Anatomical and Physiological Studies of Water Balance in the Millipeds, Pachydesmus C. Crassicutis (Polydesmida) and Orthoporus Texicolens (Spirostreptida)

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1972 Anatomical and Physiological Studies of Water Balance in the Millipeds, Pachydesmus C. Crassicutis (Polydesmida) and Orthoporus Texicolens (Spirostreptida). Thomas Charles Stewart Louisiana State University and Agricultural & Mechanical College

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Recommended Citation Stewart, Thomas Charles, "Anatomical and Physiological Studies of Water Balance in the Millipeds, Pachydesmus C. Crassicutis (Polydesmida) and Orthoporus Texicolens (Spirostreptida)." (1972). LSU Historical Dissertations and Theses. 2249. https://digitalcommons.lsu.edu/gradschool_disstheses/2249

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STEWART, Thomas Charles, 1937- ANATOMICAL AND PHYSIOLOGICAL STUDIES OF WATER BALANCE IN THE MILLIPEDS, PACHYDESMUS C. CRASSICUTIS (POLYDESMIDA) AND ORTHOPORUS TEXICOLENS (SPIROSTREPTIDA).

The Louisiana State University and Agricultural and Mechanical College, Ph.D., 1972 Zoology University Microfilms, A XEROXCompany, Ann Arbor, Michigan ANATOMICAL AND PHYSIOLOGICAL STUDIES GF WATER BALANCE IN THE MILLiPEDS PACHYDESMUS C. CRASSICUTIS (POLYDESMIDA) AND ORTHOPORUS TEX I COLENS (SPIROSTREPTIDA)

A D issertat i on

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillm ent of the requirements for the degree of Doctor of Philosophy

i n

The Department of Zoology and Physiology

by Thomas C. Stewart B.S., Stephen F. Austin State College, 1959 M.S., Louisiana State University, 1966 May, 1972 PLEASE NOTE:

Some pages may have

indistinct print.

Filmed as received.

University Microfilms, A Xerox Education Company ACKNOWLEDGMENT

I would like to thank Dr. J. P. Wood ring, my faculty advisor and major professor, and the other members of my graduate committee for their invaluable help in the preparation of this dissertation.

* t TABLE OF CONTENTS

ACKNOWLEDGMENT i i

TABLE OF CONTENTS...... i i i

LIST OF TABLES...... iv

LIST OF ILLUSTRATIONS ...... v

ABSTRACT...... vi

INTRODUCTION ...... I

MATERIALS AND METHODS ...... 6

RESULTS ......

Pachydesmus crass icut i s ...... 1*+

Orthoporus texicolens ...... 21

DISCUSSION ...... 28

LITERATURE CITED ...... 38

VITA ...... ^2 LIST OF TABLES

TABLE 1. Sulfuric acid concentrations to give desired

relative humidities and saturation deficits at

two temperatures ...... 8

TABLE 2. Effects of desiccation on Pachydesmus

crass icut. is ...... 17

TABLE 3. Metabolic rate of Pachydesmus crassicutis .... 20

TABLE k. Effects of desiccation on Orthoporus tex icolens . . 2k

TABLE 5- Metabolic rate of Orthoporus t e x i c o l e n s...... 27 LIST OF ILLUSTRATIONS

Plate I Pachydesmus crass icut i s ...... 15

Figure 1. Ventral view of parts of 3 sequential

d i plosegments.

Figure 2. Internal medial view of tracheal pocket.

Figure 3. Sagittal section of tracheal pocket and

spi racle.

Figure Cross section of firs t tracheal pocket

and spiracle.

Plate 2 Orthoporus texicolens ...... 22

Figure 5. Ventral view of 3 sequential diplosegments.

Figure 6. Enlarged ventral view of spiracular lips

and sp i racle 1 .

Figure 7- Enlarged ventral view of spiracular lips

and spi racle 2.

Figure 8. Sagittal section of tracheal pocket 1

and part of tracheal pocket 2.

Figure 9. Sagittal section of tracheal pocket showing

lever-muscle mechanism. ABSTRACT

1. A spiracular closing mechanism is lacking in the hygric

Pachydesmus crassicutis and in the mesic Orthoporus texicolens, but in the latter the spiracles can be closed by a combination of diploseg- mental overlap and close appression of the coxae.

2. The rate of water loss at several temperatures is much less in

0. texicolens than in £. crass icut is. and neither is affected by elevated carbon dioxide levels.

3. Because of the control of respiratory water loss in 0. texicolens, cuticular water loss accounts for about half the daily water loss under desiccating conditions; but in £. crass icut i s so much water is lost via the respiratory system that cuticular water loss is minor in comparison.

k. £. crass icutis has a greater desiccation tolerance (by main

taining a constant hemolymph osmolarity) than 0. texicolens. but

0. texicolens (by restricting spiracular water loss) has a greater

tolerance to desiccating conditions than £. crass i cut i s.

5. Both species are oxygen conformers, but £. crass icut is has a much higher rate of oxygen consumption than 0. texicolens.

6 . Both species are ammonotelic, but £. crass icut is feces also contains urea (urea/ammonia ratio of 0 .0^7) and 0. tex icolens feces also contains uric acid (uric acid/ammonia ratio of 0 .50). INTRODUCTION

Water loss is a major problem to all terrestrial life , millipeds being no exception, but a search of the literature did not reveal conclusive evidence as to the major site of water loss and the means of

its control in millipeds. The purpose of this investigation was to determine the morphological and physiological means that representative millipeds use to prevent water loss. The animals selected were from

two different habitats and represented two different phylogenetic

lines: the polydesmoid Pachydesmus crassicutis crassicutis (Wood) from a hygric habitat and the spirostreptoid Orthoporus texicolens

Chamberlin from a mesic habitat.

Previous workers are contradictory in their opinions of the role of the integument and the respiratory system in water loss in m illi peds. Verhoeff (1928-32), Edney (1957), Kevan (1962), Dwarakanath and

Job (1965), and Toye (1966) reported that the tracheal system is the major site of transpiration, while Clouds ley-Thompson ( 1950) reported

that the major site is the cuticle. O'Neill (1969) agreed that the primary site of water loss is the tracheal system, but stated that water is also taken up through the cuticle when a desiccated animal

(Narceus americanus) comes in contact with a wet sponge. This means of water uptake demonstrates the permeability of the cuticle to water, and

thus I assume that water can be lost via the cuticle. Edney (19^9) and

Clouds ley-Thompson (1950) did not find a wax layer in the epicuticle of

the Diplopoda, and the millipeds did not demonstrate a " c ritic a l" or

"transitional" temperature characteristic of those insect species with 2 a distinct wax layer. This transitional temperature Is the temperature at which the epIcuticular wax layer suddenly becomes more permeable to water, thus permitting a greatly Increased rate of cuticular transplra- tion. Mead-Briggs (1956) stated that although the absolute permeabil ity to water of some arthropod cuticles without a wax layer in the epicuticle is high, there is a low increase in the permeability with a temperature increase. Cloudsley-Thompson (1950), by means of selected solvents, found a lipid in the epicuticle of Oxidus qraci1 is that he presumed to be an inefficient waterproofing agent of the cuticle. When this lipid was removed by hot chloroform the rate of transpiration increased slig h tly. Wigglesworth (19^5) reported a constant relation ship between weight and surface area for a number of arthropod species, and gave a rate of evaporative loss from the cuticle for each stage of development of 0. graci1 is , the rate being greatest in the earlier stages.

Most of the descriptions of milliped respiratory morphology are based upon whole mounts and not sectioned material, and thus the structures are not clearly understood. Verhoeff (1926-28) describes the structure of the tracheal apparatus of various millipeds and summarizes all previous work in this area. In the Proterospermophora

(= Polydesmida) he found a tracheal pocket consists of three regions

(p. 1221, Fig. 708), the outermost of which is part of the integument.

Unbranched tracheae arise only from the middle region of the tracheal pocket. Instead of trachea«, many coxal muscles originate on the walls of the third (innermost) region, indicate that tracheal pockets are basically muscle apodemes. He described in the second region 3 of the tracheal pocket a small lever attached to a cuticular ring in the wall, the center of which Is a very thin cuticular window. He thought that perhaps when the muscle connected to the lever contracts, the wall of the pocket might expand, thus causing the inhalation of a ir. He thought that this muscle is homologous to the closing muscle of the spiracles of the Opisthospermophora (= Julida, Spirobolida,

Spirostreptida, and Cambalida). He did not think that the Polydesmida can close their spiracles. In the Opisthospermophora, Verhoeff

(op c i t .) described an outer region fu lly within the integument, open ing to the outside through a closing mechanism (p. 1189, Figs. 672-

675» 680-682). This closing mechanism was thought to operate through a lever by a muscle whose origin is at the base of the coxa of the nearest leg. The middle region of the tracheal pocket is a thick- walled cylinder (its walls continuous with the integument) and does not receive muscles or tracheae. The third region is expanded in various directions (depending on the species) and receives both muscles and tracheae. Owarakanath and Job (1965) reported structures at the spiracles that look like guard cells in Spi rostreptus sp. O'Neill

( 1969) reported that coiling (spiraling) effectively closes the spiracles of Narceus americanus and prevents tracheal transpiration.

Without elaboration, Kevan (1962) reported a "more efficient closing mechanism of the spiracles" in millipeds than in centipeds.

Adaptation to a dry habitat often includes the excretion of nitrogenous waste products in a comparably insoluble form such as uric acid or guanine. Adaptations to a moist habitat usually include elimination of the nitrogenous catabolites in the more soluble form of ammonia and/or urea. Hubert (1965, 1968, 1969) and Hubert and Razet I*

(1965) found ammonia and uric acid in all 11 miltiped species repre

senting the Orders Glomerida, Chordeumida, Polydesmida, Julida, and

Spirostreptida that they investigated. The ratio of ammonia to uric

acid varies between 2:3 and 1:1 in the 11 species. In one of the 11 species Hubert studied (Glomeris marginata). she also found traces of

urea (1969), but she did not feel that urea is an important nitrogenous catabolite in vivo. Bennett (1971), in his analysis of non protein

nitrogen in the excreta of Cy1indroiu1 us londinensis, found both ammonia and uric acid in the ratio of 5:1 and greater. He also found

arginine in the excreta of this species and thought that its presence would argue against the presence of an ornithine cycle in millipeds.

Hydroreception has been attributed to vesicular receptors on the sternite of the firs t (collar) segment of Cingalobolus bugnioni

(Rajulu, 1964). Most authors, including C louds ley-Thompson (195 0 ,

Shelford (1913), Perttunen (1953), and O'Neill (1967, 1969), agree that millipeds perceive and move toward a moist substrate, presumably to escape desiccation. Since millipeds are primarily nocturnal

(Clouds Iey-Thompson, 1959; Paulpandian, 1966; Brandon, 1967; and

Stewart, 1969), it is presumed that the avoidance of diurnal activity

is an important means of controlling water loss.

Previous workers have employed both time of death at lethal low

relative humidities and percent weight loss at various nonsaturated

relative humidities to describe desiccation resistance in millipeds.

Clouds 1ey-Thompson (1950) reported about 5%/day weight loss due to

desiccation in Ox idus grac ?1 is at 20% relative humidity (25°C).

Dwarakanath and Job (1965, 1966) reported a *+%/day weight loss at 5

8 mm Hg saturation deficit in Splrostreptus asthenes. At t ’'.- of death due to desiccation at 0% R.H., Toye (1966) reported a total weight loss of 30-UO% in S. asthenes. Concerning the length of time that 0. gracilus can live at low relative humidities, Causey (19^3) reported death within 15 hours at 0% R.H. (25°C), and Perttunen (1953) reported death only after 60-130 hours at 0% R.H. (26°C) for the same species.

Toye (1966), using Spirostreptus assiniensis, reported death in 9-50 hours at 0% R.H. (30°C).

In any study of water balance and desiccation tolerance it is

important to know the metabolic rate and oxygen consumption of the animal, especially if the respiratory system is suspected of being the major site of water loss. The rate of oxygen consumption seems to vary between species. Paulpandian (1966) reported only 3-^jul/g/hr O2 consumption for the p ill milliped Arthrosphera dalzi. Dwarakanath and

Job (1966) reported a consumption of 39-69 ^il/g/hr for the spirostreptoid Spirostreptus asthenes. Gromez-Kalkowska (1966) reported a

relationship between metabolism and weight in 0. graci1 is wherein the metabolic rate increased as a function of body size and temperature. MATERIALS AND METHODS

Living £. crass icut is , taken from East Baton Rouge Parish,

Louisiana, and 0. tex icolens, taken from Cameron County, Texas, were

kept in 2 to 10 gallon covered terraria on a substrate of leaf mold and

leaf lit te r . For £. crass icut is , the leaf litte r and leaf mold were

the sole source of food. Thin, fresh slices of Irish potato in addi

tion to the leaf mold and litte r were present as food for 0.

tex icolens.

To understand fully the anatomy of the tracheal pockets of the

test millipeds, it was necessary to study dissected preserved animals

and histological sections of the sterna with the attached respiratory

apparatuses. The animals were killed and fixed by injecting

Duboscq-BasiI's modification of Bouin's fixative (Romeis, 1968) with

subsequent immersion in the solution overnight. The region of the

body wall containing 2 tracheal pockets was carefully dissected from

the body. For both whole mounts and for subsequent sectioning, the

dissected parts were dehydrated with a series of ethanol-water solu

tions. The tissue was then placed in xylene or tetrahydrafuran over

night. For whole mount studies, this tissue was further cleared with

methyl salicylate and mounted with Permount. Tissue to be sectioned

was left overnight in a 50-50 mixture of xylene and 60-62°C melting

point paraffin. Vacuum in filtra tio n was accomplished at 10-15 mm

mercury and 65°C. The tissue was sectioned at 8-12 microns, stained with Mayer's hematoxylin (Romeis, 1968) and eosin, and mounted with

Permount. 7

Normal values of oxygen consumption, carbon dioxide production,

and respiratory quotient were determined by the use of a Gilson

Differential Respjrometer. Single side arm flasks with a center well were used to contain the animals. A 20% solution of potassium

hydroxide on a f ilt e r paper wick was placed in either the side arm or

the center wel1.

In order to test whether the 2 experimental animals were oxygen

regulators, the oxygen consumption was determined at lower pPC^. Low

oxygen content air mixtures for the respirometer were prepared by

diluting air with nitrogen and collecting the mixture in a 19 liter

bottle by water displacement. This mixture was forced into the gassing manifold inlet of the respirometer according to the manufacturer's

operating instructions to purge the reaction flasks of their previous

gaseous content, leaving the desired gas mixture in the flask.

Weight loss due to desiccation was determined by placing the

experimental animals in 250 ml beakers which were then placed in a

200 mm diameter desiccator. The volume of the desiccator was about

5 lite rs , and it contained 200 ml of a sulfuric acid-water mixture on

the bottom to control the saturation deficit. The acid-water mixture

to obtain these saturation deficits are given below in Table 1, which

was adapted from Welsh, et al_., 1969. In cases where a carbon

dioxide and air mixture was used in the desiccator, C0£ and air were

collected by water displacement in a 19 lite r bottle. Dry ice was used

as the CO2 source. The O2-CO2 mixture was forced from the bottle by

water, bubbled through a sulfuric acid-water mixture, and introduced by

air displacement into the desiccator containing the animals and the 8

TABLE 1

Sulfuric Acid Concentration to Give Approximate Relative

Humidities and Saturation Deficits at Two Temperatures

Vol . H2SO/+ Sp.Grav. °//o S.D. S.D. per 100ml of relat i ve at 25°C at 30°C solut ion solut ion humid i ty mm HG mm Hg

NONE 1 .00 100.0 0.0 0.0 8.2 1 .09 9k.8 1.2 1.6 12.1+ 1 .11+ 89.9 2.1+ 3.2 16.1+ 1. 8 81+.0 3.8 5.0 18.3 1 .20 80.5 k.S 6.1 21.3 1.23 Ik.S 6.0 8.0 23.1 1.25 70.1+ 7.0 9.3 21+.6 1.27 65.5 8.2 10.9 27.0 1.29 60.7 9.3 12.1+ 29.6 1.315 55.0 10.7 11+. 2 32.2 1.3^0 50.0 11.9 15.8 35.0 1.361 1+5.0 13.1 17.0 1+2.3 1.1+38 29.5 16.8 22.0 kk.S 1.1+56 25.0 17.8 23.1+ 1+7.0 1.^79 21.5 18.7 21+.3 51.6 1.524 15.5 20.1 26.5 57.0 1.529 10.5 21.3 38.0 67.0 1.662 k.S 22.6 29.8 100.0 1.81+0 0.0 23.8 31.5 9 desiccanl. The 19 liters of gas mixture purged the desiccator of its b liters of a ir. The millipeds were weighed daily, and after each weighing they were returned to the desiccator and the desiccator again purged with 19 liters of the gas mixture. Desiccators for studies at

29°C were kept in an air conditioned laboratory where the temperature was maintained at 24°*1°C. Higher temperatures were obtained in constant temperature cabinets.

The procedure for determining evaporative water loss in the 2 experimental animals was to weigh the millipeds at a given saturation deficit and gas mixture on a daily basis. To determine the extent of cuticular transpiration, selected animals had all of their spiracular openings covered with a coat of rubber cement. They were then weighed daily at various saturation deficits and the weight loss compared with the weight loss of animals with unblocked spiracles.

To determine if coiling resulted in telescoping of the diplosegments to the extent of covering the spiracles, both preserved and living animals were made to coil and the amount of telescoping observed under 6-50X stereoscopic magnification. Since extensive telescoping did occur in 0. tex icolens, an effort was made to determine if coiling aided in the retardation of water loss via transpiration through the spiracles. Living 0. tex icolens were straightened out and fastened to small diameter wooden sticks with 2 or 3 rubber bands. When so pre pared they were restrained from coiling, thus exposing the spiracles at all times to the drying atmosphere.

To determine if heat affected the waterproofing qualities of the cuticle, some animals were heated tokb°Z in a beaker placed in a hot 10 air oven for 10 minutes. They were placed in the appropriate desic cator at a given saturation d eficit and temperature and the rate of weight loss determined.

To determine the effect of abrasion on the waterproofing qualities of the cuticle, some animals were coated with about 400 mesh hone glass compound and gently rubbed between the thumb and index finger for about 1 minute. These animals were placed in desiccators at given saturation deficits and temperatures. As the animats moved about, they continued to abrade the exoskeleton due to friction between body parts. The rate of weight loss was determined and compared to the weight loss of nonabraded animals.

In order to determine the change in hemolymph osmolality resulting from evaporative water loss in the 2 study milliped species, the following procedure was employed. Hemolymph was collected from

laterodorsal punctures on the exoskeleton and diluted to determine the osmolal concentration with an Osmette Precision Osmometer. The minimum dilution possible to obtain the required amount of liquid from 1 animal for this instrument was 6.7:1. To see if a mixture of different compounds such as would be present in hemolymph would give correct reading with high dilutions on the osmometer, solutions of sodium chloride, sodium bicarbonate, and magnesium sulfate were mixed in various proportions and in various dilutions. Readings of dilutions of these mixtures were inversely proportional to the dilution factor.

To check the amount of amino acid in the hemolymph, a ninhydrin spectrophotometric analysis was performed according to the procedure of 11

Meites, £t a ^ ., 1962. One tenth ml hemolymph was collected from a

laterodorsal puncture and diluted to a total volume of I .5 ml with d istilled water. A standard of 2% alanine was used. The percent

transmission at 570 i^M using a Perkins Elmer spectrophotometer was read

for both samples. A calibration curve was not prepared or needed (see

results).

A quantitative analysis of ammonia and urea in the feces of both milliped species was performed by the microdiffusion technique of

Conway (1942). One gram of feces was dissolved in 1 ml of biphosphate- dihydrogen phosphate buffer, pH 7, and then diluted to a total volume of 10 ml. To determine the amount of ammonia nitrogen present, 0.5 ml saturated potassium carbonate solution and 0.5 ml of the dilute sample were mixed in the outer chamber of a Conway microdiffusion c e ll. One

tenth ml of an 0.05 N sulfuric acid solution with an indicator was added to the inner well of the c e ll. After sealing the top of the cell with its cover, the cell was rotated to mix the solutions in the outer chamber. The cell was allowed to stand at room temperature for 1 hr, after which time the lid was removed and the acid titrated with 0.05 N sodium hydroxide solution. The micrograms nitrogen from ammonia were calculated from the equation k (A-B) 0 jjgN/g feces « ------where K is the atomic weight of nitrogen times the pi acid in the inner well times the acid normality; A is the volume acid in the inner well;

B is the volume base at the same normality as the acid used in the titration; and D is the dilution factor of the buffer-feces solution.

To determine the amount of nitrogen derived from ammonia and from urea, 12

0.5 ml of the dilute buffer-feces solution was added to the outer cell with 0.5 ml urease-buffer solution (I mg/ml), the cell was covered, and the mixture allowed to digest for 1 hr at room temperature before the potassium carbonate solution was added. Titration and calculations were the same as for the ammonia determination. To adjust for any ammonia that might have been given off by the urease-buffer solution, a control was run with only buffer-urease and potassium carbonate solu tions in the outer well and the indicator-acid solution in the center well. Nitrogen liberated in this manner on a per gram basis was subtracted from the amount found in the ammonia plus urea determina tion. The amount of urea nitrogen equaled the nitrogen from ammonia and urea minus the nitrogen from ammonia and the control.

A quantitative analysis of the uric acid in the feces of the 2 test millipeds was accomplished by the method of Folin and Wu (1919).

One-half gram of feces was added to 1 ml of 10% sodium tungstate in

2/3 N sulfuric acid to precipitate any protein present. This mixture was added to 1 ml saturated lithium carbonate solution to dissolve the uric acid. The total volume of the resulting solution was brought to

5 ml by the addition of distilled water. Three ml of this solution was used as an unknown and 3 ml d istilled water served as a blank. Five cuvets were prepared that contained standard uric acid concentrations of .05 mg/ml to 0.000*+ mg/ml. To each cuvet, 1 ml Folin-Wu reagent and

1 ml 1*+% sodium carbonate solution was added. The percent transmission at 710 nui using a Spectronic 20 spectrophotometer was used to prepare a standard curve against which the concentration of the unknown was determi ned. 13

All statistical analysis was calculated as the 95% fractiles of ihe variance distribution (F test). RESULTS

Pachydesmus crass icut is

Anatomical studies of the tracheal apparatus, or tracheal pockets as Verhoeff termed them, revealed that each tracheal pocket consists of

3 regions (Figs. 1-4, A, B, C) with a total length of 1.45 mm in an adult female 68 mm long. The large oval spiracle (SP) opens into a rugose cup (A), the inside of which is covered with a g rill work which

I presume function$as dust catchers (Figs. 3, 4, G). The second region (B) has unbranched tracheae (T) arising from it. At approxi mately the junction of the firs t and second region is a lever-like cuticular process with a muscle (.02 mm diameter), which originates at the coxal base of the leg nearest to the spiracle (Fig. 2, L, LM). The second region is enlarged at the level of the lever where approximately

300 tracheae arise. Another mass of about 150 tracheae arise at different points on the second region. The third region (C) is a tapering continuation of the second chamber, which is about .70 mm in length. Muscles (M) that insert on the coxae and other respiratory apodemes (tracheal pockets) originate from the wall of the third region. The cuticular wall of the tracheal pocket is thickest (.05 mm) in the firs t region around the spiracle and in the area of the lever like process (L) (.05 mm). The sclerotized ring (Figs. 2, 4, R) to which the lever attaches is .065 mm thick, which is thicker than the wall of the second region. The thinnest sclerotized areas are the wall of the second region where the tracheae arise and the window (W) in the center of the ring (R), both being only .005 mm thick. The muscles 15

Plate I. Pachydesmus crassIcutis.

Fig. I: Ventral view of parts of 3 sequential diplosegments

(labelled |, ||, and III) with the right legs removed. Anterior is up. The limit of telescoping overlap of the posterior edge of a diplosegment onto the diplosegment posterior to it, upon coiling of the millipede, is shown by the heavy dashed line (LT). Neither the legs nor the act of coiling can close the spiracles (SP) (see text).

Fig. 2: Internal medial view of tracheal pocket (TP 1) to show complete ring structure (R), lever (L), and window (W). Compare this wi th figure k.

Fig. 3: Sagittal (line "a" in fig. 1) section of tracheal pocket and spiracle (SPI). Note the large opening into the tracheal pocket and the dust catchers on the g r ill work (G) , which arise from the wall of the outer region of the tracheal chamber (A).

Fig. k: Cross section of first tracheal pocket and spiracle showing an angled cut of the ring structure (R) and window (W).

Other abbreviations are: Outer region (A), Middle region (B),

Inner region (C), Coxal cavity (CC), Coxa (CX), Body wall (BW),

Tracheae (T ), Lever muscle (LM), Coxal muscles (M). PLATE I

ju a'ii inirff 16 with origin on the third region indicates that the whole apparatus is originally evolved as an apodeme.

When £. crass icut is is coiled the anterior portion of the sternum of each diplosegment is partially covered by the posterior portion of the sternum of the next anterior diplosegment (Fig. 1, LT). Because the sternum is elevated at the union of the coxae and the sternum, and the spiracles are lateral to the coxae, the telescoping cannot possibly cover the spiracles in this species. The articulation of the various leg segments is such that they cannot be moved in such a manner that an article of the leg can cover the spiracle.

The results obtained from the desiccation of this animal in air at

2 mm Hg, 4 mm Hg, and 6 mm Hg saturation deficit for 5 days revealed that the average weight loss per day for a total of 27 specimens was

6.05%, 18.4%, and 26.7% respectively at 24°C (Table 2), and for a total of 17 specimens, 6.73%, 20.8%, and 26.2% respectively at 30°C (Table 2).

The differences of the two temperatures are not significant (P>.05).

The rate of desiccation was highest for the smaller sized adult (range

62 to 78 mm) and immature (range 32 to 57 mm) animals. The average time to death for animals at 24°C was in excess of 5 days at 2 mm Hg

S.D., 3.5 days at 4 mm Hg S.O., and 2.4 days at 6 mm Hg S.D. (Table2).

At 0% R.H. (24 mm HgS.D., 24°C) animals died within 36 hrs. The average weight loss at the end of the 5-day test period was 30.25% at

2 mm Hg S.D., at which time all animals were living. The weight loss at death at 4 mm Hg S.O. and 6 mm Hg S.D. was 64.40% and 64.08% respectively. The weight loss at 0% R.H. (24 mm Hg S.D., 24°C) averaged only 56% of the original body weight. This low figure was 17

TABLE 2

Effects of Desiccation on Pachydesmus £. crass icut is

Average Average Av. Av.% No. of In itia l % Weight Days Wei ght Spec i- Weight Loss/day to Loss at Cond it i ons mens(n) (-Isd.dv.) (-Isd.dv.) Death Death

In a ir, 24°C 2mm Hg S.D. 10 1.39x0*08 6.05x0.90 5* 30.25** 4mm Hg S.D. 9 1.43-0.07 18.4 ±2.51 3.5 64.40 6mm Hg S.D. 8 1.46-0.10 26.7 ±4.37 2.4 64.08 24mmHg S.D. 4 1.44-0.10 44.8 - 7.02 1.25 56.04 Teneral Form, Air, 24° C 2mm Hg S.D. 1 1.97 41.2 1 41.2 4mm Hg S.D. 1 2.03 49.3 1 49.3 In Air, 30°C 2mm Hg S.D. 6 1.44x0.09 6.73J1.08 5* 33.65v* 4mm Hg S.D. 5 1.38x0.12 20.8 ±3.00 3.7 65.04 6mm Hg S.D. 6 1.46-0.11 26.2 ±4.93 2.2 64.92 Air, 24 In 5% C02, 95% ° C + 2mm Hg S.D. 4 1.43x0.10 6.5 ±1.03 5- 32.50>-- 4mm Hg S.D. 4 1.37-0.09 17.4 ±2.97 4.1 72.34 Abraded, in Air, 24°C 2mm Hg S.D. 2 1.49x0.12 6.01±1.19 5* 30.05** 4mm Hg S.D. 2 1.43-0.13 19.00±3.7 1 3.6 68.40 Heated to 45°C , in Air , 24°C 2mm Hg S.D. 2 1.32±0.10 6.15±0.99 5* 30.75** 4mm Hg S.D. 2 1.42±0.09 I9.30±3.02 3.6 69.48 6mm Hg S.D. 2 1.36- 0.10 25.40±5.03 2.8 71.12 Spiracles Closed with Rubber Cement 4mm Hg S.D. 3 1.37x0.11 1.5 ±0.04 1. 5** * 6mm Hg S.D. 2 1 - 39-0.13 3.0 ±0.11 1.5*** --

* After 5 days all animals were s t ill alive and were returned to the terrarium. ** Average weight loss at the end of 5 days. * * * All animals died within 36 hours, probably of anoxia. 18

probably due to the rapid desiccation, causing death at a lesser body

wci(|ht loss compared to the weight loss at death at 2 mm Hg and 4 mm

Hg S.D. The average time to death for animals at 30°C was in excess of

5 days at 2 mm Hg S.D., 3.7 days at 4 mm Hg S.D., and 2.2 days at 6 mm

Hg S.D. (Table 2). The average weight loss at the end of the 5-day

test period was 33.65% at 30°C and 2 mm Hg S.D., at which time all

animals were living. The weight loss at death at 4 mm Hg S.D. and

6 mm Hg S.D. was 65.04% and 64.92% respectively (Table 2). The average weight loss of animals at 24°C and 30°C at 2 mm Hg S.D. and 4 mm Hg

S.D. with 5% CO2 is not significantly different (P>.05) from the weight

loss of animals under the same conditions without the CO2 (Table 2).

Animals that were abraded before being subjected to desiccating condi

tions also had the same rate of weight loss (P>.05) as the unabraded animals (Table 2). Animals that were firs t heated to 45°C before being subjected to desiccating conditions had the same rate of weight loss

(P>.05) as the unheated animals. With the spiracles plugged with

rubber cement, the average weight loss per day at 24°C was 1.5% at

4 mm Hg S.D. and 3.0% at 6 mm Hg S.D. (Table 2), which is considerably

less than the weight loss of animals with unblocked spiracles.

Defecation seldom occurred during desiccation, but when it occurred, the water loss was calculated as part of the total water

loss. The feces of an animal from the 100% R.H. terrarium contains about 85% water, with the water content dropping to 65% during desic- cat ion.

In two instances teneral forms of £. crassicutis were found emerging from their molting chambers and they were subjected to drying conditions. The cuticle of these animals was s t ill soft, not yet

KNiiplolcly tanned or calcified. When they were exposed to saturation deficits of 2 mm Hg and *4 mm Hg they died within 2*4 hrs, having lost over *40% of their body weight (Table 2).

The rate of oxygen consumption at I5°C, 2*4°C, and 35°C was

*46.0 pl/g /h r, 10*4.6 pl/g/hr, and 256.3 jul/g/hr respectively, with a

Qjg of 2.01 between 15°C and 2*4°C, and a of 1.86 between 2*4°C and

35°C. The respiratory quotient for 15°C was . 788, for 2*4°C .759, and for 35°C .801 (Table 3). At 2*4°C the average oxygen consumption was

85.*+ pl/g/hr in 15% 02, and 65.*+ >jl/g/hr in 10% 02 (Table 3).

The osmolal concentration of the hemolymph for three normally hydrated adults averaged 180 mi 11iosmoles. This value did not change after 3 days desiccation at 6 mm Hg S.D.

The analysis of the hemolymph for the presence of amino acids involved a determination of the concentration of the carboxyl groups.

Since other compounds such as proteins or organic acids contain this radical, the test was not specific for amino acids. However, if no light was absorbed at 570 mp, it can be concluded that no amino acids

(or other carboxyl containing compounds) were present. This was the case. No amino acids were present in a concentration that could be detected by this means of analysis in either species of milliped. The

2% alanine standard did indicate a drop in transmission, indicating that a concentration of amino acids of this magnitude could have been detected. 20

TABLE 3

Metabolic Rate of P. c. crass icut is

Average 02 Average wt. Consumpt i on Average Cond i t ions Spec imens (-Isd.dv.) AJl/g/hr RQ

Air, 15°C L♦ 1.47^0.12g 46.0 .788 Air, 24 C 16 1.U6J0.07 104.6 .759 Air, 35°C 8 1.51x0.09 255.7 .801 15% 02, 85% N2, 24°C 5 l . W O . l l 85.4 — 10% 02 , 90% N2, 24°C 5 1.39-0.11 65.4

Teneral form 15% 02 + 25'o/ »I ,24°C 1 1.72 169.0 10% 02 + 90%N2,2l+°C 1 1.95 126.1 —

<*! ?5°C to 24°C = 2.01 24°C to 35°C = 1.86 21

Analysis of the fecal material of £. crassIcutls on the day that

they were taken from their native habitat of moist leaf litte r and leaf mold revealed the presence of ammonia and urea. No uric acid was

found. The concentration of nitrogen from urea was 0.004 mg/g feces, and the nitrogen from ammonia was 0.09 mg/g feces. These concentra

tions represent a urea/ammonia ratio of 0.047. The total determined non-Krotein nitrogen represents only 0.06% of the total dry weight of

the feces.

Orthoporus tex icolens

Anatomical studies of (). tex icolens reveal a tracheal apparatus

(tracheal pocket) of three regions whose total length is .75 mm in a

112 mm female (Fig. 9, A, B, C). The firs t region is within the confines of the integument (A); the second region is a simple straight cylinder (B), and the third region expands both medially and laterally

region also is the point of origin of muscles (M) that insert on the

nearest leg coxa. The thickness of the walls of the three regions varies from .4 mm in the second region to .05 mm in the third region.

In a medial groove near the opening of the spiracle is a lever

(Fig. 9, L ), and attached to this lever is a small muscle (LM) (.05 mm

diameter) which originates at the base of the nearest leg coxa. It

appears that if the muscle could contract with enough force it would

cause the closure of the spiracle by moving the wall to which it was

attached to the opposite wall (Fig. 9, L, LM). Supposedly, this is the muscle and lever that Verhoeff thought might be used to close the

spiracles in the Opisthospermophora. Physiological evidence 22

Plate 2. Orthoporus texIcolens.

Fig. 5: Ventral view of parts of 3 sequential diplosegments

(labelled l t I I, and I I I ) with the right legs removed on diplosegment

II. Anterior is up. The limit of telescoping overlap of the

posterior edge of a diplosegment onto the diplosegment posterior to it,

upon coiling of the millipede, is shown by the heavy dashed line (LT).

Note the depressions (D) for the close application of the coxae of the

legs to the body wall. Both the legs and the act of coiling contribute

to closure of the spiracles (see text).

Fig. 6: Enlarged view of spiracular Up (SL) and spiracle (SP1) showing position of lever (L ).

Fig. 7: Enlarged view of spiracle lips and spiracle 2 (SP2).

Note the different configuration of the spiracular slit.

Fig. 8: Sagittal section of tracheal pocket 1 (TP1) and part of

tracheal pocket 2 (TP2). Posterior is to the left. Note the cuticular

ridge (R) and compare to fig. 5 for better orientation.

Fig. 9: Sagittal section (higher modification) taken slightly

mesad of fig . 8 to show connection of lever (L).

Other abbreviations are: Outer region (A), Middle region (B),

Inner region (C), Coxa I cavity (CC), Coxa (CX), Body wall (BW),

Tracheae (T ), Lever muscle (LM), and Coxal muscles (M). PLATE 2

0.5mm 0.2 mm

V TP-I TP-I

SP-I SP-I

CX LT SP-2

CC CX

SP-2 I mm 23

indicates that closure in this manner is not possible (see discus sion) .

When 0. texicolens coils, the posterior edge of the pre ceding diplosegment covers the anterior portion of the following diplosegment, including at least part of the anterior pair of spiracles

(Fig. 5, LT). In addition, the coxae of the posterior leg pair of each

diplosegment is moved upon coiling into a position to cover the

anterior spiracles of the following diplosegment (Fig. 5, 0). The coxae of the firs t leg pair of each diplosegment cover upon coiling

the posterior pair of spiracles of that diplosegment. Finally, when coiled, the flattened coxa of each leg fits tightly against the coxa of

the leg anterior and posterior to it forming a comparatively complete cover over the spiracles on the sternum of each diplosegment.

The results obtained from the desiccation of 0. tex icolens at

2 mm Hg, 4 mm Hg, and 6 mm Hg saturation d eficit for 5 days gave an average weight loss per day for a total of 48 animals of 2.80%, 2.35%,

and 2.48% respectively at 24°C (Table 4), and for a total of 36

animals 3.53%, 4.40%, and 5.28% respectively at 30°C (Table 4). Only

the rates of desiccation at 6 nn Hg S.D. were significantly different

(P<.05) at the two temperatures. At the end of 10 days of dehydration

no animals had died and they were returned to the terraria. When 4 animals were placed in an 0% R.H. (24 mm S.D. at 24°C) desiccator, they died in 17 days, with an average weight loss of 40.3%. Animals that were abraded, and animals that were firs t heated to 45°C had the same

rate of water loss (P>.05) as control animals not heated or abraded.

The animals that had their undersides coated with rubber cement lost 2*+

TABLE *+

Effects of Desiccation on Orthoporus texicolens

Average Average Av. Av. % No. of In itia l % Weight Days Weight Spec i- Wei ght Loss/Day to Loss at mens(n) (-Isd.dv.) ( - lsd.dv) Death Death

In a ir. 2*+°C 2 mm Hg S.D. 16 *+.50jl .09 2.80- .85 -- *+ mm Hg S.D. 16 *+.06-0.78 2.35x -83 — 6 mm Hg S.D. 16 3.*+7x1-20 2.1+8- .82 — 2k mm Hg S.D. k *+.31-0.98 2.37- .91 17 *+0.3 In a ir, 30°C 2 mm Hg S.D. 12 3.69x0.68 3.53x1.20 -- *+ mm Hg S.D. 12 3.26J1.30 *+.*+0x0.70 — 6 mm Hg S.D. 12 3.5*+-0.79 5.28-1.00 -- In a ir, 2*+°C, Restrained from coiling 2 inn Hg S.D. 6 *+.23x0.99 *+.00x 1.10 *+ mm Hg S.D. 6 3.96x1.10 5.90x1.21 — 2k mm Hg S.D. k *+.17-0.93 13.70-1.03 3 *+1.0 In 5% C02 + air, 2k°C, Restrained from coi 1 ing 2 mm Hg S.D. 6 *+.03x0.9*+ 3.70x0.9*+ — k mm Hg S.D. 6 *+.93x1.32 5 .*+0x1 .*+0 — 2k mm Hg S.D. k *+.82-1.09 12.90-1.11 3 *+1.7 Abraded, 2*+°C 2 mm Hg S.D. 3 *+.2lJo.89 2. 7*+x 1.00 — *+ mm Hg S.D. 3 *+.09-0.97 2.93-0.98 -- Heated to *+5°C, in a ir, 2*+°C 2 mm Hg S.D. 6 *+.3270.99 2.87x0.91 k mm Hg S.D. 6 1+.82-1.02 2.36x0.85 — 6 mm Hg S.D. 6 *+.11 -0.73 2.51-0.73 -- Spiracles closed with Rubber Cement, in a ir, 2*+°C k mm Hg S.D. 3 3.97x0.87 1 .*+2x0.80 1.5* 6 mm Hg S.D. 3 *+.02*0.91 1.38*0.98 1.5*

* All animals died within 36 hours, probably of anoxia.

Means animals did not die within 5 day test period. 25 only I . k2% and 1.38% at U mm Hg and 6 mm Hg S.D. respectively and did so at all saturation deficits used. When restrained from coiling, 16 animals lost k.O%, 5.9%, and 13.7% per day at 2 mm Hg, k mm Hg, and

2k mm Hg S.D. compared to 2.8%, 2.k%, and 2.k% when not restrained.

Sixteen animals that were restrained from coiling in 5% C02 - air mixture did not lose more weight per day (P>.05) than those restrained

in air at the same saturation deficit.

The water content of the feces of 0. texicolens was about ^5% while feeding on potatoes, dropping to about 28% during 5 days dehydra tion at 2^ mm liy S.D., during which time they did not feed.

The rate of oxygen consumption at 15°C, 2.k°C, and 35°C was 32.8 p l/g /h r, 65.0 p l/g /h r, and 106.8 p l/g /h r respectively (Table 5).

Respiratory quotients at 15°C, 2k°C, and 35°C were .65, .87, and .86 respectively. In 15% O2 the oxygen consumption was ^7.0 p l/g /h r, and in 10% 02, 3^.0 pl/g/hr (Table 5). The Q.j0 was 2.04 between I5°C and

2k°C and was 1.56 between 2k°C and 35°C.

The osmolal concentration of the hemolymph of 6 well fed specimens of 0. texicolens taken directly from the litter in the terraria (0 mm

Hg S.D.) averaged 193 mOsm, which increased to an average of 302 mOsm after 5 days desiccation at 6 mm Hg S.D. No amino acids were detected in the hemolymph by the method of analysis employed.

Analysis of the fecal material of 0. tex icolens reared in the terraria revealed the presence of both uric acid and ammonia. No urea was found. The concentration of nitrogen from uric acid was .07 mg/g feces, and the nitrogen from ammonia was .\k mg/g feces. These 26 concentrations represent a uric acid/ammonia ratio of .50. The total determined non protein nitrogen represents only .028% of the total dry weight of the feces. 27

TABLE 5

Metabolic Rate of Orthoporus tex icolens

Average Average 02 Number of Wei ght Consumpt i on Cond i t i ons Spec imens (±1 sd.dv.) pl/g/hr

Air, 15°C 10 •30g|0.19 32.8 .650 Air, 24°C 10 .31gJo.17 65.5 .871 Air, 35°C 10 •33g^O.19 106.8 .865 15%02 + 85%N2,24UC 5 .30gj0.l8 47.0 10%02 + 90%N,,24°C 5 .29g-0 . l 8 34.0

to 24°C - ?. 04 24°C to 35°C = 1 .5 6 DISCUSSION

The tracheal apparatus of the two millipeds studied differs both morphologically and functionally. In the polydesmoid p. crass icut is , the tracheal pockets consist of three regions with no mechanism pro vided for closure (Figs. 1-4). The tracheal pocket is unbranched and does not communicate with other tracheal pockets. The lever-muscle arrangement of region B thought by Verhoeff to be a possible aid in ventilation has been shown on the basis of experimental evidence to be unable to pump air in or out of the tracheal pocket. The thickness of the tracheal pocket walls and the small size of the muscle attached to the lever indicates that this structure is an unlikely pump. It seems improbable that the more massive coxal muscles attached to the third region of the tracheal pocket would provide enough movement to aid significantly in forced ventilation. The manometric measurement of oxygen consumption (Table 3) indicates a smooth and continuous rate with no peaks or burst of oxygen consumption even with elevated levels of CO2 in the a ir, supporting the idea that £. crass icut is lacks a ventilation or closing mechanism associated with the tracheal pockets.

Ventilation is probably passive, a simple diffusion of respiratory gases in and out of the spiracle. The approximately 500 tracheae, each

.02 mm in diameter, found attached to the four tracheal pockets per diplosegment would provide an effective diameter sufficiently large for the passive exchange of 104 ^jl/g/hr oxygen that is needed by the animal at 24°C. Prosser and Brown (1962, p. 154) pointed out that 0^ diffuses 10 m illion times faster in air than in muscle. At this low 29 metabolic rate the tissue would be the limiting area in gas transport and not the tracheal system. If there had been active ventilation in this animal, it is presumed that the ventilation rate would have been increased when the animal was exposed to low oxygen pressures. This is not the case, for £. crass i cut i s proved to be a metabolic conformer

(Table 3) when placed in 10% and 15% 0 atmospheres. Had ventilation increased, the oxygen volume per gram per hour would have remained more constant.

In 0. tex icolens a three-chambered tracheal pocket is also found, but in contrast io £. crass icu tis, the third region is expanded anteriorly and medially (Figs. 5-9)• The left and right tracheal pockets of each pair are continuous via this medial expansion. The arch so formed is the point of origin for massive muscles that enter into the coxae of the legs immediately posterior to it . The tracheae of this species, about 300 per pocket (each .02 mm diameter), join the third region of the tracheal pocket and not the second region, as found in P. crass icut is . Near the boundary of region 1 and region 2 (just internal to the spiracle) is a lever-muscle mechanism situated in such a manner it would seem that if sufficient tension on the lever is effected it would cause the posterior or medial lip to close (at least partially) against the lateral or anterior lip of the spiracle.

However, the thickness of the cuticle of the tracheal pocket at this point (about .3 mm) would indicate that the lever muscle could not close the spiracle. Morphologically it is not possible to conclude that this mechanism is inoperative. 30

Sim ilarities in the muscle-lever mechanism of the two species investigated seem to substantiate Verhoeff's idea that they are homologous in these two milliped groups. Both muscles originate at the base of the coxa of the nearest leg and insert on a lever derived from either region 1 or region 2 of the tracheal pocket. The fact that the lever is associated more with the second region of the tracheal pocket of P. crass icut is and more with the firs t region of the tracheal pocket of 0. tex icolens does not present a problem in homology, for the entire tracheal apparatus is thought to be derived from a single invagination of the body wall. Even though this lever-muscle mechanism is not functional in the two species under consideration, it is entirely possible that it might be functional in other milliped taxa.

If either species had a valving mechanism associated with the spiracles, one would expect to obtain results similar to those found in insects when comparing a CO2 rich atmosphere to an atmosphere low in

CC^. With a closing mechanism one expects the rate of water loss and oxygen consumption to be irregular under conditions of high saturation d eficit and low CO2 concentrations. Conversely, one expects then an elevated rate of water loss and uniform oxygen consumption when CO2 concentration is raised, assuming the CO2 stimulates opening of the spiracles. These phenominae were not observed in the 2 species of millipeds studied, because rate of water loss and rate of oxygen consumption remained the same at CO2 concentrations of air (.5%) and at

5%. I interpret these results as being further evidence of the lack of a functional valve in either species. 31

Coiling in P. crass icut is does not block the spiracles and there

fore coiling cannot retard respiratory water loss. Coiling does

effectively retard water loss in 0. tex icolens. When the animal was

restrained from coiling at 24 mm Hg S.D., its rate of desiccation was

13.7%/day as compared to 2.37%/day when permitted to co il. When the

animal is permitted to co il, the fittin g of the leg coxae over the

spiracles, the telescoping of the sterna so as to cover the anterior

spiracles of each diplosegment, and the close contact of the leg coxae

to each other provides a sufficient cover for the spiracles to greatly

reduce the water loss through the respiratory system.

The metabolic rates of the two experimental animals also account

for some of the difference observed in the rate of desiccation. With

the spiracles uncovered in a drying atmosphere, the animal with the

higher metabolic rate has a higher exchange rate of gases and therefore

loses more water vapor through the respiratory system. Pachydesmus

crass icut is has a higher metabolic rate and a higher water loss rate

than does 0. tex icolens. When 0. texicolens is permitted to co il, it

can keep its spiracles closed for a long period of time due to its

lower metabolic rate. This fact plus the fact that the low metabolic

rate causes less gaseous exchange accounts in part for the lower rate

of desiccation for this animal compared to P. crassicutis at all condi

tions tested.

Cuticular transpiration accounts for a portion of the total water

loss per day in the two test species. P. crass icutis lost about

1.5%/day of its total body weight in this manner at 2U°C, U mm Hg S.O., which represented 8% of the total water loss per day. Orthoporus 32

tex icolens lost only about 1 .*4% of its total body weight per day by

cuticular transpiration at 2k°C, 4 mm Hg S.D., but because the total water loss per day only represented 2.8% of the total body weight, the

cuticular water loss was 50% of the total water loss. This high figure

is not due to a more permeable cuticle but to a lower rate of water

loss through the respiratory system.

Neither the abrasion caused by rubbing the animals with an

abrasive powder, nor the continued abrasion caused by friction between

cuticular body parts coated with the abrasive caused any measurable

increase in water loss. It was found that the same treatment applied

to those species of insects which have a waxy layer on the epicuticle

lose water several times as fast (Richards, 1953, p. *+6). As is seen,

the waterproofing material of the integument must not be part of the epicuticle in these millipeds. Whatever the waterproofing agent is in

the millipeds investigated, it is not affected by heat of ^5°C, shown by a lack of an increase in the rate of desiccation in animals that had

firs t been heated to this temperature. Richards (1953, p. t*i*) reported

that in those insects which have a waxy layer on the epicuticle, the

rate of desiccation was greatly Increased after heating to 1+5°C. It

appears that in the species investigated the cuticle is rather imper

vious to water even though it does not have a waxy layer on the epi

cuticle. This is the same situation found in many insect species.

As pointed out in the literature, the nocturnal habit of millipeds

accounts for some water conservation because the saturation d eficit in

the air above the litte r layer Is lower at night than during the day.

Other behavioral patterns of £. crassIcut is were observed that also aided in lowering water loss. During dry periods of the year P. crassicut is would congregate in the more moist areas by either moving several meters or by burrowing deeper into the leaf mold. At times of severe drought many of the animals formed molting chambers. When some of these chambers were brought into the laboratory and placed in moist

terraria, the millipeds within emerged from the molting chambers within two weeks. A check at the site of collection revealed that none of the chambers there had opened. It was not determined if adults could build chambers for protection, but since the Polydesmida do not continue to molt and add segments after reaching sexual maturity, it is considered doubtful. Field observations were not available for 0. texicolens, but

in the laboratory these animals burrow deeper into the soil as it dries on the surface. Since the Spirostreptida continue to molt and add segments throughout the total life span, it is considered possible that both immature stages and adults of 0. texicolens might molt in times of water stress. O'Neill (1969) has reported molting to be a mechanism to

reduce water loss during times of drought in the spirobolid Narceus amer icanus.

If the temperature is raised, and if the absolute water content of the air remains the same, the relative humidity drops. At the same time, the saturation deficit is increased due to the increased satura

tion vapor pressure possible in air at the higher temperature. In this

investigation the saturation deficit was kept the same at Zk°C and

30°C to study 'the effects of temperature increase on the rate of desic cation. In £. crass icut is it is seen that the rate of desiccation remained statistically the same at each respective saturation deficit 3**

.it the two temperatures. In 0. tex Icolens there is an indication that the rate of water loss is higher at the higher temperature at each respective saturation d e fic it. In £. crass icut is the constant rate of desiccation can be explained by the fact that the spiracles are open at all times. At each saturation d e fic it, water exits via the spiracles at its maximum rate for that S.D. The net rate of water loss is equal to the number of molecules of water leaving the spiracles per unit time minus the number of molecules of water entering the spiracle per unit time. According to Graham's Law, the rate (p) that a gas (water vapor) escapes through an o rifice is dependent on the pressure (P) of the gas

(partial pressure of water vapor) and the density (d) of the gas,

and is independent of the temperature (Williams and Williams, 1967. p. 18). In the case of 0. tex?colens in which the rate of water loss increased somewhat with a rise in temperature, the increase of water loss resulted from opening the spiracles more often to obtain more oxygen. With the spiracles closed by coiling, the body tissues were not exposed to the drying atmosphere of the desiccator.

Desiccation tolerance and tolerance to desiccating conditions are sometimes used interchangeably, but for the purpose of this discussion they w ill be given different definitions. Desiccation tolerance

(* tolerance to desiccation) refers to the amount of internal water loss an organism can withstand. Tolerance to desiccating conditions denotes the a b ility of an organism to withstand external drying condi tions. It has been shown that the weight loss at death from water loss 35 was about 56% in £. crass icutIs and about ^0% in 0. tex icolens when desiccated at 2k mm Hg S.D. (0% R.H.) at 24°C. These data were explained when I determined the osmolality of the hemolymph of the two animals from the time they were firs t placed in the desiccators until the time of death. The osmolality of the hemolymph of P. crass icut i s

remains about the same during the entire test period, indicating that this animal is capable of reducing the circulating salts as the total water content of the hemolymph is reduced. In doing so, it prevents the rapid reduction of cellular water because of osmosis, and ultimately a rapid death because the animal cannot control water loss through the spiracles. This is the condition found in most sod -litter

inhabiting animals (Prosser and Brown, 1962, p. 3k). Analysis of the osmolality of the hemolymph of 0. texicolens revealed that the osmola

lity increased as the animal lost water. This increase indicates that the animal does not have the a b ility to reduce its circulating salts,

therefore the cellular water was quickly reduced by osmosis upon the

loss of hemolymph water, accounting for the lower weight loss at death due to desiccation. Because 0. texicolens can control its water loss via the respiratory system and by other systems that are discussed

later, its tolerance to desiccating conditions is much greater than in

the polydesmoid. In conclusion, £. crass icut is has a greater desicca

tion tolerance than 0. texicolens, but 0. texicolens has a greater tolerance to desiccating conditions than P. crass icut is.

The correlation between the ava ilab ility of water to an animal and

the form of the nitrogenous waste product of that animal is with some exceptions very uniform throughout the Metazoa; however, no group of 36 animals has its nitrogenous excretion limited to one product (Prosser and Brown, 1962, p. 239). Those animals that have available an ample supply of water to remove the soluble but toxic ammonia usually excrete ammonia as their main nitrogenous catabolite, but they may excrete smaller percentages of nitrogen in other forms at the same time. These animals are termed ammonotelic. Pachydesmus crassicutis from its hygric habitat and 0. texicolens from its mesic habitat are both found to be ammonotelic. Pachydesmus crassicutis has traces of urea in its excreta. I did not detect uric acid in the excreta ofJP. crass icut i s, but did find it present in the excreta of 0. texicolens. My results of a ratio of 1:2 uric acid/ammonia do not compare to Bennett's (1971) f i nd i ng of a 1:5 uric ac id/ammon i a in Cylindroiulus lond i nens is , a mesic juloid, or to Hubert's (1965) finding a ratio of 1:4 in the same species. The ratio of urea to ammonia in P. crass icut is or the ratio of uric acid to ammonia in 0. texicolens did not change as the animals were desiccated. Prosser and Brown (1962, p. 146) reported that many animals change their excretory products from the more soluble forms to the less soluble forms as the availability of water becomes less. The implications of no change occurring in the ratio of excretory products in these millipeds under differing desiccating conditions was not pursued. The important fact is that the hygric £. crass icut is has ammonia and urea as its metabolic waste products and the mesic 0. texicolens has ammonia and uric acid as its metabolic waste product.

The uric acid-ammonia excreted by 0. tex icolens seems to indicate this species is better adapted to a drier environment than the urea-ammonia excreting £. crass icut is. 37

An investigation of the water content of the feces indicated the

a b ility of the digestive system (hindgut) and malpighian tubules to

reabsorb water. Pachydesmus crass icut is is less able to conserve water

in this manner than is 0. tex icolens. As £. crass icutIs becomes

desiccated the feces change from a very fluid state (85% water) to a

semifluid state (65% water). In contrast, 0. texicolens always has

fecal pellets, varying from 45% water when in 100% R.H. to 28% when dehydrated. This also indicates that 0. texicolens is better adapted

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insects. J. Expl. Biol. 21:97-115. VITA

Thomas Charles Stewart was born in Tyler, Smith County, Texas,

1 January 1937, of Clarence P. and Annie L. (Haley) Stewart. He attended elementary and high school in Tyler, graduating from John

Tyler High School in May, 1955- He attended Tyler Junior College and

Stephen F. Austin State College, graduating from the latter with a B.S. degree in biology. He taught chemistry and biology in Tyler High

School, leaving in 1964 to enter Louisiana State University. He graduated with an M.S. in zoology in August, 1966. From September,

1966, to July, 1968, he was an instructor of biology at Stephen F.

Austin State College. During that period he was married to Linda S.

Carlson. He returned to Louisiana State University in September, 1968, and is applying for the degree of Doctor of Philosophy in the

Department of Zoology and Physiology in May, 1972. EXAMINATION AND THESIS REPORT

Candidate: Thomas C. Stewart

Major Field: Zoo logy

Title of Thesis Anatomical and Physiological Studies of Water Balance in the Millipeds Pachydesmus c. crassicut i s (Polydesmida) and Ortho porus tex ? co lens "(Spi rostreptida) . Approved:

Major Professor ancr'Chairman

Dean /of the Graduate School

EXAMINING COMMITTEE:

Date of Examination:

k February 1972