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Xerox UnhrortRy Microfilms 300 North ZM b Road Ann Aibor, Michigan 40100 ELLINGSEN. Inger Johanne, 1944- COMPARISON OF ACTIVE AND QUIESCENT PROTONYMPHS OF THE AMERICAN HOUSE-DUST MITE. The Ohio State University* Ph.D.* 1974 Biology

Xerox University Microfilms,Ann Arbor, Michigan 46106

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED, COMPARISON OF ACTIVE AND QUIESCENT PROTONYMPHS OF

THE AMERICAN HOUSE-DUST MITE

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By Inger Johanne Ellingsen, Cand. mag., Cand. real., M. S.

The Ohio State University 1971*

Reading Committee: Approved by

Professor G. W. Wharton Professor F. W. Fisk Ad/M Advisor Professor R. P. Holdsworth Department of Entomology ACKNOWLEDGEMENTS

X would like to thank my advisor, Professor G. W.

Wharton, for guidance and encouragement throughout the investigation and preparation of this manuscript, and for assisting me in preparing programs for drawing graphs by the aid of a programable calculator. Thanks are due to the Department of Entomology for generously supporting me as a teaching associate during my stay at The Ohio State

University. The Acarology Laboratory is acknowledged for providing facilities for the investigation and the use of drawings by Dr. W. Bruce. Special thanks are due to

Professor F. W. Fisk and Professor R. P. Holdsworth for serving on my reading committee and for their review and criticism of the manuscript. VITA

February 13, 1944...... Born, Namsos, Norway

1966 ...... Cand. mag. The University of Oslo, Oslo, Norway

January, 1969...... Cand. real. The University of Oslo, Oslo, Norway

1969-1970...... Lektor at Tromso Gymnas Tromso, Norway

1971 ...... M.S., The Ohio State University, Columbus, Ohio

1971-1974...... Teaching Associate at The Ohio State University, Columbus, Ohio

PUBLICATIONS

"Fecundity, aphid consumption and survival of the aphid predator Adalia bipunctata L. (Col., Coccinellidae)". Nor. Entomol. Tidsskr. 16: 91-95, 1969.

"Effect of constant and varying temperature on development, feeding and survival of Adalia bipunctata L. CCol., Coccinellidae)". Nor. Entomol. Tidsskr. 16: 121-125, 1969.

Holdsworth, R. P., Jr. and I. J. Ellingsen. "Role of stigmaeid mites in the biological control of European red mited in Ohio apple orchards." Proc. N. C. Branch, Entomol. Soc. Am. 28: 104-105, 1973.

FIELDS OF STUDY

Major Field: Entomology Studies on biological control. Professor R. P. Holdsworth Studies on acarology. Professor G. W. Wharton.

iii TABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS...... ii

VITA...... iii

LIST OF TABLES...... vi

LIST OF PLATES AND FIGURES...... vii

CHAPTER

I INTRODUCTION...... 1

II IDENTIFICATION OF A LONGTERM QUIESCENT PROTONYMPH...... 9

Introduction...... 9 M e t h o d s ...... 11 Results and Interpretations...... 12

III OXYGEN CONSUMPTION...... 21

Introduction...... 21 M e t h o d s ...... 22 R e s u l t s ...... 23 Interpretations ...... 23

IV WATER B A L A N C E ...... 27

Introduction...... 27 Methods ...... 30 R e s u l t s ...... 33 Water mass of quiescent protonymphs at zero water vapor activity .... 33 Water mass of active and quiescent protonymphs at 0.75 water vapor a c tivity...... 3*1 Tritium loss and decrease in specific activity at av ** 0.75 of active and quiescent protonymphs . * 34 Interpretations ...... 59 TABLE OF CONTENTS, CONT'D.

Page

CHAPTER

V DISCUSSION...... 69

LITERATURE CITED ...... 77 LIST OF TABLES

Table Page

1. - Quiescent protonymphs and tritonymphs collected from the top of the culture j a r ...... 19

2. Dry weights of active and quiescent protonymphs killed in various ways .... 20

3. Oxygen consumption by active and quiescent protonymphs at a„ = 0.7S and 2 5 ° C ...... 26

4. Mean masses (in yg) of quiescent proto­ nymphs in zero water vapor activity . at 25°C...... 35

5. Quiescent protonymphs: mean measurements of water mass, tritium content and specific activity at a^ = 0.75 and 25 C. . 41

6. Active protonymphs: mean measurements for change in water mass, tritium content.and specific activity at a = 0.75 and 25°C...... v . . . . 43

7. Dead quiescent protonymphs (frozen for 24 hours): mean measurements of tritium content at ay = 0.75 and 25°C...... 44

8. First order rate' constants for tritium loss, km., and decrease in specific activity, k , Cboth with percentage of explained smms of squares of variation) at av = 0.75 and 25 C ...... 60

9. Half-lives...... "67

10. Constant rates of sorption (m_ = k m . x at a„ = 0.75 and 25°C...... 68 V

vi ’ LIST OF PLATES AND FIGURES

Plate Page

I* • The protonymph CA) and the tritonymph (B) of the American house-dust mite. Arrows are pointing to the genital field...... 15

Figure

1* Water loss in quiescent protonymphs at 0.00 water vapor activity and 25 C. The mites were assumed to die at day 19 so observations preceding day 19 are considered separately as live mites and observations succeeding day 19 as dead ...... 36

2. Water mass of active and quiescent protonymphs at 0.75 water vapor activity and 2 5 ° C ...... 38

3. Tritium loss in active protonymphs in an untritiated atmosphere of 0.75 water vapor activity and 25°C. Data from two separate experiments are plotted...... 45

4. Decrease in specific activity in active protonymphs in an untritiated . atmosphere of 0.75 water vapor activity and 25 C ...... 47

5.. Tritium loss in quiescent protonymphs in an untritated atmosphere of 0.75 water vapor activity and 25°C. The regression line is drawn according to a one rate model...... 49

6. Tritium loss in quiescent protonymphs in an untritiated atmosphere of 0.75 water vapor activity and 25 C. The regression line is drawn according to a changing rate at day 16 ...... 51 vii Decrease in specific activity of quiescent protonymphs in an untritiated atmosphere of 0 .75 water vapor activity and 25 C. The regression line is drawn according to a one rate model......

Decrease in specific activity of quiescent protonymphs in an untritiated atmosphere of 0.75 water vapor activity and 25°C. The regression lines are drawn according to a change in rate at day 16. . . .

Tritium loss in dead quiescent mites (frozen for 24 hours) in an un­ tritiated atmosphere of 0.75 water vapor activity and 25°C......

viii CHAPTER I

INTRODUCTION

The .life-cycle of arthropods consists of embryonic development within an egg and a series of instars that culminate in the adults. In the mite group Acaridei the overt preadult instars may include a larva, a protonymph, a deutonymph and a tritonymph. Each of these instars appears to consist of an active and an intermediate inactive period.

If we consider the time periods and processes within one instar they are: a pharate period during which the newly formed instar is located within the cuticle of the pre­ ceding instar; the pharate period ends by the process of ecdysis when the new instar emerges and the active period begins; the active instar is usually mobile and feeding, it

lasts through a certain number of degree-hours or until it has gained sufficient energy reserves to form the next

instar; the active period is succeeded by a short inter­ mediate inactive period during which the process of molting

occurs or a longer period of minimum metabolic activity

results in a quiescent period.

Under constant environmental conditions there is an

obligatory sequence of instars. In the Acaridei this sequence includes a period within the egg-shell during which a prelarva iB formed; it.molts into a larva which hatches. The larva after hatching feeds and molts.' The protonymph feeds and molts. If the deutonymph is present, it is.called a hypopus; it is different from the preceding and succeeding instars both in morphology and behavior. The hypopus does not feed but it lives on the reserves from the protonymph. In the cases where it is obligatory for all individuals in the life-cycle, it is specialized for phoresy

• providing a means of getting from one ephimeral habitat to the next. In other cases it is obligatory in a small but constant percentage of the population or facultative. The hypopial cuticle encloses the partially regressed organ- systems and some functioning hypopial systems (Wallace,

1960). The hypopus molts into a tritonymph. The tritonymph feeds and molts and the males and females emerge. After mating eggs are produced and the cycle repeats itself.

Under variable environmental conditions the life-cycle patterns of various kinds of mites are modified. In addition to instars whose function is feeding, developing

and reproducing, there will occur forms that are adapted to survive unfavorable conditions for example by phoresy or

long-term stress resistance. This may occur as a special­

ized or arrested period within an instar or as a whole

generation adapted for a special function. Examples of adaptations within an instar is a period of arrest in the over-wintering and aestivating eggs of Panonychus ulmi

(Koch), (Beament, 1951) and in Halotydeus destructor (Tucker)

(Wallace, 1970). Likewise, the adult female may be in a

diapausing condition through the winter as in Tetranychus

urticae Koch, (Dubynina, 1965) and in Neoseiulus fallacis

(Garman), (Rock et al., 1971). Examples of whole genera­

tions being specialized for seasonal changes >is observed

in eryophyid mites. At the end of summer special mummylike

females are formed that will not reproduce until next

summer (Keifer, 1952).

Diapausing and aestivating forms occur with seasonal

changes in the environment. With aperiodic environmental

changes, there are facultative modifications in the life­

cycle. The formation of specialized forms is induced by

stimuli changing development from a form adapted to one

environmental condition to a form adapted to another environ­

mental condition. Examples of environmental stimuli that

are reported to induce hypopus formation have been.reviewed « by Kuo and Nesbitt (1970). They are: extreme conditions

of temperature and relative humidity; the quality (for

example, comrent of vitamins like ergosterol) and quantity

of food; overcrowding, and waste-product accumulation.

Examples of stimuli triggering continuation of the molting

process in the hypopus are: favorable thresholds of humidity and or temperature, a temperature shock, addition of fresh food, or a special salt concentration. The hypopus must receive a molting stimulus before a certain critical period is reached, because it does not feed and must develop into a tritonymph before it exhausts its reserves from the proto­ nymph (Kuo and Nesbitt, 1970).

Hypopi are found in the freeliving Acaridei in the superfamilies Anoetoidea, Acaroidea and one family in the

Canestrinoidea. The rest of the Acaridei are infectious parasites of vertebrate animals with the exception of one family, the Pyroglyphidae (Krantz, 1970). Fain C1971) dis­ cusses the evolution of the different types of hypopi.

How do these forms, that are adapted to withstand environmental stress, manage to survive for long periods of time? They have the twin problems of budgeting their energy reserves and satisfying their oxygen requirements without losing too much water. Water loss is serious because biological membranes are known to be more permeable to water than to respiratory gases (Waggoner, 1967).

These specialized forms are non-feeding. They keep their Energy consumption down to a minimum. Reserves have been reported in hypopi of Caloglyphus mycophagus (Megnin),

CKuo and Nesbitt, 1971) where the parenchymateous tissue had Bpecial ovoid cells similar to the fat body of other arthropods. Eggs of P. ulmi have an accumulation of sorbitol, this, however, is interpreted as having a supercooling effect (Somme, 1965).

The degree of metabolic arrest is important for re­ sistance and duration of survival. Cryptobiotic cysts of the crustacean Artemia salina Leach are reported to have their metabolism completely arrested. Thus, they can afford

4 to be completely impermeable because respiratory exchange can be suspended (Morris and Afzelius, 1967). Near imper­ meability to water has also been found in eggs of P. ulmi

CBeament, 1951). This is not a problem for the overwinter­ ing eggs as their metabolism is almost entirely arrested

(Dittrich, 1972). The summer eggs, however, have a problem.

In T. urticae the maturing eggs at the prelarval stage have, underneath the transparent shell, an air-filled duct system and perforation organs to puncture the shell; the whole organ functions for exchange of respiratory gases

(Dittrich, 1972). Similar structures have been observed in the eggs of Dermatophagoides pteronyssinus (Trouessart),

(Spieksma, 1967), and in Dermatophagoides farinae Hughes

(Hodgson, pers. communication).

Hater conservation in metabolically active arthropods is dealt with in various ways. The problem becomes extreme as size decreases and the surface to volume ratio increases.

Most works on water stress in arthropods have considered cuticular permeability. Generally water proofing is accomplished by extracuticular wax-layers of very special structure (Locke, 1964). An outer wax layer was observed on the hypopus of Lardoglyphus kanoi CSasa and Asahuma),

(Vijayarabike and John, 1973), but not on C. mvcophaguB, (Kuo and Nesbitt, 1971). Both hypopi, however*, were reported to have thick cuticles and the hypopus of the former species

• was reported to be many times more resistant to penetration of fixative fluids than the active instars. Some arthropods exhibit decrease in cuticular permeability under dehydra­ ting conditions, and these supposedly lack extracuticular lipids, but possess endocuticles that are rich in lipo­ proteins CEdney, 19S7). Bursell (1955) found that when terrestrial isopods are exposed to dehydrating conditions, the rate of evaporative water loss falls for some time before it reaches steady state. He suggested that dehydra­ tion of the cuticle of isopods brings endocuticular lipids

in close proximity to each other, which results in a per­ meability decrease. Tardigrades (Macrobiotus areolatus

Murray) have special .permeable areas of the cuticle that were discovered by using dyes. These areas were observed to retract under desiccating conditions at the beginning of tun formation (Crowe, 1972). Tuns are barrel-shaped

resistant forms of tardigrades. During dehydration at low

humidities, the permeability coefficient of tuns decreased

a hundred fold; the tuns can be reduced to 2-3% water con­

tent in dry air without being killed (Crowe, 1972). The

impermeable P. ulmi eggs have.an inner shell layer enclosing

the living material which is extremely resistant to penetration and to attack by chemicals or solvents; it appears to be composed of a material similar to keratin

(Beament, 1951). The cryptobiotic cysts of Artemia also possessed very resistant membranes (Morris and Afzelius,

1967). Ghiradella and Radigan (19710 suggested that col- lembolan cuticle (Tomocerus flavescens Tullberg) of active individuals is impermeable to water, and they held that the gas exchange takes place elsewhere than through the cuticle.

Waggoner (1967) wrote that evolution has favored gas

t and water exchange from the moist surfaces on the bottom of pores, examples being the stomata Of plants, lungs of vertebrates and the tracheae of insects. It is known that active uptake of water can sometimes occur in cavities, i.e., the rectum of Thermobia, (Noble-Nesbitt, 1970) possibly also in the rectum of Tenebrio (Grimstone et al.» 1968), and in the mouth of the tick Amblyomma variegatum (Fabricius)

(Rudolph and Knulle,.197*4). Mechanisms suggested are: the humidity in the air spaces which form in the rectum is kept low by activity of the rectal epiteliura, as in Tenebrio

(Grimstone et al., 1968), then the opening of the anus would provide a channel for uptake of atmospheric water, provided that the humidity of the atmosphere was high enough.

Rudolph and Knulle (1974) reported that the saliva of A.

variegatum contains a hygroscopic salt; and the saliva

remained liquid above the critical relative humidity for

the tick. In D. farinae it is conjectured that a secretion 8 from the supracoxai gland coming down to the mouth may exhibit a similar pattern (Brody et al., in press).

This paper is going to discuss: (1) recognition of quiescent nymphs in the life-cycle of D. farinae; (2) their energy consumption and reserves in comparison with the active nymph at the protonymphal stage; and (3) equilibrium and non-equilibrium water exchange. CHAPTER II

IDENTIFICATION OF A LONGTERM QUIESCENT PROTONYMPH

Introduction

A modification of the relative duration of instars and the sequence of processes in them has been observed in the

American house dust mite (Dermatophagoides farinae, Acarina:

Pyroglyphidae) under low temperature conditions. At 15°C development is slow, mostly affecting the duration of the

intermediate inactive period of the protonymph, increasing it to an average of 143.8 days (Furumizo, 1973). In addi­ tion to the normal process within the intermediate inactive period, there must have been a period of metabolic rest or quiescence prior to ecdysis.

The process of molting starts with apolysis. In the

legs of the Acaridei the mechanism is different from what

is seen in.most insects. In the latter, the leg epidermis

separates from the cuticle and a new cuticle is secreted

within the old leg hull. In the Acaridei, the old leg

tissue dedifferentiates and regresses back into a limb bud

in the coxal region; the new limb grows from the bud into

the exuvial space between the old body cuticle and the new

CWoodring, 196 9)*

9

r 10

For comparison of the above mentioned modification in the life-cycle of D. farinae under low temperatures, the duration under optimum temperature conditions (25°C) as reported by Furumizo ..(1973) is: an egg (8 days), an active

4 larva (5.4 days), a first internediate period (2.8 days), an active protonymph (5.6 days4), a second intermediate period (2.7 days), an active tritonymph (4.4 days), a third intermediate period (.3.3 days), followed by adult males and females. Total duration of immature development is 32.2 days. Larson (1971) reported an average of 30 days at 25°C with a range from 24 to 35 days.

In what follows, mites with an extended second inter­ mediate period will be termed quiescent protonymphs or quiescent tritonymphs as the caBe may be. Special proper­ ties of these forms other than immobility and long duration are not known. However, as is found in other resistant forms in the Arthropoda, structural modifications could be expected, together with physiological adaptations.

The fine structure of the cuticle of a mite Laelaps echidnina Berlese has been described by Wharton et al.

(1968). Their identification of the layers was based on an

account of insect cuticle (Locke, 1965). Wharton et al.

(1968) described: a granular Schmidt's layer or subcuticle,

a nonsclerotized endocuticle, a sclerotized exocuticle, and

an epicuticle consisting of a dense layer with closely

packed thin lamellae, a cuticulin layer, a wax layer and a 11

cement layer. Brody aiid Wharton (1970) have an electron micrograph of a cross- Section of the cuticle of an active

instar of D. farinae. Measuring from their illustration,

the proportion of the. hickness of the epicuticle: exo-

cuticle: endocuticle i 3 approximately 1: **: 32.

Quiescent nymphs In the l4ife-cycle of D. farinae have

been observed* This c tiapter will discuss their frequency

of occurrence, their d|ur»ation and a comparison of wet and

dry weights of quiesce at protonymphs with those of active

protonymphs.

Methods

D. farinae was cu ltured in 0.5 oz. vials. The vials

were covered by cigare tte paper that was held in place by

a plastic snap cap hoi lowed out to a ring. The mites were

fed finely ground dry dog food. Human hair served as a

substrate and possibly also as a nutrient. This technique

has been described by Larson et al. (1969).

Active mites were collected from the bottom of the

culture vials where the food source was. The larger and

plumper individuals we=re chosen for experimentation,

Quiescent nymphs were brushed off from the lip of the vial

and the covering paper.

Quiescent nymphs were desiccated in completely dry

air over ^ ^ C it took months to obtain completely

dry mites, but raising the temperature to 60 C speeded up 12 the process considerably. Active mites were killed first, either by immersion in alcohol for 24 hours or by freezing for 24 hours. Then they were dried.

The mites were weighed on a model G-2 Cahn Electro-

i ’ balance which has a sensitivity of 50 nanograms. The weighings were done on the 0.S? mg range, and the balance was calibrated with class M weights. The point of the beam

balance which is at the null point of the balance was

found to drift at the 0.5 mg range. Because of this, zero

was rechecked after every weighing.

All weighings of dry mites were done in groups of 5

or 10 because the balance did not give reproducable

weighings for individual dry mites.

Results and Interpretations

In a typical crowded culture of D. farinae, two con­

centrations of mites were found. One, consisting of eggs

and all the active instars was at the bottom of the vial

with the food source. The second concentration was found

at the top of the culture, consisting of all instars but

mostly of immobile quiescent protonymphs. This character­

istic of the american house-dust mite to have concentrations

of mites at the top of the culture under crowded conditions

has been discussed by Wharton (1971) when comparing spatial

distributions of housedust mites. The protonymphs of D.

farinae showed thigmotatic behavior before becoming 13 quiescent. They crawled into corners of the covering paper and onto the rim of the culture vial where the snap cap fitted tightly* These mites anchored themselves to the glass or the paper by means of a sticky secretion.

The immobile mites were brushed off the paper and the

4 lip of the culture vial, and were kept for a couple of days.

After this period of time, the mites that were molting could be selected out, since ecdysis would occur in 2.7 days for the tritonymphs and in 3.3 days for the adults. If forma­ tion of new tissues was in progress in the immobile mite, the propodosoma became transparent as compared to the milky color of the hysterosoma. The developing appendages of the next instar can be seen in the propodosomal region of the previous instar when observed with transmitted light under the microscope. At this stage of development, the old legs were only cuticle with no muscle tissue evident.

Dead mites also appeared immobile. However, dead quiescent mites can be distinguished from living quiescent mites. The former have a dull appearance as opposed to the

shiny look of live mites. The dead quiescent mites were

also much less firm.

The rest of the immobile mites collected were uni­

formly milk-white in colors They stayed immobile for up

to two months or more, and some molted into tritonymphs.

Longlasting quiescent nymphs of two kinds exist: one is a protonymph and the other is a tritonymph. The latter are usually larger. For exact identification the quiescent mites were cleared in lactic acid and mounted on microscope slideB. Protonymphs have one pair of genital papillae

Whereas the tritonymph has two pairs; and the number of setae in .the genital field is -one pair in the protonymph and two pairs in the tritonymph (Plate I). Longterm • quiescent nymphs collected from the top of the culture consisted of 88.6% protonymphs and 11.*+% tritonymphs (Table

1). In a sample of 12 quiescent tritonymphs, three of them developed into pharate males, no pharate females were observed.

The fact that predominantly it is the protonymph that becomes quiescent, and that if it is a quiescent trito­ nymph, it produces a male when reactivated, may be connected with energy expenditure. The quiescent period consumes energy, so there will be less reserves for the molting process. More energy is expended to develop a female than a male, and more to produce an adult than a tritonymph.

Another possibility is that the protonymph may be more

susceptible to the stimulus that induces quiescence than the tritonymph.

When quiescent protonymphs are mounted on a microscope

slide in mineral oil under a cover slip, the pharyngeal

pump can be seen contracting, and the protonymphal leg

tissue is still present. No cast skins indicating a molt PLATE I, The protonymph (A) and the tritonymph CB) of the American house-dust mite. Arrows are pointing to the. genital field. M cn 16 before going into quiescence were observed. These observa­ tions indicate that the mites undergo the quiescent period while they,are still protonymphs.

The formation of.quiescent protonymphs was not obliga-

i tory in the life-cycle of D. farinae, only a small portion of the mites in a culture woul'd become quiescent. Quiescent mites were found under optimum conditions of temperature, relative humidity and food. It is conjectured that their formation was triggered by a crowding factor. This is different from Furumizo's observations where the long- lasting intermediate period waB observed only under low temperatures. It is the same stage that is being effected, however. Furumizo (pers. comm.) did not find any longterm protonymphs at 2S°C, but he reared D. farinae under un­ crowded conditions. The only way these forms were observed to be induced to molt was by repeated disturbance. In weighing experiments at 75% relative humidity (where the same mites were used), 7 mites out of 10 molted within 8 days. They had been disturbed and handled 4 times.

Mechanical disturbance may serve as a stimulus to reactivate development during the prolonged second intermediate period of the quiescent protonymphs* If we imagine the natural habitat of the housedust mite, quiescent protonymphs may be induced to continue development when a temporarily un­ used chair or bed is reoccupied by the human host. 17

Dried quiescent protonymphs appeared thick and white,

whereas dried active protonymphs were transparent, thin and

fragile. Desiccated active protonymphs contained white-

yellow solids that are not seen in quiescent mites. It

could be accumulated waste products, nutrients or some

metabolite. The dry weight of quiescent protonymphs was

1*1 yg and the dry weight of active protonymphs was 0,7 yg

Cof which 0.46 yg was soluble in alcohol) (.Table 2).

The cuticle of the quiescent mite was thicker. Whether

the specialization consists of an extracuticular lipid

layer or endocuticular lipoproteins has not been determined.

Probably another cuticular specialization is the secretion

of a sticky substance, possibly wax, for anchoring the

, quiescent nymphs.

When the quiescent mites were desiccated to dry weights,

they died in the process. Although it is not possible to

say when, they probably lived at least until half of their

water was gone. D . farinae females lasted until they had

lost 52% of their original water content CArlian, 1972).

However, it is known that stress resistant forms survive

severe dehydr.ation. Extreme examples are anhydoobiotic

cysts of the brine shrimp (life without water) (Morris and

Afzelius, 1967), and tuns of tardigrades can be reduced to

2-3% water content without being killed (Crowe, 1972). The

last 0.2 yg of water mass remained in these mites at least one month at 25°C. If the cuticle was ruptured, this residue appeared to have an oily consistency. Suggestions as to what it contained would be a fatty reserve, a carbo­ hydrate solution or a.glycerol solution. 19

TABLE 1. QUIESCENT PROTONYMPHS AND TRITONYMPHS COLLECTED FROM THE TOP OF THE CULTURE JARS

# of # of quiescent quiescent % of % of Total # proto- trito- proto­ trito- of Mites nymphB nymphs nymphs nymphs

Mites recovered 216 204 12 94.6 5.6 from water balance studies

Others 1899 1692 217 88.6 11.4 20

TABLE 2. DRY WEIGHTS OF ACTIVE AND QUIESCENT PROTONYMPHS KILLED IN VARIOUS WAYS

Mean Type # of Masses in Standard of Mite Mites pg Error

Active protonymphs freeze dried 130 0.70 ±0.01

Active protonymphs killed in alcohol 100 0.24 ±0*03

Quiescent proto­ nymphs desiccated at 60°C over P20g 90 1.07 ±0.04 I

CHAPTER III

OXYGEN CONSUMPTION

Introduction

Energy requirements of mites have been measured as oxygen consumption by: Arlian C1972), Berthet (1964),

Engelmann (1971), Kanungo (1965), LeBrun (1971)., Wallwork

(1967), Webb (1969 and 1970) and Wood and Lawton (1973).

Some of the mentioned works were done by indirect measure­ ments based on the general assumption that there exists proportionality between live weights of small arthropods and their oxygen consumption (Keister and Buck, 1964). Formu­ las for converting width and length into weight and then to oxygen consumption were worked out by Berthet (1964) and

LeBrun (1971). Direct and comparative studies of oxygen consumption, however, have also been done. Examples are comparisons of the different life-stages by Webb (1969) and of mites occupying different ecological niches within an ecosystem by Wood and Lawton (1973). Wood and Lawton found that active predators consume more oxygen than

sluggish detritus feeders.

Oxygen consumption has been measured for different diapausing insects. Plots with time on the x-axis of

21 22 respiration rates of the diapausing stage plus its pre­ ceding and succeeding active stages produced a typical U- shaped curve with a minimum in the diapausing range. An example of this U-shaped curve is given by Villacor et al.

(1972) from works on diapausing sawflies. Oxygen consump­ tion is low and stable during insect diapause. This low respiration rate in diapausing insects, however, signals a qualitatively different sort of metabolism (Keister and

Buck, 196U).

Here a comparison of the oxygen consumption of the two forms of the protonymph will be investigated in order to estimate the degree of metabolic arrest in the quiescent form compared to the respiration rate of the active form.

Methods

Oxygen consumption was measured by the Cartesian diver technique. Small divers to accommodate these mites were made according to Arlian (1973). Differences in pressure required to make the divers float at a constant level were recorded by an assembly described by Kanungo (1965).

Activity of the water vapor inside the divers was main­ tained at 0.75 by the concentration of a KOH seal in the neck of the diver (22.25 gramB of KOH per 100 gram solu­ tion) . Temperature of the floating vessels was maintained constant within 0.1° Celsius by a water jacket circulating water from a 'Magni Whirl* utility bath. Both humidity 23 and temperature conditions were optimum for D. farinae

(Furumizo, 1973). A diver with all the seals but no mite, a thermo-barometer, was floating together with the experi­ mental divers. Pressure changes observed for the experi­ mental divers were corrected according to the thermo-barome­ ter (Arlian, 1972). From this the actual pressure change due to 02 -consumption was obtained.

Time intervals for reading the divers for active and quiescent nymphs were different. Active protonymphs were observed every two hours for an average of 8 hours, as they rarely survived overnight. Quiescent mites were kept in the divers for a week and measurements were taken daily.

Any mite that had been wetted by the KOH seal was discarded.

Results

Active protonymphs consumed 0.0114 microliters of oxygen per hour per mite, whereas quiescent protonymphs consumed 0.0027 microliters per hour per mite. This is a fourfold difference. When volume of oxygen is converted into calories', the energy consumption of active protonymphs -5 is 5.7 x 10 calories per hour per mite, and for quiescent protonymphs it is 1.4 x 10*’’® calories per hour per mite

(Table 3).

Interpretations

High: oxygen consumption in active protonymphs and low consumption in quiescent protonymphs follows the same 24 pattern as described for comparisons of diapausing and non- diapausing stages of insects. The decrease in oxidative respiration was not as dramatic as have been reported for insects (Keister and Buck, 1964), however, with the mites, the possibility of metabolism other than oxidative was not investigated.

The energy consumption of a population of mites of different developmental stages and in different physiologi­ cal conditions measured by indirect measurements gives only rough estimates. As demonstrated above, there can be a fourfold difference in energy consumption in two different physiological forms of the same instar.

The active protonymph of D. farinae also consumes energy at a higher rate than a standardized non-egglaying female under the same temperature and humidity conditions, the females lasted longer in the diver. They consumed

0.0083 pl/hr/mite (Arlian, 1972) compared to the active protonymph's consumption of 0.0114 pl/hr/mite. The low survival rate, of active protonymphs may have been due to exhaustion of energy reserves because of their high metabo­ lism per unit body-weight. If all the alcohol soluble dry weight of the active protonymph (Table 3) is assumed to be carbohydrate that is metabolized at the rate of 4.5 x 10“5 calories per hour per mite, their entire dry weight would be consumed in 32 hours. That it all should be carbohy­ drate is not possible since a mite must contain a number of 25 other dry substances besides its protein and cuticle, and it also accumulates waste products in the form of guanine.

Webb Cl969) reported that the protonymph of Nothorus silvestris Nicolet, has the highest respiration rate per unit body weight of all the instars, and generally the growing and the reproducing mites consume the most energy. -5 Quiescent mites consumed 1.4 x 10 calories per hour.

In order to last for 60 days with the same and constant —3 respiration rate* they would need 2.02 x 10 calories. — 3 Since 1 yg of fat yields 9 x 10 calories when oxidized, the quiescent mites would need 2.2 yg of fat reserves to satisfy a constant energy consumption as reported. For this reason, the reported energy consumption cannot be a correct average of the requirements for the whole period.

The mites were measured for a week only. The whole experi­ mental procedure of loading the divers might have caused enough stress or damage to raise the metabolic activity of the mite. As mentioned earlier, mechanical disturbance may be a stimulus for triggering onset of the molting process. TABLE 3. OXYGEN CONSUMPTION BY ACTIVE AND QUIESCENT PROTONYMPHS AT 25 C and ay = 0.75

Mean 02 , °2 Energy # of Consumption — Consumption Consumption b/ Mites in yl/hr/mite Standard in in (pl/hr/pg) Error pl/hr/mite cal/hr/mite

Active protonymphs 55 0.0114 ±0.0013 3.5 x 10'** 5.7 x 10“7 (0.0032)

Quiescent protonymphs i*8 0.0027 ±0.0006 1.1 x 10“U 1.4 x 10“7 (0.00077)

—^ A test for difference between the two means gave t s 6.522, which corresponds to a difference on the 0.1% conficence level.

—^ This column is calculated according to 1 ml of 02 corresponding to 5 calories (Bayliss, I960, vide Kanungo, 1965). CHAPTER IV

WATER BALANCE i

Introduction

The tendency of water to move by diffusion from one

compartment to another is conveniently expressed in terms

of the activity of water because activity can be used for

all three phases: vapor, liquid and solid. The activity is

proportional to the molar concentration of water: no water has an activity of 0 and pure water has an activity of 1.

Thus, water will diffuse from compartments of high

activity to compartments of low activity.

Activity of water vapor in the air (av ) is numeri­

cally equal to the relative humidity of the air divided

by 100. The water activity of the haemolymph of the mite •

is designated a... The a., of D. farinae is 0.987 (Wharton

and Arlian, 1972). This value must be maintained within

narrow limits, or metabolism will slow down or stop. Edney

(1968) discusses the effect of water loss on the haemolymph

of the cockroaches Arenivaga sp. and Periplaneta americana

(L.). The haemolymph osmotic pressure increased during

dehydration, but was subject to strong osmoregulation

mostly by removal of chloride ions.

27 ‘ 28

Water getting out of a mite (transpiration) is forced by the vapor pressure of the water pool of the mite: Py x aw .

Where Py is the vapor pressure of water in the air at saturation at that particular temperature. Transpiration is resisted by the impermeability of the barrier between the water pool of the mite and the atmosphere. Water enters the mite in two ways, actively and passively. The force responsible for the passive sorption by diffusion is directly proportional to the vapor pressure of the air: Py x ay . Sorption is also held back by the impermeability of the interface.

The critical equilibrium activity (CEA) is the lowest activity at which the organism can maintain a constant water mass. When ay is less than aw , an active mechanism is required. At constant activities above the CEA the water mass of the mite remains the same and we say that the conditions are at equilibrium. This is a dynamic equili­ brium where the amounts of water transpired or sorbed must be equal to each other and constant, because the water mass is constant. This means that at equilibrium conditions transpiration and sorption rates are zero-order processes in time.

A solid initially in equilibrium with a given ay will gain or lose weight when placed in an atmosphere at a

different ay . The relations for evaporation or condensation 1

29 by a small surface limited solid which had been in equili­ brium with one vapor pressure and then was plunged into another at.some constant vapor pressure reduce to first order kinetics (Wharton and Devine, 1968):

Cm^. - m^) = (mQ - mB ) e"1^* (l) or

In (mt - mw )/Cm9 - mB )' = -J^t (2) where m. is water mass at time t, m„ is water mass at equilibrium (or time infinite), mQ is water mass at time zero, kjg is the rate constant for net change in water mass, and t = unit time.

Analysis of data on loss of tritum and decrease in specific activity of a mite that was in equilibrium with a tritiated atmosphere, and then is placed into an untritiated atmosphere of the same av , have been based on the above model. That tritium loss is a first order rate process means that every molecule of HTO inside the mite has the same chance of getting out. Rate of loss can also be expressed as a constant percentage of what is left.

The above mentioned concepts and models on water balance.in mites have been worked out by Wharton and Devine

(1968), Wharton and Arlian (1972), Knulle and Devine (1972),

Devine and Wharton (1973), and by Arlian and Wharton (1971).

Some other works on water-balance and cuticular permeability of acarines include: Hafez et al. (1970), Knulle (1965), 30

Lees (1947), Madge (1964), McEnroe (1961), and Winston and

Nelson (1965).

Half-lives of water loss of some mites have been summarized by Wharton and Arlian (1972). Short half-lives are connected with permeable cuticles. Different measures have been used to express permeability of an organism to water. Crowe (1972) used the same permeability factor as used for diffusion through cell membranes. Arlian and

Wharton (1974) have a dimensional analysis of the rate constant for transpiration and for the constant rate of sorption. In the present study, rates of tritium loss are used to compare the permeability to water in active, quiescent and dead forms of the protonymph of D. farinae.

Methods

During these experiments constant temperatures were maintained within one tenth of a ° Celsius in BOD incu­ bators. Constant water vapor activities were maintained in desiccators over saturated salt solutions. An ay ~ 0.75 was obtained by saturated sodium chloride solutions and completely dry air by p2°5 (Winston and Bates, 1960).

Cannibalism is common in D . farinae (Brody et al.,

1972). For this reason the active mites had to be caged during experimentation. Cages were made from 5 mm diameter aluminum disks, with a circular depression 3 mm wide and

1 ram deep. A hole was made through the thin bottom of the I 31 depression with a minuten needle. The hole was too small for the mite to escape, and large enough to allow exchange with the ambient air. Once a mite was placed in the depression, the cage was sealed off by a 5 mm diameter D piece of parafilm .

When being weighed, the active mites were slowed down by placing their cages on a pan in ice-water.

In the experiments using tritiated water, the dry mass' of individual mites could not be obtained. Therefore mean dry masses of 1.1 yg and 0.7 yg were subtracted from the wet masses of quiescent and active protonymphs respectively to obtain net water mass per individual mite.

The equilibrium chamber for loading mites with triti­ ated water was a small desiccator. It contained a saturated sodium chloride solution (ay = 0.75 at 25°C) made by using tritiated water. The specific activity of the tritiated water used was about 1 mCi/ml. The water vapor in the air that is in equilibrium with this salt solution contained the same proportion of labelled molecules as the salt solution.

Hites that were exposed to this atmosphere exchanged unlabelled water for labelled water. Active mites con­ tained a measurable amount of water with a tritium label after 24 hours exposure time. Quiescent mites need a longer time to taJce up significant amounts. If they had to take up HTO as quiescent mites, they would not last long

enough afterwards for completion of a tritium experiment.

Por this reason whole culture jars were kept in the exposure

chamber. These cultures were put in with the top free of quiescent mites. Quiescent mites harvested from the top of

these cultures and would then contain the same proportion

of tritium as the other water compartments in the chamber.

After predetermined periods of time in an untritiated

atmosphere, samples of mites were put individually into

vials containing scintillator solution. These vials were

assayed for tritium activity by a Tricarb liquid scintilla­

tion spectrometer, Model No. 12003 (Packard Instrument,

Inc.). The cocktail which was found to be the most effi­

cient for tritium counting (Cutcher, 1970), and which was

used throughout the investigation consisted of: 1000 ml

toluene, 10 ml absolute methanol, and 7.5 g butyl-PBD

(Packard). Ten ml of the cocktail were used to count each mite, and each vial was counted long enough to get about

10,000 counts above background (i.e., 10 minutes). Once a

sample was placed in the spectrometer, a period of roughly

three hours was required for the cpm to reach a constant

level. The normal background was determined by including

a control cocktail with each series of samples. The back­

ground count, consistently about 20 cpm, was subtracted

from each sample to obtain the net count. Quiescent mites were allowed to dry out, then they were assayed for tritium content. The obtained radioactive count represented tritium not in the water but in a toluene soluble substance of the mite. It was subtracted from each observed tritium count to obtain net counts for the water pools alone. This concerned the mites grown in a tritiated atmosphere only.

Results

Water mass of quiescent protonymphs at zero water vapor activity.

Quiescent protonymphs lost significant amounts of water when exposed to completely dry air (Table 4). At equilibrium or time infinite all the water mass had dis­ appeared (i.e., mw = 0). If we put = 0 into equation

(2) we get:

In mt/m0 = or In m t = + In m Q C3).

The latter is the formula for a straight line where In mQ is the Y intercept and -1^ is the slope of the line. A plot of the natural logarithm of the observed masses (In m^) against time fits a straight line. ThiB can be interpreted as water loss being a first order process in time. A regression analysis gives km = 0.0017 per hour (or 0.0H06 per day) and 93% of the sums of squares of the variation can be explained by the relationship expressed in equation 31+

The above results are from mites that must have died during the experimentation. They probably remained alive for 19 days at least, when about 60% of their water mass was gone. We can consider the first 19 days separately as

i live mites, and the last 24 days separately as dead mites.

By treating the data in the same way as above, we get km

Clive mites) = 0.0014 per hour, and km (dead mites) =0.0033 per hour.

Water mass of active and quiescent protonymphs at 0.75 water vapor activity.

The experiments were done under equilibrium water vapor conditions. According to Larson (1969) the CEA for

D. farinae is 0.70. The weight of the mites stayed close to constant. Water mass of quiescent mites (Table 5) had a total mean of 2.3 + 0.012 yg, and the water mass of active mites (Table 6) stayed at about 3.6 yg. A test for significance in regression based on the null hypothesis that the slope is not different from zero, gave no signifi­ cance in regression. Generally, the water mass of individ­ ual active mites vary much more than the water mass of quiescent mites (Figure 2).

Tritium loss and decrease in specific activity at av = 0.75 of active and quiescent protonymphs.

When the mites that were in equilibrium with a tritiated 35

TABLE 4. MEAN MASSES Cin ug) OF QUIESCENT PROTONYMPHS IN ZERO WATER VAPOR ACTIVITY AT 25°C

Time held in desiccating atmosphere in # of • Total weights Water Mass hours (days) Mites ± S. E. l S. E.

0 (0) 15 3.5 ± 0.1 2.4 ± 0.1

192 (8) 10 CO • ± 0.1 2.1 ± 0.1

264 (11) 10 2.8 ± 0.2 1.7 ± 0.2

384 (.16 ) 10 2.5 ± 0.1 1.4 ± 0.1 o CM 456 (.19) 10 2.2 ± * 1.1 ± 0.2

576 (24) 10 2.1 ± 0.1 1.0 ± 0.1 00 696 (29) 10 1.9 ± 0.1 o . ± 0.1

960 (40) 15 1.3 ± 0.0 0.2 ± 0.0 4

JPIGURE 1. Water loss in quiescent protonymphs at 0.00 water vapor activity and 25°C. The mites were assumed to die at day 19; observations preceding day 19 are analysed separately as live mites, and observations succeeding day 19 as dead mites.

36 LN Dr HATER HR55E5 b B til. 0 0 2 0 1 R5 N R RR “ R1R DRY IN DRY5 IS 30 4

FIGURE 2 .Water mass of active (0) and quiescent (X) protonymphs at 0.75 water vapor activity and 25°C.

38 HERN NRTER MRS5E5 IN HICRDGRRHS fH Ln ni‘ l i t tii it L 0 0 S 0 S 0 S 0 HE H0 3S 30 2S 20 IS 10 TIME IN TIME S 7 . 0 WATER VAPDR ACTIVITY 0 ACTIVE HITES (TIME IN ACTIVEHITES (TIME HC11R5) 0 QUIESCENTHITES (TltiE INX DAYS) i i i i to CO 40 atmosphere are plunged Into an untritiated atmosphere, they lose measurable amounts of tritium (Tables 5, 6, and 7)*

Since the mites are killed once they are put into the scintillator fluid, at each time interval a different sample was assayed.

Tritium loss and decrease* in specific activity (cpm/pg water mass) of active protonymphs seemed to fit a first order model (Figures 3 and 4)'. The rate of tritium loss

was 0.034 per hour; and the rate of decrease in specific activity ke was 0.41 per hour. The two slopes are not significantly different from each other (Table 8).

Plots of n'atural logarithm of tritium content or of specific activity against time for quiescent mites do not fit a straight line very well (Figures 5 and 7). The data can be analyzed as a first order process where tritium is being lost at a constant rate, or as tritium being lost at an early fast rate, and then at a later slow rate (Figures

6 and 8).

If the quiescent mites are assumed to lose water at a constant rate, by regression analysis, the rate constant of tritium loss k^. is 0.0006 per hour (or 0*015 per day); and the rate constant for decrease in specific activity kg is

0.0007 per hour (or 0.017 per day) (Table 8). These two slopes are not significantly different from each other. ui

TABLE 5. QUIESCENT PROTONYMPHS. MEAN MEASUREMENTS OF WATER MASS, TRITIUM CONTENT AND SPECIFIC ACTIVITY AT WATER VAPOR ACTIVITY 0.75 AND 25°C

Tritium Specific Time out of Water Content Activity in # of Tritium in Mass in in cpm cpm/yg of Water Mites hours (days) yg l S.E. ‘ 1 S.E. ± S.E.

8 0 CO) 2.4*0.07 2605.0*134.8 1047.3*61.9

9 145 (2) 2.410.06 2283.31141.9 925.2141.8

9 95 C4) 2.310.07 2222.11119.6 982.7147.0

8 145 C6) 2.2810.03 2523.91172.1 1106.5174.8

8 189 C8.) 2.3110.05 1978.5198.1 850.9137.8

9- 239 (10) 2.2910.09 1899.11167.8 838.9177.6

8 287 (12) 2.2910.05 1533.31128.9 671.1154.8

5 336 (14) 2.3410.05 1694.31172.5 735.7170.2

6 384 (16) 2.3410.04 1086.3171.2 455.9129.3

7 432 (18) 2.3810.07 1237.41181.2 500.4168.3

7 480 (20) 2.3810.06 1102.01104.5 419.1144.9

9 529 (22) 2.3410.05 1142.8174.7 491.5137.1

7 574 (24) 2.2310.07 1225.01163.4 481.4158.9

8 622 (26) 2.3710.04 1062.8154.5 446.4118.4

9 673 (28) 2.3810.05 1000.0193.5 420.8137.7

5 743 (31) - 982.8156.7 435.1116.5

# 9 793 (33) 2.3410.04 1037.91100.3 440.3138.5

8 841 (35) 2.4110.05 1567.51128.3 577.3169.6 42

TABLE 5. CONT’D.

Tritium Specific Time out of Water Content Activity in # of Tritium in Mass in in cpm cpm/yg of Water Mites hours (days) yg 1 S.E. ± S.E. ± S.E. i •

9 889 (37) 2.28±0.03 1217.0±106.8 534.0150.0

8 936 (.39) 2.38±0.03 1344.41125.9 570.3156.4

8 984 (41) 2.2710.05 970.7169.9 428.5128.4

9 1032 (43) 2.33±0.08 1265.81163.6 555.4166.3

8 1080 (45) 2.34±0.03 1364.41129.2 588.5156.4

9 1126 (47) 2.23±0.05 828.2H00.0 372.2145.3 TABLE 6. ACTIVE PROTONYMPHS, MEAN MEASUREMENTS FOR CHANGE IN WATER MASS, TRITIUM CONTENT AND SPECIFIC ACTIVITY AT WATER VAPOR ACTIVITY 0.75 AND 25°C

# of Time out of Water Mites Tritium in Mass in Tritium Content Specific Activity hours lig ± S.E. in cpm 1 S.E. cpm/yg l S.E.

6 0 3.410.5 295.2161.3 100.3122.1

6 5 3.6±0.3 173.6128.0 56.11 9.3

5 10 4.410.3 203.4144.4 49.21 6.7

5 15 3.410.2 149.9111.8 51.01 4.5

4 20 4.110.4 134.7120.4 * 37.51 2.6

2 25 2 .610.1 65.9121.2 38.91 7.9

4 1 - 204.4126.8 -

5 6 - 224.1135.6 - -

4 11 - - 221.1169.3 -

4 16 - 117.3123.1 -

3 21 - 123.9134.9 -

1 26 - 91.0 -

-p CO 44

TABLE 7. DEAD QUIESCENT PROTONYMPHS (FROZEN FOR 24 HOURS) MEAN MEASUREMENTS OF TRITIUM CONTENT AT WATER VAPOR ACTIVITY 0.75 AND 25°C

Time out of Tritium Content # of Tritium.in in opm Mites ( .... hours (days) i S. E.

9 .0 (0) 2678.5^103.2

9 24. (1) 2428.81138.9

9 47 (2) 2220.1±123.9

9 72 (3) 2381.31121.7

a 97 (4) 2050.91129.7

9 120 (5) 1957.51 92.1 » 9 144 CO 1685.91145.3

9 168 (7) 1923.7H41.1

9 192 a> 1747.71211.2

9 216 (9) 1847.6H23.2

9 240 (10) 1723.41117.0

9 312 (14) 1269.51147.3

9 384 (18) 491.1H53.8

9 456 (22) 466.91106.5

4 504 (25) 175.11 54.2

9 576 (29) 0 FIGURE 3, Tritiujn Loss in Active Protonymphs in an Untritiated Atmosphere of 0.75 Water Vapor Activity and 25°C, Data From Two Separate Experiments are Plotted. LN DF TRITIUM CONTENT IN CPH a i i t ut l i t l i t 0 0 £ 0 S 0 25 20 IS 10 HOURS OUT OF TRITIUM OF OUT HOURS

DAYS DUT DF TRITIUM os FIGURE 6. Tritium Loss in Quiescent Protonymphs in an Untritiated Atmosphere of 0.75 Water Vapor Activity and 25°C. The Regression Lines are Drawn According to a Change in Rate at Day 16.

51 * UN OF TRITIUM CONTENT CPM IN 7.0 m ea ca ui iv i m ta

DAYS OUT OF TRITIUM ZS * FIGURE 7. Decrease In Specific Activity of Quiescent Protonymphs in an Atmosphere of 0.75 Water Vapor Activity and 25 C. The Regression Line is Drawn According to a One Rate Model.

53 LN OF SPEC.RCTIV.(CPM/WHTER MR55; 0 I I 2 2 3 3 M MS MB 3S 30 2S 20 IS IB S 0 . ca til 4 f Y OUTDflY5 TRITIUM OF -P cn FIGURE 8. Decrease in Specific Activity of Quiescent Protonymphs in an Untritiated Atmosphere of 0.75 Water Vapor Activity and 25°C. The Regression Lines are Drawn According to a Change in Rate at Day 16.

55 UN DF 5PEC.HCTIV.(CPM/WRTER HR55> 83 83 us i/i to r* DAYS OUT TRITIUM DF cn Cn FIGURE 9., Tritium Loss in Deaid Quiescent Protonymphs CFrozen for m Hours) in an Untritiated Atmosphere of 0.75 Water Vapor Activity and 25 C.

57 LN Dr TRITIUM CONTENT K P M J ID til til I>HY5 DEAD AND OUT TRITIUM OF 59

However, only U5% of the sums of squares of the variation in tritium content can be explained as due to dependence in time.

If changing slopes are postulated, the rate constant for tritium loss k^. for the first 16 days was 0.0032 per hour Cor 0.076 per day); and the rate constant for decrease in specific activity k was 0.0026 per hour (or

0.063 per day). The explained sums of squares are 76% for tritium loss and 66% for decrease in specific activity.

After day 16 the rate constant for tritium content had a positive value of 0.00018 per hour, however, only if% of the sums of squares of the variance could be explained as due to dependence of time. This means that the slope is not significantly different from zero.

Tritium loss in dead quiescent protonymphs seemed to fit the first order model (Figdre 9). The rate constant for tritium loss k^. was 0.0Q*+1 per hour (or 0.098 per day) (Table 8).

Interpretations

It is assumed that the pool from which tritium is lost behaves as a single well-mixed pool containing normal and tritiated water of known specific activity. At equi­ librium conditions, the amount of water sorbed is equal to the amount transpired. When water is sorbed, the concen­ tration of tritium (specific activity) in the mite will 60

TABLE 8. FIRST ORDER RATE CONTANTS FOR TRITIUM LOSS, k-,., AND DECREASE IN SPECIFIC ACTIVITY, k , (BOTH 1 WITH PERCENTAGE OF EXPLAINED SUMS OF SQUARES . OF VARIATION) AT a = 0.75 AND 25°C.

Decrease in Explained Tritium loss Explained Specific Sums of -kj. per Sums of Activity Squares hour Cday) Squares -k3 per hour

Active proto­ 0.0344 78.2% nymphs £/ 0.0343 ■78.3% 0.0412 81.0%

Quiescent 0.0006 45.3% 0.0007 50.6% protonymphs— C0.Q15) CO. 017) Cone pool model)

Ctwo rate, 0.0032 76.4% 0.0026 66.6% initial) (0.076) (0.063)

Ctwo rate, 0.00018—^ 7.1%* -0.00028 25%

second (-0.0043) (-0.0068) slow) Quiescent protonymphs 0.0041 89.5% ' frozen dead (0.098) •

a / — ' Not significantly different from zero; all other k's are significant.

—^ No significant difference between k^. and kQ . 61

decrease, so that the rate of decrease in specific activity

is an indication of water moving into the mite. If trans­

piration is the only process, the amount of tritium will be

reduced, but specific- activity will not change. It was f observed that the rate of decrease in specific activity, ks ,

4 is in close agreement with the rate of tritium loss, kp.

(Table 8). This is interpreted as meaning that the observed movement of tritium is indicative of the movement of water.

Therefore whenever patterns of tritium loss are considered,

gross transpiration of water has the same characteristics.

Transpiration of tritium and thus water, as well as

decrease in specific activity fit a first order model in

time for active and quiescent protonymphs above the CEA.

Loss of exchangeable water by quiescent protonymphs in * completely dry air and dead quiescent protonymphs at ay

0.75 also (mathematically) followed first order kinetics.

Similar models have been proposed for the spiny rat mite,

ticks arid female D. farinae (Wharton and Devine, 1968;

Knulle and Devine, 1972; and Arlian and Wharton, 197*0.

Arlian and Wharton (197*0 discussed water loss from a two

pool system under non-equilibrium conditions. Their fast

pool was suggested to be leakage from the inactive water

pump. Both pools obeyed the first order rate hypothesis.

However, if the data on tritium loss of quiescent

protonymphs (above the CEA) are analyzed as a first order 62 process In time, only half of the variation can be explained as due to dependence on time. If the data are analyzed in this way, the variation not due to dependence on time needs to be explained. The mites that were used for the experi­ mentation were not of the same age; it can be conjectured that the variation was due to differences in physiological condition with age. Another possibility was that some of the material might have been dead or dying and therefore losing water at a different rate. To investigate whether some of the data on quiescent mites could be disregarded as

» coming from dead mites, tritium loss in dead mites was studied. Tritium loss in dead and live (disturbed) qui­ escent mites (Tables 5 and 7) can be compared: At time zero they are equal; from day 2 to m tritium content of dead mites (average values) was about one standard error less than for live (disturbed) quiescent mites. Thereafter, the dead mites kept losing tritium by the same constant percentage of the remaining tritium (the explained sums of squares were 89.5%); whereas the tritium Io s b in live

(disturbed) quiescent mites approached zero and showed considerable variation; Kate of water loss (Figure 1) in dying mites increased with time while change in rate of tritium loss in live (disturbed) quiescent mites decreased.

No conclusion on the viability of the mites for the first m days can be made, however, the considerable variation 63 thereafter can not be considered as due to dead mites.

It seems preferable to analyze the data on tritium loss in quiescent mites as having two rate constants; an initial rpaid rate by which about 60% of the tritium content is lost, and a subsequent zero rate such that the last 40% of the original tritium content remained in the mite for at least another 30 days. A broken line (Figures 6 and 8) fits the data points better. For the initial fast rate the explained sums of squares was 77.4%. The slope of the line from 16 to 47 days is not significantly different from zero. * Why a change in rate constant is plausible can be explained in several ways. One suggestion is that the mites exchanged water with the atmosphere for the first 16 days, and then they became impermeable. However, since the mites were a mixture of all ages, they needed to be synchronized by an external factor such as being disturbed when they were brushed off the lip of the culture vials where they were attached. The sides that were attached might be more permeable until new secretion was produced.

They could also have been injured, and lost water until they were repaired. Thus, suggesting that normally (when undisturbed) quiescent protonymphs are practically im­ permeable to water loss. An alternative suggestion is that after, a certain time, the disturbed quiescent protonymphs started to metabolize a reserve that contained tritium, 64 thus radioactive metabolic water would add to the specific activity of the exchangeable water in the mite. A third suggestion is that the tritiated water in the mite could be in two pools that were not exchanging water with each other.

One of these pools Cconnected to the water pump) exchanged water with the atmosphere, whereas the other did not. The non-exchangeable tritium is thought to be in the isolated water pool, rather than in some touluene soluble substance.

The reason being that dead quiescent mites that dry out completely have only 35 cpm above background. Of the three explanations, the first one is preferred.

Since aM of the haemolymph is 0.987 and the experi­ mental ay was 0.75, passive sorption driven by the water vapor pressure of the ambient air cannot possibly be equal to the transpiration from the mite. There has to be active uptake of water into both active and quiescent mites above the CEA. Quiescent protonymphs kept in ay = 0 where sorption is'impossible, did lose water. The work of active uptake is done against at least 288.6 atmospheres of dif­ ference in osmotic pressure Cconversion tables for ay into osmotic pressure in Wharton and Devine, 1968).

At equilibrium when the rate constant for tritium loss and decrease in specific activity were found to be equal, the constant rate of sorption can be expressed as ng = fcj,. x m w CWharton and Devine, 1968). The m s for active 65 protonymphs was 0.095 pg per hour (Table 10). Assuming that the driving force for passive sorption is directly pro­ portional to ay , and given that at equilibirum sorption is equal to transpiration, we can find the passive component of sorption at ay s 0.75 by the proportions: ay/aw = (pas­ sive sorption at ay)/(total transpiration at aM>. The passive component of sorption in active mites was 0.0722 pg per hour, and the active component was 0.023 pg per hour.

The minimum energy needed to actively sorb one mole of water from ay = 0.75 to aw = 0.987 is given by the formula: 6 =

-RT ln(ay/aw >. The energy needed for active sorption in active mites was at least 2.1 x 10 calories per hour, or

0.3% of their total energy intake. The constant rate of sorption in quiescent mites (initial rate for disturbed mites) is 0.0073 pg per hour, of which the active component is 0.0018 Pg per hour; this uptake requires minimum 0.11% of the total energy consumption (Table 10). Energy required for active uptake of water to keep steady water mass in mites was discussed by Kanungo (1965) and Arlian (1972).

They also concluded that the energy needed for active up­ take of water was only a small percentage of their total energy consumption.

Half-life of tritium and exchangeable water loss can also be used to compare water exchange (Table 9). If we insert raQ - 1 and mt = 1/2 into equdtion (3) we get the

* 66 formula for half-life t^yg * ^t av» no water is lost and ms s m^. Water is exchanged, however, and in one half-iife as measured by tritium loss or decrease in specific activity 1/2 of the water originally in the mite will have been replaced by water from the surrounding air.

Therefore half-lives are equally as good as rate constants for comparing water kinetics of different forms of mites. 67

TABLE 9. HALF-LIVES

ay hours (days)

Tritium loss in active protonymphs 0.75 20.4

Tritium loss in quiescent protonymphs Cone rate model) 0.75 1155 C46.9)

Cof fast rate in a two rate model) 0.75 218.7 C9)

Tritium loss in dead quiescent protonymphs 0.75 169 C7.1)

VaterlosB in quiescent proto- , nymphs Clive part of experiment) 0.00 489.3 C20.4)

Cdead part of experiment) 0.00 207.9 C8.7) TABLE 10. CONSTANT BATES OF SORPTION (me = km. x m ) a t a = 0.75 AND 25 C s i v

Quiescent Quiescent Active Protonymphs Protonymphs Protonymphs (one rate) (fast rate of 2)

Calculated as yg/hour Cday) total 0.095 0.0016 (0.039) 0.0073 (0.175) active 0.023 0.0001* 0.0018 passive 0.0722 0.0012 0.0055

Calculated as ymoles/hour (day) total 5.3 x 1 0 ' 3 9 x 10“5 4 x 10“* active 1.3 x 10"3 l.»* x 10"5 1 x 10”* Energy required for active uptake in calories 2.1 x 10"7 i* x 10“10 1.6 x 10"8

Energy in %.of total consumption of 02 0.3% 0.003% 0.11%

o> oo CHAPTER V

DISCUSSION

•» In this investigation it has been established that quiescent nymphs sometimes occur in the life-cycle of

* American house-dust mites. These quiescent nymphs are pre­ dominantly protonymphs. They will be compared on the one hand with the active protonymphs} and on the other hand with the hypopial stage of other related mites.

Before discussing the differences between active and quiescent protonymphs, the physiological condition of the quiescent mites used should be considered. In all cases they were removed from their attachment to the substrate at the beginning of a period of observation. By being brushed off the lip of the culture jars, the mites probably suffered a loss of wax on one side that left them more permeable during a period of repair that required a higher metabolic rate. As a result the metabolic rate was found to be too high to be maintained constant throughout the whole qui­ escent period. Further, it was observed that while water exchange in disturbed quiescent mites was ten times faster than in active ones after about 16 days water exchange in quiescent mites seemed to stop altogether. Thirdly, re­ peated handling Cfour times) at optimum and temperature 66 70 seemed to trigger continuation of the molting process. In summary, then, it appears that undisturbed -quiescent proto­ nymphs are completely impermeable and that some 16 days are required for the disturbed quiescent form to regain its i impermeability.

4 The active protonymph is adapted for feeding and accumulation of reserves that are utilized in the production of the tritonymph; whereas the quiescent pootonymph is adapted to long-term survival in unfavorable conditions and possibly also to phoresy. This quiescent form requires reserves for long-term maintenance in addition to those utilized in the production of the tritonymph. The reserves are accumulated while the protonymph is still active. The most obvious difference between active and quiescent forms are that one form is active while the other is immobile, and the latter endure at least ten times longer than the former.

These observations are reflecting a difference in metabolic rate. The active protonymph feeds and has a higher oxygen consumption. In an atmosphere in equilibrium with a saturated NaCl solution at 25°C, at least 0.3% of the total energy consumption of this form is used for active uptake of water. If it is confined without food as in the divers or in the cages, it dies in 8 to 2U hours. The disturbed quiescent mite is non-feeding and has at least a four-fold lower metabolic rate; it uses 0.11% of its energy 71 for active uptake of water. Thus the energy requirement for uptake of water by the quiescent form is about 3 times less, than that required for the active protonymph.

The energy expenditure of the impermeable undisturbed qui­ escent protonymph must be much lower than required by dis­ turbed quiescent mites.

The .permeability of the interface between the mite and the environment will be different in the various forms. It is the general rule in arthropods that long-term resistant forms decrease their permeability in one way or another.

For a minute animal like the quiescent protonymph of the

American house-dust mite, which has a volume of about 3.5 nl, the danger of desiccation is critical. The quiescent mites

Cdisturbed) showed a 10-fold slower rate of loss of tritium than the active mites, and undisturbed quiescent mites are practically impermeable to water.

The degree of anhydrobiosis, or lowering of water content in quiescent protonymphs was not spectacular. The percentage of water mass in active mites was 83.7 and in quiescent mites 68.6. These values are based on the water content that is free to evaporate in dry air at 60°C.

Tolerance to water loss of the latter is expected to be higher than in active mites. Quiescent protonymphs must be well protected.

The measure of permeability that was used to compare water exchange in the two forms was the initial rate of 72. tritium loss. The dimension of the rate constant is per unit time. To show that it is a reasonable measure for comparing permeabilities, a full dimensional analysis of the transpiration rate constant (Arlian and Wharton, 197*0 pvAxV 1 1 = 1 is given: k = y jx ^ x ^ ^ where P is unit pressure,

A is surface area of the mite,4 V is volume Cor mass) and t is time. t Most of these units cancel out and the rate con- stant is given per unit time. The formula consists of two parts, the driving force is pressure per units of the sur­ face to volume ratio. Then there is the permeability factor which is volume transpired (or mass) per surface t area, per unit pressure and per time. When the experiments were done at the same av , temperature and atmospheric pressure, the driving force out of the mites CP x a ) did V w not change significantly, neither did the area of the volume of the mites. Thus, the observed difference in the rate constant for tritium loss must be due to a change in permeability. Therefore, Jop. is a suitable measure for the comparison of the permeability of the two forms of the protonymph.

Considering the high impermeability to water of the quiescent nymphs, it seems they would have a problem in satisfying their oxygen requirements. According to the -U given calculations, 1.1 x 10. micromoles per hour per mite

— ii % of oxygen, and x 10 micromoles per hour per mite of

* water Cinitial) are exchanged Cthese are observations on 73 disturbed mites, undisturbed quiescent mites may not ex­ change water at all). The pressure of water to get out of the mite (Py x aw > at 25°C is 23.5 mm of Hg; the mole fraction of O2 in the.atmosphere is 20.95 which gives a partial pressure of 159.2 mm of Hg. Thus, the partial pressure of oxygen on the mite4 is 7 times greater than the water vapor pressure from within the mite. When the partial pressures for water vapor and oxygen are considered, we see that Waggoner'b (1967) assumption that biological membranes are more permeable to water than to respiratory gases can hold, and still provide sufficient Og for respiration of quiescent protonymphs.

The eggs of the house-dust mite may be impermeable to water and oxygen. It seems, then, that this could also be the case of the cuticle of the protonymphs. Instead of the cuticle which has protective functions, the other parts of the interface with the environment, the openings, can be considered as a site for exchange with the environment. The openings of D. farinae nymphs are the mouth, the anus, the openings of the lateral opisthosomal and the supracoxal glands and possibly the genital papillae. The buccal cavity is the site for active uptake in ticks (Rudolph and

Knulle, 1974); there are indications that the mouth is important in D. farinae also. Cross-sections through the gnathosoma of D. farinae (Brody et al., 1972) show that

/ 74

the prebuccal cavity has a large surface area that could be

the exchange site. That something goes on in this cavity may be suggested from the observation that the pharyngeal

I>ump is working in quiescent mites mounted in mineral oil.

' Changes in the water vapor activity of the ambient 4 air seem to influence the permeability of the interface

between the mites and the environment. The half-life of

water loss in completely dry air of disturbed quiescent

protonymphs was 20 days, whereas half-life of tritium loss

(initial) in ay of 0.75 was 9 days. Several explanations of

how the decrease in permeability of arthropods is accomp­

lished exist. In tardigrades it is shown that when exposed

to desiccation, special permeable areas of the cuticle

were retracted from contact with the air (Crowe, 1972);

drying out of the cuticle of terrestrial isopods is thought

to bring endocuticular lipids closer to each other, and

thus reduce the permeability of the cuticle (Bursell, 1955);

thirdly, there is the possibility of secretion of more wax

layers. Devine (1969) examined the trace of k^,. versus ay

and derived an eighth order equation. This indicated a

complex relationship. However, he worked with a mite that

possessed a tracheal system which addB to the complexity of

the system.

.How do the quiescent protonymphs compare with other

stress resistant forms of arthropods? Disturbed proto­

nymphs respire for at least eight days after the 75

disturbance, in constrast to anhydrobiotio cysts and winter-

eggs that do not respire. The main structural modification

observed was the thicker cuticle. In this respect they

compare well with hypopi. They also have in common with

the. hypopus of being non-feeding. The quiescent proto-

nymphs occur in the life-cycle in the same place where the

hypopus would have been, succeeding the active protonymph

and preceding the active tritonymph. The quiescent proto­

nymphs may also have dispersal function, like the hypopus,

because they are able to stick to surfaces. However, these

mites are mere analogs of the hypopus* Because there is no

molt preceding the stage; the protonymphal leg-tissue is

still present, which shows that apolysis has not yet started

and the mite is still a protonymph. In addition to a

quiescent protonymph in the life cycle where the hypopus

would have been, quiescent tritonymphs also occur; however,

much less frequently.

The superfamilies of the Acaridei that have a true

hypopus are mostly free-living in stored products or in

nests. These are environments that will change when the

'food reserve is gone; or the animals in the nest leave.

These mites need a stage to endure unfavorable conditions,

and to get a ride on an animal to another nest; the hypopus

is adapted for this function. Hypopi do not occur in the

life-cycle of Acaridei that are infectious parasites on

vertebrate animals and reproduce on the host. Such mites 76 do not have a need for a specialized resistant phoretic form.

The Pyroglyphidae is the only family in the parasitic super­ family Psoroptoidea that is free-living in nests. They may be a link between the ancient free-living forms from which the■Psoroptoidea evolved; or they may have evolved from parasitic mites and are going back to the nest habitat.

When the .parasitic part of the Acaridei evolved from the free- and nest-living forms, they lost the hypopus. The quiescent nymphs of D. farinae are not hypopus homologs, but mere analogs. Since evolution does not go backwards

(Dollo's Law)., the fact that when resistant forms were needed again, hypopus analogs occurred, indicates that the

Pyroglyphidae evolved from parasitic Psoroptoidea, rather • than bieing a link between the ancient free-living forms possessing hypopi and the parasitic psoroptoids. LITERATURE CITED

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