable com- HETZ S.K. 1994: Untersuchungen zu Atmungs, Kreislauf und ments on this manuscript. The research was supported by the Säure-Basen-Regulation an Puppen der tropischen Schmet- Estonian Science Foundation (grant number 9449), State Forest terlingsgattungen Ornithoptera, Troides und Attacus. 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104 TERBLANCHE J.S. & CHOWN S.L. 2010: Effects of flow rate and WASSERTHAL L.T. 1996: Interaction of circulation and tracheal temperature on cyclic gas exchange in tsetse flies (Diptera, ventilation in holometabolous insects. Adv. Insect Physiol. 26: Glossinidae). — J. Insect Physiol. 56: 513–521. 297–351. VAN DER STERN J.J.M. 2001: Review of the methods to deter- WOODS H.A. 2011: Breathing, bugs, and brains: conceptual uni- mine the hazard and toxicity of pesticides to bumblebees. — fication? — Funct. Ecol. 25: 1161–1162. Apidologie 32: 399–406. YARKOV D., PAVLOV D., YOTOVA I. & GAHNIAN R. 2003: Impact VANATOA A., KUUSIK A., TARTES U., METSPALU L. & HIIESAAR K. of Fastac 10 EC (alphacypermethrin) and artichoke (Cynara 2006: Respiration rhythms and heartbeats of diapausing Colo- scolimus L.) extract on the laying hens blood parameters. — rado potato beetles, Leptinotarsa decemlineata, at low tem- Trakia J. Sci. 1(1): 72–74. peratures. — Entomol. Exp. Appl. 118: 21–31. ZAFEIRIDOU G. & THEOPHILIDIS G. 2004: The action of the insecti- WANYIKA H.N., KARERU P.G., KERIKO J.M., GACHANGJA A.N., cide imidacloprid on the respiratory rhythm of an insect: the KENJI G.M. & MUKIIRA N.J. 2009: Contact toxicity of some beetle Tenebrio molitor. — Neurosci. Lett. 365: 205–209. fixed plant oils and stabilized natural pyrethrum extracts ZAFEIRIDOU G. & THEOPHILIDIS G. 2006: A simple method for against adult maize weevils (Sitophilus zeamais Motschul- monitoring the respiratory rhythm in intact insects and sky). — Afr. J. Pharm. Pharacol. 3: 66–69. assessing the neurotoxicity of insecticides. — Pestic. Bio- chem. Physiol. 86: 211–217.

Received May 28, 2012; revised and accepted August 17, 2012

105

V Effects of larval food plants on the development and diapause of the cabbage moth, Mamestra brassicae L.

Luule Metspalu, Katrin Jõgar, Aare Kuusik, Marika Mänd, Ingrid H. Williams, Eve Veromann, Eha Švilponis, Angela Ploomi, Külli Hiiesaar, Irja Kivimägi and Anne Luik

Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences, 1 Kreutzwaldi St., 51014 Tartu, Estonia

Abstract 1. To clarify the influence of food plants on some biological parameters of Mamestra brassicae, the larvae were reared in environmental test chambers, at 21 °C, 75% RH, and LD 12 : 12 h on five food plants: Brassica oleracea, B. napus, Pisum sativum, Beta vulgaris and Allium cepa. 2. The food plants influenced larval development rate, body mass and mass loss, mortality and intensity of diapause. Larval period on B. oleracea was shorter (26.2 ± 2.2 days) than on B. napus (31.8 ± 1.72 days), B. vulgaris (34.2 ± 1.7 days), A. cepa (35.6 ± 2.3 days) or P. sativum (mean 45.6 ± 2.6 days). Larval and pupal mortality was higher on P. sativum than on the other test plants. The largest pupal mass loss occurred on B. napus which was significantly higher than on other food plants. 3. Standard metabolic rate in diapausing pupae was lowest –1 –1 with B. oleracea (mean 0.04 mL O2 g h ) and highest with –1 –1 P. sativum (mean 0.067 mL O2 g h ). Discontinuous gas exchange bursts were more frequent on P. sativum (mean 9 h) than with B. oleracea (mean 18.8 h). Supercooling points of pupae did not vary with food plant. 4. We concluded that one of reasons for the great decrease in M. brassicae abundance following mass reproduction is that larval viability is lower on less valuable food plants and the pupae perished during winter. Keywords Mamestra brassicae, food plants, development rate, supercooling, discontinuous gas exchange, respiration.

Introduction Cabbage moth, Mamestra brassicae L. (Lepidoptera: Noctuidae) is a serious pest throughout the world. In northern areas, it usually has one full generation, although, in more favourable years, a second generation may occur (Finch & Thompson, 1992). It has a wide range of host plants, including both cultivated crops and wild plants. The larvae are pests on cabbage, Brassica oleracea (L.), but also occur on a wide range of other vegetable crops causing substantial economic losses (Turnock & Carl, 1995). Although, in the past, it has been a sporadic pest in the Baltic countries,

108 in recent years it has gradually become more widespread and damaging, probably due to climate warming. For polyphagous insects, the availability of different host plants plays an important role in triggering population outbreaks (Singh & Parihar, 1988) and study of the effects of food quality on the biology of insects is important for understanding host suitability of plant infesting insect species (Xue et al., 2010). Food plants affect insect development and reproduction and food quality is a key determinant of the fecundity of herbivorous insects (Awmack & Leather, 2002). Furthermore, food quality may interact with photoperiodic and temperature responses to influence diapause induction, as demonstrated pupal diapause in Hyphantria cunea (Morris, 1967), Helicoverpa armigera (Liu et al., 2009, 2010) and larval diapause in Choristoneura rosaceana (Hunter & McNeil, 1997). Many lepidopteran species can tolerate unfavourable periods by entering diapause, which is induced by various environmental cues and represents a complex dynamic process characterized by several specific physiological and behavioural features (Tauber et al., 1986; Danks, 1987; Denlinger, 1991). According to Harvey (1962), diapause is a state of developmental arrest of insects, characterized by minima in both endergonic biosynthetic activities such as protein synthesis and exergonic energy trapping activities such as metabolic rate and gas exchange patterns. The best known cues associated with diapause are photoperiod and temperature (Tauber et al., 1986); other factors, such as food quality of larvae (Hunter & McNeil, 1997; Liu et al., 2009, 2010), humidity (Lenga et al., 1993) and predation (Kroon et al., 2008) have been found to influence the apparent intensity of diapause of various insect and mite species. Mamestra brassicae exhibits pupal diapause 3–5 cm deep in the soil to survive the winter. Some information is available on induction and development of this diapause, showing the major role of day length and temperature (Goto & Hukusima, 1995; Hodek, 1996) but, in spite of its economic importance, little information exists on the nutritional indices of this pest on different food plants. However, the effects of larval food plants on pupal discontinuous gas exchange cycle (DGC) phases, haemolymph circulation and water loss of diapausing Pieris brassicae L. and M. brassicae have been dealt with in earlier publications (Metspalu et al., 2003; Jõgar et al., 2004, 2005; Jõgar, 2006). Similar studies, performed in recent years by Liu et al. (2007, 2009, 2010) on the cotton bollworm H. armigera, established a direct correlation between larval food quality and the duration of development, pupal mass as well as the number of progeny. They showed, that high quality larval food plants provided a better preparation for diapause, which appeared to be a prerequisite for successful overwintering and increased survival. Denlinger (2011) found that diapause is such an important aspect of the life cycle that disruption of its timing, e.g. by making the insects go into diapause at the wrong time or by breaking them out too early when no food is available, has potential as an effective tool for a pest control. Thus, factors inhibiting the induction of diapause or preventing diapause from becoming more intense will, at the same time, reduce the survival of insects in winter (Metspalu, 1976). The present study aimed to investigate whether development of M. brassicae larvae on less acceptable food plants had potential for decreasing population size the following year. To assess the influence of food plant quality on M. brassicae,

109 the duration of larval development, pupal mortality, pupal body mass and mass loss, sex ratio when larvae were fed on different food plants were measured and standard metabolic rate (SMR), discontinuous gas exchange (DGE) and supercooling points (SCP) were investigated in diapausing pupae.

Materials and Methods Design of the experiment Egg clutches of M. brassicae were collected from an experimental field of the Estonian University of Life Sciences, near Tartu in 2009 and those containing at least 100 eggs were included in the experiment. To prevent the time factor affecting the food plants, the clutches were all collected within one week. Larvae hatched from the same egg clutch were used in each replicate of the food plant species. Each food plant treatment consisted of at least 100 larvae (five replications, each 20 larvae). Larvae were divided into 5 groups, each fed on one of the following food plants: white cabbage (Brassica oleracea L. var. capitata L., variety ‘Krautman’), pea (Pisum sativum L., variety ‘Aamisepp’), red beet (Beta vulgaris L., variety ‘Bordoo’), onion (Allium cepa L., variety ‘Peipsiäärne’) and swede (Brassica napus L. var. napobrassica (L.) Rchb, variety ‘Kõpu’). The food plants were selected according to their importance as cash crops as well as their known associations with M. brassicae. All food plants were grown on the same field under uniform agronomic conditions. Newly hatched larvae were reared in Petri dishes (15 cm diameter and 2 cm deep), one replication in each dish, until the 3rd instar. The larvae were then placed in groups of five in 1 l breeding vessels covered with net and layered with sheets of filter paper to absorb excessive moisture. The food plant was replaced daily. Larvae were reared in environmental test chambers “Sanyo” with a short-day light cycle (LD 12:12 h), at 21 °C and 75% RH. Larval mortality was recorded at 24 h intervals. Before pupation, a 10 cm deep layer of peat was placed on the bottom of the vessels. To ensure that the pupae reached a stable diapause state, they were kept in the peat for one month, before collection by hand-sorting. Each pupa was classified as alive or dead according to the presence or absence, respectively, of abdominal movement in response to touch. Pupal gender was determined according to external sexual characters on the ventral side of the last abdominal segments (Sannino & Espinosa, 1999). Diapausing pupae were stored in the dark, at 21 ± 2 °C and 75 ± 10% RH in a state of ‘permanent’ diapause in standard plastic Eppendorf tubes (volume 1.5 ml, the cover of the tube was pierced with a needle). According to Jõgar (2006) such insect chambers have an extremely low water loss rate at room temperature and ambient humidity.

Pupal mass The pupae were weighed weekly for three months. Each pupa was weighing on an analytical balance to 0.1 mg (Explorer Balances, Ohaus Corporation, Pine Brook, New Jersey). To minimize manipulation stress, the handling was carried out with the Eppendorfs’s tube.

Respirometry Respirometry was conducted on 3 month-old pupae in deep diapause (Metspalu

110 -1 1976; Jõgar, 2006). Metabolic rate (O2 mL/g h ) was measured using an electrolytic volumetric manometric system characterised by a continuous (uninterrupted) O2 compensating system (for design see Kuusik et al., 1996; Tartes et al., 1999, 2002; Lighton, 2008).

Calorimetry The duration of discontinuous gas exchange (DGE) cycles was determined in ten pupae (five male and five female) from each treatment. Since the sexes produced identical results, the readings were combined for analysis. Calorimetry is the method for continuous recording of DGE for weeks in individuals without evoking stress by handling and adjusting the apparatus. A simple twin differential calorimeter was constructed of vessels made from copper foil (0.1 mm) connected with copper- constantan thermocouples, while a micro-nano-voltmeter and recorder were used (Kuusik et al., 1994; Harak et al., 1999; Jõgar et al., 2005). The volume of both the insects and reference vessels was 0.5 mL and the sensitivity of the calorimeter was 50 µV m W-1 with a detection limit of 4 µW. The calorimeter was calibrated electrically by the Joule effect (Hemminger & Höhe, 1984). The calorimeter was sufficiently sensitive to record CO2 releases by bursts and abrupt air intakes into the tracheae of the pupae.

Supercooling points (SCPs) The supercooling point of diapausing pupae was measured using a copper-constantan thermocouples-thermometer RS-232, Data logger Thermometer; TES Electrical Electronic, Taipei, Taiwan. Low temperatures were attained by deep-freeze Haier HF-103 (-30 °C). The thoracic tergite of the pupa was ﬁxed to the thermocouple, placed in a Styrofoam box and then transferred to a freezer chamber. The insect decreased its body temperature at a rate of 0.5 °C min-1. The SCP was taken as the lowest temperature before the increase in temperature caused by the latent heat of crystallization.

Statistical analysis Data was analysed using STATISTICA 9.1 (StatSoft, Inc/USA). Differences of means of replications in developmental time, larval and pupal mortality and SCP of the larvae reared on the different food plants were analyzed with one-way ANOVA, replications’ means on different food plants were compared with Fisher’s LSD post- hoc tests (P≤0.05). Two-way ANOVA was used to determine the effects of the food plants, gender and the food plant by gender interaction on these variables, pupal body mass, mass loss and supercooling points of hibernating pupae. Computerised data acquisition from the respirometer and calorimeter and the analysis were performed using the DAS 1401 A/D (analogue-digital) hardware and the TestPoint software (Keithley, Metrabyte, Cleveland, OH, USA) with a sampling rate of 10 HZ. Four bipolar channels allowed simultaneous recording of four events and standard metabolic rate (Mean±SD) was calculated automatically using a statistical program (StatSoft ver. 8 Inc/USA. One-way ANOVA and Fisher’s LSD-test was used to determine the differences between standard metabolic rate (SMR) and duration of DGE cycles in diapausing pupae.

111 Results

Development of M. brassicae larvae was significantly affected by food plant (F4,20 = 42.9, P < 0.0001, Figure 1). The development time of those reared on P. sativum was significantly longer (mean larval duration 45.6 ± 2.6 days) than of those reared on A. cepa (35.6 ± 2.3 days), B. vulgaris (34.2 ± 1.7 days), B. napus (31.8 ± 1.72 days), and on B. oleracea (26.2 ± 2.2 days). The larval period on B. oleracea was significantly shorter than on all other host plants (P < 0.05, LSD-test). Development of larvae reared on A. cepa, B. napus and B. vulgaris differed statistically from those reared on B. oleracea and P. sativum but did not differ mutually between A. cepa and B. vulgaris (P = 0.37, LSD-test) or B. napus and B. vulgaris (P = 0.13, LSD-test).

Larval and pupal mortality

Mortality of M. brassicae larvae was significantly affected by food plant (F4,20 = 32.1, P ≤ 0.0001, Figure 2) and was lowest on B. oleracea, with the result statistically different from other plant species tested (all values P < 0.05). Pisum sativum appeared to be the least suitable food plant for larval development as it induced significantly higher mortality than the other food plants (all values P < 0.05). We found that mortality of M. brassicae pupae was significantly affected by food plant (F4,20 = 8.4, P ≤ 0.0001, Figure 2). The highest average pupal mortality occurred in insects fed on B. vulgaris and P. sativum with 32% and 28%, respectively, of the pupae dying during the experimental period. Mortality on A. cepa reached 19% whereas only 15–16% of pupae died on B. oleracea and B. napus. Hence, the host plants can notionally be categorized into two groups by pupal mortality: 1. B. oleracea–B. napus–A. cepa; 2. P. sativum–B. vulgaris, with no significant differences between plants within the groups (LSD-test, P > 0.05).

Pupal mass and sex ratio Pupal mass (Table 1) of M. brassicae was signiﬁcantly affected by food plant (Two- way ANOVA: F4,60 = 3.2, P = 0.017), and the interaction of gender and food plant

(Two-way ANOVA: F4,60 = 2.97, P = 0.026) but gender alone was not signiﬁcant

(Two-way ANOVA: F1,60 = 3.073, P = 0.08). The pupae from larvae fed on B. oleracea resulted in a female-biased sex ratio (Table 1), whereas those fed on A. cepa, B. napus, P. sativum and on B. vulgaris resulted in a male-biased sex ratio.

Pupal mass loss Pupal mass loss was not significantly affected by food plant and gender, but the interaction of gender and plant was significant (Table 2). In male pupae, food plant appeared a significant factor (F4,30 = 9.37, P < 0.0001); the largest pupal mass loss of 26.1% occurred on B. napus which was significantly higher (all-values P < 0.05) than on other food plants: 6.4% on P. sativum, 4.5% on A. cepa, 4.8% on B. oleracea and 4.4% on B. vulgaris. Food plant had no significant effect on mass loss of female pupae (F4,30 = 1.362, P = 0.27); the highest mass loss appeared on A. cepa (12.2% of the initial weight), followed by P. sativum (7.5%), B. vulgaris (4.8%), B. oleracea (4.6%) and B. napus (4.4%).

112 Respiration The standard metabolic rate (SMR) of diapausing pupae was significantly affected by the food plant of the larvae (F4,37 = 8.50, P < 0.0001, Figure 3). SMR, measured –1 as rate of O2 production, was lowest on B. oleracea (mean 0.038 ± 0.006 mL O2 g –1 –1 –1 h ; n = 12) and highest on P. sativum (mean 0.067 ± 0.01 mL O2 g h ; n = 7) with statistically significant difference between the two (LSD-test, P < 0.05). The SMR of pupae from larvae fed on B. oleracea differed significantly also from those fed B. –1 –1 vulgaris (mean 0.048 ± 0.01 mL O2 g h ; n = 8) and A. cepa (mean 0.054 ± 0,01 –1 –1 mL O2 g h ; n = 8). Moreover, no significant differences in O2 consumption were found between male and female pupae.

Calorimetry The time lapse between DGE bursts of M. brassicae pupae was significantly affected by the food plant of the larvae (F4,45 = 17.58, P < 0.0001, Figure 4). Overall, the time lapse between bursts lasted longest in pupae from larvae reared on B. oleracea (mean 18.8 ± 2.2 h, n = 10) which differed from all other food plants. Statistically significantly shortest periods between the DGE bursts occurred on P. sativum (mean 9 ± 2.3 h, n = 10). Pupae from larvae fed on B. napus (mean 15.1 ± 2.9 h, n = 10), B. vulgaris (mean 15.2 ± 2.5 h, n = 10) and A. cepa (mean14.2 ± 2.5 h, n = 10) did not show any statistically significant difference within the group, but only in comparison to B. oleracea and P. sativum.

Supercooling points

SCP (Table 3) was not signiﬁcantly affected by food plant (F4,161 = 1.55, P = 0.19), gender (F1,161 = 0.33, P = 0.56) or gender and food plant interaction (F4,161 = 0.40, P = 0.8).

Discussion This study demonstrated significant differences in some biological parameters of M. brassicae larvae and pupae reared on different food plants. Overall, food plants influenced larval development rate, larval and pupal mass, mass loss, mortality and the intensity of diapause. Developmental time of larvae varied with food plant; that on B. oleracea was significantly shorter than on A. cepa, B. napus, B. vulgaris or P. sativum (Figure 1). For normal growth and development of larvae the proportions of nutritional elements in the food plant are of primary importance (Awmack & Leather, 2002; Syed & Abro, 2003). Faster development may allow a short life cycle, high reproductivity, and rapid population growth (Singh & Parihar, 1988; Liu et al., 2004). Generally, slower development or digestion and lower fertility rate in herbivorous insects are caused by lower food quality (Chen et al., 2004). To compensate for deficiency of essential nutrients, insects may start feeding voraciously, extend their feeding period or apply both strategies. Additionally, they may enhance feeding efficiency by extending the time food is in the alimentary canal or by activating digestive enzymes (Barbehenn et al., 2004). Quality food plants may give rise to the second full generation in individuals with a faster development cycle in northern regions, increasing crop damage. Food quality also affected the viability of M. brassicae. Larval mortality was higher on P. sativum than the other plants, probably because

113 it was a poorer quality host plant. Larval host plants also had a significant effect on pupal mortality; high larval mortality reduced the number of pupae from larvae fed B. vulgaris and P. sativum than B. oleracea. Body mass is an important fitness indicator in insect population dynamics (Liu et al. 2004). Pupal body mass varied with food plant. Again, the lowest mean pupal body mass was in P. sativum fed larvae. Body mass is directly dependant on reserves stored at the larval stage, and pupae with small body mass appear when growing conditions, including food quality in the larval stage are unfavourable. Pupal mass is important, since heavier female pupae lay more eggs when adult (Kramer, 1959; Haukioja & Neuvonen, 1985) consequently affecting potential growth rate of the population. Insects cannot clear the hurdle of food quality and the nutritional features are directly reflected in the abundance of progeny (Ruohomäki et al., 2000). Pupal body mass in M. brassicae differed between sexes: female pupae were lighter on all food plants than on B. oleracea, suggesting they were less adequate nutritionally. In many insect species, females are larger than males as higher body mass is biologically more important for females; it is a key precondition for the viability of the progeny, as heavier pupae become larger adults able to lay more eggs (Armbruster & Hutchinson, 2002). Moreover, nutritional requirements of female and male catepillars are somewhat different. Male larvae tend to consume more lipids than females, possibly because of their greater energy need (to enable longer mating flights), whereas females need more protein for egg production. Female fecundity increases with adult size, whereas male mating success is less dependent on size (Gotthard, 2008). For instance, unfavourable temperature affects the growth of both sexes similarly, but poor food quality affects their development and body mass somewhat differently, normally resulting in smaller females producing fewer eggs (Ohsaki & Yoshibumi, 1994). Female reproductive fitness is influenced by fecundity, which is often related to the quality and quantity of food ingested and the resulting size of adults. Male reproductive fitness is usually most closely correlated with the number of mates inseminated and is rarely directly related to the size of individuals. Such differences may explain why females are often more sensitive than males to variation in plant quality, resulting in differential survival and fecundity on hosts of different quality (see Johns et al., 2009). The sex ratio of pupae could also determine whether the population can adapt to a certain food plant. Sex ratio indicating optimal food plant quality was exhibited only on B. oleracea in our experiment, as other food plants produced a male-biased population. Merzeevskaja et al. (1976) suggested that female-bias in Noctuid butterflies implies high quality food and higher fecundity than other sex ratios. Similarily, Awmack and Leather (2002) showed that slight prevalence of females in a population results from good quality larval food plants. Morrill et al. (2000) found that host quality affects the sex ratio of both phytophagous and entomophagous Hymenoptera with more females produced on plants of higher quality. Larval food plants affected potential hibernation success of M. brassicae pupae; they affected not only diapause induction (Hunter & McNeil, 1997) but also the intensity of pupal diapause. The physiological state of an insect is usually estimated by standard metabolic rate (SMR) (Keister & Buck, 1964), commonly

114 by flow-through CO2 respirometry (Lighton, 1996; Chown & Nicolson, 2004). SMR is defined as a value measured at a particular temperature, when the insect is quiet, inactive, is not digesting a meal, nor exposed to any stress (Withers, 1992). In long cycle insects, when only about one burst is released during a day, the SMR is difficult to measure via CO2 flow-through analyser, so we measured it in diapausing M. brassicae pupae by oxygen consumption (see Slama, 2010). For the same reason, we recorded the frequency of DGE in diapausing pupae by means of our calorimetric system. We used a volumetric-manometric respirometer, which served also as an activity detector. Diapause intensity (see Belozerov, 2009; Kostal, 2006) is characterised by SMR, which in lepidopteran pupae may decrease to very low –1 –1 levels – 0.01–0.04 O2 g h (Keister & Buck, 1964; Jõgar et al., 2005, 2007). On the contrary, at the initiation of pupal diapauses of P. brassicae, a SMR 0.07–1.2 O2 g–1 h–1 may be observed (Jõgar et al., 2004, 2005, 2011). We found a similar SMR in M. brassicae pupae when the larvae were reared on less suitable food plants. Our results suggested the most suitable food plant was B. oleracea, as judged by the –1 –1 lowest level of SMR in diapausing pupae (0.04 mL O2 g h ). Such a low level of SMR points to a deep diapause which favours overwintering of the pupae (Fourche, –1 1977). On P. sativum, the significantly higher SMR in the pupae (0,067 mL O2 g h–1) was a sign of an abnormally decreased intensity of diapause. In addition to the SMR, patterns of discontinuous exchange (DGE) cycles are used to characterise the physiological state of an insect (see Kestler, 1985, 1991); during these, CO2 is expelled in bursts. The classic DGC in moth pupae consists of three phases: constriction (C) fluttering (F) and open (O) phase. To avoid losing water through respiration, they open their tracheae periodically, a phenomenon known as discontinuous ventilation. Diapausing pupae of P. brassicae and M. brassicae expel

CO2 by only 1–2 bursts per day (Metspalu & Hiiesaar, 1984; Jõgar, et al., 2005; 2007). Deep diapause of M. brassicae pupae in our experiment was characterized by DGEs with large outbursts of CO2 lasting 15–20 minutes. Time lapses between outbursts were long occurring only once or twice per 24 hours. However, on P. sativum, they were shorter (4–5 minutes) and occurred more frequently, 3–4 times per 24 h. As a rule, lower metabolic rate is associated with fewer DGE cycles per day (Slama, 2010). We obtained short gas exchange cycles and relatively high metabolic rates in diapausing pupae of M. brassicae, when caterpillars were fed on less favourable food plants (e.g. P. sativum). Resulting pupae were characterized by frequent gas emission cycles, higher aspiration rate and body mass loss. This suggests that pupal diapause had not developed normally and such a physiological state is probably unﬁt for the overwintering period. Although cold hardiness and diapause are essential for the survival of most overwintering insects, the relationship between them is less clear (Denlinger, 1991). According to Worland (2005), supercooling points have been considered an index of cold hardiness in many, but not all, insects. Tsutsui et al. (1988) and Goto et al. (2001) found that diapause and non-diapause pupae of M. brassicae did not differ in their SCP-s, and therefore M. brassicae should be considered among the species in which SCPs cannot be utilized to assess the depth of diapause. Probably the pupae of M. brassicae have a supercooling ability as a speciﬁc physiological property independent of diapause. However, SCP can be affected by other factors e.g. that of H. armigera diapausing pupae have been affected by food plant quality of the larvae

115 (Liu et al., 2007, 2009). In our experiment with M. brassicae pupae, such results were not confirmed as there was no significant difference between the different plants fed to the larvae. Nevertheless, certain trends should be pointed out since the female pupae of M. brassicae exhibited somewhat lower SCPs on B. oleracea. In male pupae, lower SCPs tended to be from B. napus fed larvae, even though this difference was not statistically significant. Therefore, considering all determined factors, the five food plants tested can be arranged in decreasing order of host suitability: B. oleracea > napus > A. cepa > B. vulgaris > P. sativum. This information helps our understanding of the mechanism of population build-up of this pest on different food plants, but field observations are also needed. We concluded from the study that one of reasons for the great decrease in the abundance of M. brassicae following mass reproduction is the reduced viability of caterpillars growing on lower quality food plants. The mortality rate of these caterpillars was high, the pupae were underweight, diapause was not as deep as expected and, in most cases, the pupae perished during winter. Different food plants obviously play an important role in triggering population increases and outbreaks. Further, as the dynamics of M. brassicae over the whole season is still unclear, further research on the effects of different food plants is needed.

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Acknowledgements This study was supported by the grants 9449 and 8895 of the Estonian Science Foundation, and Estonian Ministry of Education and Research targeted ﬁnancing project no SF 0170057s09 and the project P9003PKPK.

120 Tables

TableTables 1 Pupal mass (Mean ± SE, mg) and sex ratio of Mamestra brassicae larvae reared on five different food plants. Table 1 Food plants Pupal mass Pupal mass Pupal sex ratio Female Male Female : Male Allium cepa 417.0 ± 13.9 ab 424.8 ± 11.58 a 1 : 1.23 Brassica oleracea 461.0 ± 10.85 b 415.9 ± 13.0 ab 1 : 0.52 Beta vulgaris 399.3 ± 34.2 ab 465.0 ± 9.95 ad 1 : 1.4 Pisum sativum 346.3 ± 47.0 a 367.0 ± 42.3 b 1 : 1.32 Brassica napus 403.9 ± 16.19 a 474.5 ± 18.4 d 1 : 1.31 F 2.84 3.77 d.f. 4 4 P 0.04 0.01 Means within columns followed by different letters are significantly different at P ≤ 0.05 (LSD-test).

Table 2 Summary of two-way ANOVA of the effect of sex and treatment on pupal mass lossTable of 2Mamestra brassicae. Effect d.f. MS F P Pupal mass loss Food plant 4 121.10 2.30 0.069 gender 1 33.62 0.63 0.427 interaction 4 264.70 5.02 0.001 error 60 52.68

Table 3 Means ± SE of supercooling point (SCP) °C of Mamestra brassicae pupae from Tablelarvae 3reared on different food plants. SCP SCP Food plants (Mean ± SE) � C, n (Mean ± SE) � C, n females males Brassica oleracea -21.4 ± 0.32 25 -21.3 ± 0.35 13 Allium cepa -20.6 ± 0.25 21 -20.8 ± 0.23 25 Brassica napus -21.1 ± 0.44 16 -21.6 ± 0.31 21 Beta vulgaris -20.7 ± 0.38 10 -21.1 ±0.42 14 Pisum sativum -21.1 ± 0.41 10 -20.7 ±0.42 15 13

You created this PDF from an application that is not licensed to print to novaPDF printer (http://www.novapdf.com) 121

You created this PDF from an application that is not licensed to print to novaPDF printer (http://www.novapdf.com)

You created this PDF from an application that is not licensed to print to novaPDF printer (http://www.novapdf.com) Figures Figures Figure 1 50 d 48

40 b 38 bc 36

34 c

30 a

Average duration of larval stage (days) (days) larvalstage of Averageduration 28

26 Mean Mean±SE 24 Mean±SD 22 B. oleracea A. cepa B. napus B. vulgaris P. sativum

Figure 1 Average duration of larval stage of cabbage moth Mamestra brassicae reared on Brassica oleracea, Allium cepa, B. napus, Beta vulgaris and Pisum sativum. Columns with different letters were significantly different (P < 0.05, ANOVA, LSD-test).

Figure 2

50 c Larvae 45 Pupae

40 B 35 BC b 30 , % b y lit a 25 rt b o A M 20 A A 16

15 a You created this PDF from an application that is not licensed to print to novaPDF printer (http://www.novapdf.com) 10

0 B. oleracea A. cepa B. napus B. vulgaris P. sativum

Figure 2 Larval and pupal mortality of Mamestra brassicae reared on Brassica oleracea, Allium cepa, B. napus, Beta vulgaris and Pisum sativum. Different lowercases above the bars show significant differences between larvae and uppercase significant differences between pupae at P < 0.05, LSD-test.

You created this PDF from an application that is not licensed to print to novaPDF printer (http://www.novapdf.com) Figure 3 0.09

c 0.08

b 0.07

-1 0.06 h -1 ab ml g 2

O 0.05 a

0.04

0.03 Mean Mean±SE Mean±SD

0.02 B. oleracea A. cepa B. napus B. vulgaris P. sativum

-1 -1 Figure 3 Standard metabolic rate (O2 mL g h ) in three month old Mamestra brassicae pupae from larvae fed on Brassica oleracea, Allium cepa, B. napus, Beta vulgaris and Pisum sativum. Columns with different letters were significantly different (P < 0.05, LSD-test).

Figure 4 22 a Mean Mean±SE 20 Mean±SD b b 18 b

12 c

10 18

Time-lapse between DGE between Time-lapse (hours) 8 You created this PDF from an application that is not licensed to print to novaPDF printer (http://www.novapdf.com)

4 B. oleracea A. cepa B. napus B. vulgaris P. sativum

Figure 4 Mean duration of DGE cycles in hibernating pupae of Mamestra brassicae on different larval food plants: Brassica oleracea, Allium cepa, B. napus, Beta vulgaris and Pisum sativum. Columns bearing the same letter were not significantly different (ANOVA, LSD, P < 0.05).

123

You created this PDF from an application that is not licensed to print to novaPDF printer (http://www.novapdf.com) CURRICULUM VITAE

First name: Irja Last name: Kivimägi Date of Birth: May12th 1969 Address: Estonian University of Life Sciences, Kreutzwaldi 1, Tartu 51014 E-mail: [email protected]

Education: 2008 – 2012 PhD studies in Entomology, Estonian University of Life Sciences 2008 – 2009 Course of pedagogy, University of Tartu 2006 – 2008 MSc studies in Horticulture, Estonian University of Life Sciences (cum laude) 2003 – 2006 BSc studies in Horticulture, Estonian University of Life Sciences 2001 – 2003 Horticulture, Räpina Horticultural College 1984 – 1988 Veterinary Medicine, Väimela Agricultural Technical School 1976 – 1984 Primary School of Võnnu

Foreign languages: Estonian, Russian, English

Professional employment: 2012 – … Specialist, Department of Silviculture; Institute of Forestry and Rural Engineering, Estonian University of Life Sciences 2008 – 2012 Specialist, Department of Plant Protection; Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences 2007 – 2009 Teacher (Biology), Rannu Secondary School

Membership: 2010 – … Member of the Vanemuise Selts 2007 – … Member of the Association of Estonian Biology Educators 2005 – … Member of the Estonian Plant Protection Society

124 Academic degree: 2008 Master’s Degree, The seasonal dynamics of cold-hardiness in some ground beetles (Coleoptera: Carabidae), Institute of Agricultural and Environmental Sciences, Estonian University of Life Sciences,

Awards and grants: 2012 Travelling scholarship, ESF DoRa T8 „Participation of young researchers in international exchange of knowledge“ 2011 Travelling scholarship, ESF DoRa T8 „Participation of young researchers in international exchange of knowledge“ 2011 Travelling scholarship, Doctoral School of Earth Sciences and Ecology 2011 Travelling scholarship, ESF DoRa T8 „ Participa- tion of young researchers in international exchange of knowledge“ 2010 Travelling scholarship, ESF DoRa T8 „Participation of young researchers in international exchange of knowledge“ 2010 Raefond’s scholarship of Estonian University of Life Sciences 2010 Estonian Word Council Inc. Margot M. and Herbert R. Linna’s scholarship 2010/2011 2010 Estonian Students Fund Elsa and Edgar Mathiesen’s scholarship 2010/2011 2010 Travelling scholarship, ESF DoRa T8 „Participation of young researchers in international exchange of knowledge“ 2009 Travelling scholarship, ESF DoRa T8 „Participation of young researchers in international exchange of knowledge“ 2007 Scholarship of Estonian Students Fund in USA

Research interest: Insect physiology, sustainable plant protection

125 Dissertations supervised: 2012 Kristi Keskla, Master’s Degree, Influence of hop extract on the Colorado potato Beetle (Leptinotarsa decemlineata Say). Estonian University of Life Sciences 2011 Teele Vaarak, Master’s Degree, Influence of natural field margin on the cereal field arthropods abundance and species richness. Estonian University of Life Sciences 2011 Tuuli Vaarak, Master’s Degree, Impact of sowing field margin on the cereal field arthropods abundance and species diversity. Estonian University of Life Sciences

Target financed (TF) projects and grants of the Estonian Science Foundation (ESF):

2012 – 2015 State Forest Management forest protection project No 8-2/T12115MIMK Forest protection problems associated with reforestation in Estonian forestry and their prevention with environmentally sustainable methods. Investigator. 2009 – 2014 Target-financed project No SF0170057s09: Plant protection for sustainable crop production. Investigator, PhD student 2009 – 2013 Ministry of the Agriculture of Estonia, Bumblebee study within the framework of evaluation 2009-2013 agri-environment scheme in Estonia. Investigator, PhD student 2012 – 2015 ESF grant No 9449: Effects of pesticides on the development and overwintering ability of insects. Investigator, PhD student 2011 – 2014 ESF grant No 8895: Impact of host plants on the major pests of cruciferous plants and their parasitoids in different cropping systems. Investigator, PhD student 2007 – 2010 ESF grant No 7130: The effects of food plants and microsporidiosis (Microsporidia, Nosematidae) on development and over-wintering physiology of insect pests on vegetable crops. Investigator, PhD student 2006 – 2009 ESF grant No 6722: Delayed effects of sublethal doses of natural insecticides on pest and beneficial insects. Investi- gator, PhD student

126 2004 – 2007 ESF grant No 5736: Occurrence of carabids and hymenopterous parasitoids in different crops depending on growing technologies and field locations. Investigator, MSc student

Professional training: 2010 Course Public performance, University of Tartu 2010 Course Art of lecturing, University of Tartu 2010 Course Field observations and collections in environmental education, University of Tartu 2010 Course Conversational topics, Estonian University of Life Sciences 2009 PhD course Insect Conservation, Norwegian Institute for Agricultural and Environmental Research 2009 PhD course Mutualistic interactions, University of Copenhagen 2008 PhD course Pathogens in social Insects, Swedish Univer- sity of Agricultural Sciences 2007 PhD course Organic beekeeping; University of Helsinki 2007 PhD course Beekeeping Techniques in Cold Climates; Latvia University of Agriculture

127 ELULOOKIRJELDUS

Eesnimi: Irja Perekonnanimi: Kivimägi Sünniaeg: 12. mai 1969 Töökoht: Eesti Maaülikool, Kreutzwaldi 1, Tartu 51014 E-mail: [email protected]

Haridus: 2008 – 2012 Doktoriõpe põllumajanduse õppekava entomoloogia erialal, Eesti Maaülikool 2008 – 2009 Pedagoogika kursus, Tartu Ülikool 2006 – 2008 Magistriõpe aianduse erialal, Põllumajandus- ja keskkonnainstituut, Eesti Maaülikool (cum laude) 2003 – 2006 Bakalaureuseõpe aianduse erialal, Eesti Maaülikool 2001 – 2003 Aianduse eriala, Räpina Aianduskool 1984 – 1988 Veterinaarvelskri eriala, Väimela Näidissovhoostehni- kum 1976 – 1984 Võnnu 8-kl kool

Keelteoskus: Eesti keel, vene keel, inglise keel

Teenistuskäik: 2012 – … Spetsialist, metsakasvatuse osakond, Metsandus- ja maaehitusinstituut, Eesti Maaülikool 2008 – 2012 Spetsialist, taimekaitse osakond, Põllumajandus- ja keskkonnainstituut, Eesti Maaülikool 2007 – 2009 Rannu keskkool, pedagoog

Teadusorganisatsiooniline tegevus: 2010 – … Vanemuise Seltsi liige 2007 – … Eesti Bioloogiaõpetajate Ühingu liige 2005 – … Eesti Taimekaitse Seltsi liige

Teaduskraad: 2008 Magistrikraad,väitekiri „Mõnede jooksiklaste (Coleoptera: Carabidae) külmakindluse sesoonne dünaamika“. Eesti Maaülikool.

128 Teaduspreemiad ja -tunnustused: 2012 SA Archimedese DoRa T8 stipendium osalemiseks sümpoosiumil 2011 SA Archimedese DoRa T8 stipendium osalemiseks kongressil 2011 Maateaduste ja ökoloogia doktorikooli stipendium osalemiseks konverentsil 2011 SA Archimedese DoRa T8 stipendium osalemiseks konverentsil 2010 SA Archimedese DoRa T8 stipendium osalemiseks kongressil 2010 SA EMÜ Joosep Tootsi Fond, Raefondi stipendium 2010 ÜEKN-i Margot ja Herbert Linna nimeline stipendium 2010/2011 2010 Eesti Üliõpilaste Toetusfondi Elsa ja Edgar Mathiesen’i nimeline toetus 2010/2011 2010 SA Archimedese DoRa T8 stipendium osalemiseks kongressil 2009 SA Archimedese DoRa T8 stipendium osalemiseks PhD kursusel 2007 Eesti Üliõpilaste Toetusfondi Mathieseni nimeline stipendium 2007/2008

Teadustöö põhisuunad: Putukate füsioloogia, loodussäästlik taimekaitse

Juhendatud väitekirjad: 2012 Kristi Keskla, magistrikraad, „Humalaekstrakti mõju kartulimardikale (Leptinotarsa decemlineata Say)“. Eesti Maaülikool 2011 Teele Vaarak, magistrikraad, “Loodusliku taimikuga põlluserva mõju teraviljapõllu lülijalgsete arvukusele ja liigilisele mitmekesisusele”. Eesti Maaülikool 2011 Tuuli Vaarak, magistrikraad, „Külvatud põlluservariba mõju teraviljapõllu lülijalgsete arvukusele ja liigilisele mitmekesisusele”. Eesti Maaülikool

129 Osalemine uurimisprojektides ja programmides: 2012 – 2015 RMK projekt 8-2/T12115MIMK „Metsakultiveerimi- sega seotud metsakaitseprobleemid Eesti metsanduses ning nende vältimine keskkonnasäästlike tõrjevõte- tega“. Täitja. 2009 – 2014 EV Haridusministeeriumi sihtfinantseeritav teadus- teema „Taimekaitse jätkusuutlikule taimekasvatusele“. Täitja, doktorant 2009 – 2013 Maaelu arengukava (MAK) põllumajandusliku kesk- konnatoetuse (PKT) seire ja hindamine „Kimalaste mitmekesisuse ja arvukuse uuring aastatel 2009-2013“. Täitja, doktorant 2012 – 2015 ETFi grant nr 9449 „Pestitsiidide toime putukate arengule ja talvitusvõimele“. Täitja, doktorant 2011 – 2014 ETFi grant nr 8895 „Peremeestaimede mõju ristõieliste õlikultuuride võtmekahjuritele ja nende parasitoididele erinevates viljelustingimustes“. Täitja, doktorant 2007 – 2010 ETFi grant nr 7130 „Toidutaimede ja mikrosporidioosi (Microsporidia, Nosematidae) mõju köögiviljakultuuride putukkahjurite arengule ja talvi- tusfüsioloogiale“. Täitja, doktorant 2006 – 2009 ETFi grant nr 6722 „Looduslike insektitsiidide subletaalsete dooside järeltoime kahjuritele ja kasuritele“. Täitja, doktorant 2004 – 2007 ETFi grant nr 5736 „Jooksiklaste ja kiletiivalistest parasitoidide esinemine erinevates kultuurides sõltuvalt kasvatustehnoloogiast ning põllu äärealadest“. Täitja, magistrant

Erialane täiendamine: 2010 Avalik esinemine, Tartu Ülikool 2010 Loengupidamise kunst õppejõududele, Tartu Ülikool 2010 Välivaatlused ja kollektsioonid loodushariduses, Tartu Ülikool 2010 Inglise keel õppetöös, Eesti Maaülikool 2007 Organic beekeeping; Helsingi Ülikool 2007 Beekeeping Techniques in Cold Climates; Läti Põllu- majandusülikool 2007 Statistikapaketi STATISTICA kasutamine, Tartu Üli- kool

130 LIST OF PUBLICATIONS

1.1. Publications indexed in the ISI Web of Science database:

Kivimägi I., Kuusik A., Ploomi A., Metspalu L., Jõgar K., Williams I.H., Sibul I., Hiiesaar K., Luik A., Mänd M. 2012. Gas exchange patterns in Platynus assimilis (Coleoptera, Carabidae): respiratory failure induced by a pyrethroid. European Journal of Entomology. (Accepted August 17th 2012). Veromann E., Metspalu L., Williams I.H., Hiiesaar K., Mänd M., Kaasik R., Kovács G., Jõgar K., Švilponis E., Kivimägi I., Ploomi A., Luik A. 2012. Relative attractiveness of Brassica napus, Brassica nigra, Eruca sativa and Raphanus sativus for pollen beetle (Meligethes aeneus) and their potential for use in trap cropping. Arthropod-Plant Interac- tions, 6(3), 385–394. Jõgar K., Kuusik A., Ploomi A., Metspalu L., Williams I.H., Hiiesaar K., Kivimägi I., Mänd M., Tasa T., Luik A. 2011. Oxygen convective uptakes in gas exchange cycles in early diapause pupae of Pieris brassicae L. (Lepidoptera, Pieridae). Journal of Experimental Biology, 214, 2816–2822. Hiiesaar K., Williams I.H., Mänd M., Luik A., Jõgar K., Metspalu L., Švilponis E., Ploomi A., Kivimägi I. 2011. Supercooling ability and cold hardiness of the pollen beetle. Entomologia Experimentalis et Applicata, 138, 117–127. Kivimägi I., Kuusik A., Jõgar K., Ploomi A., Williams H.I., Metspalu L., Hiiesaar K., Sibul I., Mänd M., Luik A. 2011. Gas exchange patterns of Pterostichus niger (Carabidae) in dry and moist air. Physiological Entomology, 36, 62–67. Kivimägi I., Ploomi A., Luik A., Jõgar K., Sibul I., Kuusik A. 2008. Cold-hardening of the ground beetle Carabus granulatus L. (Coleop- tera, Carabidae). Zemdirbyste-Agriculture, 95, 428–432.

131 1.2. Papers published in other peer-rewiewed international journals with a registered code:

Metspalu L., Jõgar K., Ploomi A. Hiiesaar K., Kivimägi I., Luik A. 2010. Effects of biopesticide Neem EC on the Mamestra brassicae L. (Lepi- doptera, Noctuidae). Agronomy Research, 8, 465–470. Kivimägi I., Ploomi A., Metspalu L., Švilponis E., Jõgar K., Hiiesaar K.; Luik, A.; Sibul, I.; Kuusik, A. 2009. Physiology of a carabid Platynus assimilis. Agronomy Research, 7, 328–334. Jõgar K., Metspalu L., Hiiesaar K., Ploomi A., Švilponis E., Kuusik A., Menšhykova N., Kivimägi I., Luik A. 2009. Influence of white cabbage cultivars on oviposition preference of the Pieris rapae L. (Lepid- optera: Pieridae). Agronomy Research, 7, 283– 288. Ploomi A., Jõgar K., Metspalu L., Hiiesaar K., Švilponis E., Kivimägi I., Menšhykova N., Luik A., Sibul I., Kuusik A. 2009. Effect of cultivar on oviposition preference of the cabbage moth, Mamestra brassicae L. (Lepidoptera: Noctuidae). Agronomy Research, 7, 451–456. Metspalu L., Hiiesaar K., Jõgar K., Švilponis E., Ploomi A., Kivimägi I., Luik A., Menšhikova N. 2009. Oviposition preference of Pieris brassicae (L) on different Brassica oleracea var. capitata L. cultivars. Agronomy Research, 7, 406–411. Ploomi A., Jõgar K., Metspalu L., Hiiesaar K., Loorits L., Sibul I. Kivi- mägi I., Luik A. 2009. The toxicity of neem against to the snail Arianta arbustorum. Sodininkyste ir Darzininkyste (Horticulture and Vegetable Growing), 28, 153–158.

3.4. Articles/presentations published in conference proceedings not listed in Section:

Jõgar K., Metspalu L., Hiiesaar K., Ploomi A., Mänd M., Kivimägi I., Tasa T., Luik A. 2011. The influence of the larval food plants on microsporidia (Nosema mesnili P.) infection in diapausing Pieris brassicae L. pupae. Bulletin IOBC/wprs: (Eds.) Ehlers R.U., Crick- more N., Enkerli J. et al. IOBC/WPRS, 249–253.

132 3.5. Papers in Estonian and in other peer-reviewed research journals with a local editorial board:

Jõgar K., Metspalu L., Hiiesaar K., Kivimägi I., Ploomi A., Tasa T., Luik A. 2012. Suur-kapsaliblika (Pieris brassicae L.) arvukus toidutaimedel. Agronoomia 2012, 125–128. Kivimägi I., Ploomi A., Mänd M.; Kruus M.; Jõgar K.; Metspalu L.; Hiiesaar K.; Luik, A.; Vaarak, T.; Vaarak, T. 2012. Rohumaariba mõju jooksiklaste ja ämblike arvukusele talinisupõldudel. Agro- noomia 2012, 135–140. Metspalu, L., Švilponis E., Jõgar K., Hiiesaar K., Ploomi A., Kivimägi I. 2012. Sordid mõjutavad porgandikahjurite valikuid. Agronoomia 2012, 151–56. Ploomi A., Kivimägi I., Mänd M., Kruus M., Jõgar K., Metspalu L., Hiiesaar K., Luik A., Sibul I. 2012. Maapinnal liikuvate lülijalgsete arvukus ja mitmekesisus rohumaaribadega ääristatud talinisupõldu- del. Agronoomia 2012, 161–68. Metspalu L., Jakin E., Jõgar K., Kivimägi I., Ploomi A. 2011. Kapsa- öölase (Mamestra brassicae L.) arvukus erinevatel ristõielistel köögivil- jakultuuridel. Agronoomia 2010/2011, 177–182. Ploomi A., Metspalu L., Jõgar K., Kivimägi I., Jakin E. 2011. Kapsakoi toidutaimede eelistused. Agronoomia 2010/2011, 193 –196. Jõgar K., Metspalu L., Ploomi A., Hiiesaar K., Kivimägi I., Tasa T. 2011. Väike-kapsaliblika (Pieris rapae L.) arvukus erinevatel kultuuridel. Agronoomia 2010/2011, 161 –164). Metspalu L., Hiiesaar K., Jõgar K., Kivimägi I., Miil T., Ploomi A., Švilponis E., Veromann E. 2009. Naeri-hiilamardika (Meligethes aeneus Fab.) peremeestaimede eelistused. Agronoomia 2009, 204– 209. Hiiesaar K., Metspalu L., Jõgar K., Švilponis E., Ploomi A., Kivimägi I. 2009. NeemAzal T/S mõju kartulimardika (Leptinotarsa decemlineata Say) käitumisele. Agronoomia 2009, 192–197. Kivimägi, I., Ploomi, A., Luik, A. 2008. Süsi-ketasjooksiku (Platynus assimilis Payk.) külmakindluse sesoonne dünaamika. Agronoomia 2008, 139–142. Ploomi A., Kivimägi I., Luik A. 2007. Mõnede jooksiklaste allajahtu- misvõime. Agronoomia 2007, 137–140.

133 5.2. Conference abstracts:

Kivimägi I., Ploomi A., Metspalu L., Jõgar K., Švilponis E., Hiiesaar K., Luik A., Sibul I., Kuusik A. 2011. Impact of alpha-cypermethrin on the physiology of carabid beetle Platynus assimilis. Programme and Abstracts. Global Conference on Entomology, March 5-9, Chiang Mai, Thailand, 544. Century Foundation, India. Jõgar K., Metspalu L., Hiiesaar K., Ploom, A., Kivimägi I., Starast M., Tasa T., Luik A. 2011. Insect pests on different cabbage varieties. Pro- gramme and Abstracts. Global Conference on Entomology, March 5-9, Chiang Mai, Thailand, 202. Century Foundation, India. Kivimägi I., Ploomi A., Jõgar K., Metspalu L., Hiiesaar K., Luik A., Mänd M., Kuusik A., Sibul I. 2011. Supercooling capacity and cold hardiness of the Carabus granulatus L. (Coleoptera, Carabidae). Next generation insights into geosciences and ecology, 61. Doctoral students’ conference. Kivimägi I., Ploomi A., Metspalu L., Jõgar K., Hiiesaar K., Švilponis E., Veromann E., Mänd M. 2011. The influence of acclimation of the carabid beetle Platynus assimilis physiology. 4th International Sym- posium on the Environmental Physiology of Ectotherms and Plants, 84. Oxford University Press. Kivimägi I., Ploomi A., Metspalu L., Švilponis E., Jõgar K., Hiiesaar K., Veromann E., Mänd M., Kuusik A. 2011. Does Alpha-cypermethrin have influence on body mass of the non-target predacious carabid beetle? VIIth Arthropods, 18-23 September, Bialka Tatrzanska, Poland, 93. Ploomi A., Jõgar K., Metspalu L., Hiiesaar K., Sibul I., Kivimägi I. 2011. The effects of some biopesticides on cabbage pests. Interna- tional Conference on Biopesticides 6 (ICOB 6) „Biopesticides Shap- ing World Agriculture, Public Health and the Environment“, 11-16 December 2011, Chiang Mai, Thailand, 184. Hindawi Publishing Corporation. Ploomi A., Kivimägi I., Metspalu L., Jõgar K., Sibul I., Hiiesaar K., Kuusik A. 2010. The seasonal cold-hardening of the carabid beetle Platynus assimilis Payk. In: Abstract book: XXVIII Nordic-Baltic Congress of Entomology, Birštonas, Lithuania, 2.-7. August, (Eds.) Adrius Petrašiunas. 66. Ploomi A., Kivimägi I., Metspalu L., Švilponis E., Jõgar K., Hiiesaar K., Sibul I., Kuusik A. 2010. Cold-hardiness of the carabid beetle

134 affected by alpha-cypermethrin. Programme and Book of Abstracts, IXth European Congress of Entomology, 22-27. August, Budapest, Hungary, 186. National Organizing Committee of ECE 2010. Jõgar K., Metspalu L., Hiiesaa, K., Ploomi A., Kivimägi I., Luik A., Tasa T. 2010. Relationship between feeding on different plants and hibernation physiology in Mamestra brassicae. Programme and Book of Abstracts. IXth European Congress of Entomology, 22-27. August, Budapest, Hungary, 183. National Organizing Committee of ECE 2010. Kivimägi I., Ploomi A., Metspalu L., Švilponis E., Jõgar K., Hiiesaar K., Kuusik A. 2010. Impact of alpha-cypermethrin on body mass of the carabid beetle Platynus assimilis. Programme and Book of Abstracts. IXth European Congress of Entomology, 22-27. August 2010, Budapest, Hungary, 222. National Organizing Committee of ECE 2010. Metspalu L., Hiiesaar K., Jõgar K., Švilponis E., Ploom, A., Kivimägi I., Luik A. Menšhikova N. 2009. Oviposition preference of Pieris brassicae on different Brassica oleracea cultivars. NJF Report. NJF Seminar 422. International Scientific Conference „Fostering healthy food systems through organic agriculture - Focus on Nordic-Baltic Region“, Tartu, Estonia, August 25-27th, 41. Jõgar K., Metspalu L., Hiiesaar K., Ploomi A., Švilponis E., Kuusik A., Kivimägi I., Luik A. 2009. Influence of white cabbage cultivars on oviposition preference of the Pieris rapae L. NJF Report. NJF Seminar 422. International Scientific Conference „Fostering healthy food systems through organic agriculture - Focus on Nordic-Baltic Region“, Tartu, Estonia, August 25-27th, 41. Ploomi A., Jõgar K., Metspalu L., Hiiesaar K., Švilponis E., Kivimägi I., Luik A., Sibul I., Kuusik A. 2009. Effect of cultivar on oviposition preference of the cabbage moth, Mamestra brassicae L. NJF Report. NJF Seminar 422. International Scientific Conference „Fostering healthy food systems through organic agriculture - Focus on Nordic- Baltic Region“, Tartu, Estonia, August 25-27th, 40. Kivimägi I., Ploomi A., Metspalu L., Švilponis E., Jõgar K., Hiiesaar K., Luik A., Sibul I., Kuusik A. 2009. Physiology of carabid beetle Platynus assimilis. NJF Report. NJF Seminar 422. International Sci- entific Conference „Fostering healthy food systems through organic agriculture - Focus on Nordic-Baltic Region“, Tartu, Estonia, August 25-27th, 54.

135 Ploomi A., Jõgar K., Metspalu L., Hiiesaar K., Loorits L., Kivimägi I., Švilponis E., Luik A., Sibul I. 2009. The toxicity of neem against to the snail Arianta arbustorum Linnaeus, 1758. Abstracts of Inter- national Scientific Conference „Development of integrated plant protection strategies in horticulture“, September 17-18th, Babtai, Lithuanian Institute of Horticulture, 35. Jõgar K., Metspalu L., Hiiesaar K., Loorits L., Ploomi A., Kivimägi I., Švilponis E., Kuusik A., Luik A. 2009. Influence of NeemAzal- T/S on Mamestra brassicae L. (Lepidoptera: Noctuidae). Abstracts of International Scientific Conference „Development of integrated plant protection strategies in horticulture“, September 17-18th, Babtai, Lithuanian Institute of Horticulture, 23. Kivimägi I., Ploomi A., Luik A. 2008. Cold hardening of ground beetle Carabus granulatus L. International Plant Protection Conference Advances in Plant Protection Strategies, September 10-12th, 37. Kivimägi I., Ploomi A., Luik A., Jõgar K., Sibul I. 2008. Cold hardening of some ground beetle species. International Conference Diversifying Crop Protection, July 12-15th, 75.

136

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