0 Captions for Drawings Figorh - The apparatus used to investigate temperature re- sponses in the laboratory. Fig. 5 - Six stages through which te development of eighteen broods was followed. The seventh stage counted was emergence from the marsupium. Behavioral and Physiological Adaptations of Female P. scaber Within the Crustacean genus Isopoda, females of most spe¬ cies brood their young in a ventral marsupium, formed by five pair of oostegites which cover the first five thoracic segments, The young emerge as fully developed, miniature adults, except that they lack the last pair of thoracic legs. Speculations have been made about the kind and degree of protection offered by the brood pouch (Verhoeff 1920), but little is actually known. Young isopods, especially these in the marsupium, can¬ not move from adverse microclimates as readily as adults. They have a narrower range of tolerance to stress, for example, they are more susceptible to desication than older individuals, prob- ably because of their small size (Heeley 1911). Paris (1963) found heavy mortality among young in the terrestrial species Ar¬ madillidium vulgare as a result of desication. Verhoeff (1920) suggests that the female is able to regulate the microclimate of the brood pouch to protect the developing embryos from desica¬ tion. I now report on work indicating that the female has be - havioral and physiological adaptations which combat environmental stress to provide for the development of her brood. The terrestrial isopod Porcellio scaber is abundant within the Monterey Peninsula. I looked for evidence of any difference in natural distribution among three classes of P. scaber: brood- ing females, males, and non-brooding females. Three general types of habitat were sampled: (1) a predominately shaded area, cool and moist, where isopods were found in the surface duff and under rot¬ Behavioral and Physiological Adaptations of Female P. scaber ting logs and boards; (2) under rocks of surface contact area ranging fromm 200 to 800 square centimeters, resting on fine, silty soil. These rocks were shaded about half the day, and underneath them the relative humidity ranged from 60 to 80 percent. (3) Isopods were found on moist, decaying humus un¬ der iceplant on slopes exposed to the sun. The surface temperatures under a roek, board or litter were measured by slipping a glass-bulb mercury thermometer underneath and checking the temperature in more than one place. The temper¬ ature varied little with the position of the thermometer. Rela- tive humidity was measured by slipping cobalt chloride papers un¬ der the rock (Bedford 1955). Following these measurements all isopods were collected, counted, sexed, and measured. Each rock or board was treated as a separate sample. In areas where there were no rocks or boards to deliniate a sample, a 50X50 or 30X30 square centimeter quadrat was used, to insure a sample size of at least ten animals. Maximum-minimum thermometers were placed at the sample sites for a period of six to ten days, both cool and warm, from May 10 to May 25. The mean temperature was calculated as the average of the maximum and minimum temperatures for all the days. The data from these field samples indicated temperature de- pendant distribution. Since some sample sizes were small brood¬ ing and non-brooding females were grouped. Female P. scaber were found in significantly greater numbers at mean temperature from Behavioral and Thysiological Adaptations of Female P. scaber 16 - 19' C than at mean temperature from 12'- 15 C (p«.001, stud¬ ent's t-test) (Fig. 1). There was a higher percentage of females at maximum temperature from 21 - 30 C than at maximum temperature from 11 - 17 C (p£.010, student's t-test) (Fig. 2). There was a higher number of females at minimum temperature 13 C than at min¬ imum temperature 11 C (p2.005, student's t-test) (Fig. 3). To test the hypothesis that female P. scaber seek a different temperature than male, Lbuilt a temperature gradient apparatus in which the temperature response of animals of each class could be tested in individual runways. Its heavy iron base produced a smooth gradient ranging from 11' C to 30'C over a distance of 65 centimeterd. A strip of wet filter paper was laid down each run¬ way to provide 100 percent humidity. The temperature was measured at two centimeter intervals along the gradient with a TELE-THERMO- METER probe. Measuring the temperature across the gradient showed less than O.5 C variation between different runways. The tempera- ture gradient changed little during a series of five experiments. In each experiment the animals were alternated by class. Since P. scaber are photonegative, the apparatus was covered to elimi¬ nate light induced activity. After 10 minutes the cover was re- moved and the positions of the isopods were recorded. The mean temperatures sought by brooding females, non-brooding females, and males were 20'C, 18°C, and 15.1 C respectively. An analysis of variance determined that the variation of means was due to difference in sex, and not to replication of residual errer Behavioral and Physiological Adaptations of Female P. scaber (pé.Ol, F-test). There are significant differences between the mean temperatures sought by males and brooding females (p.001, student's t-test) and by males and females (pé.Ol, student's t-test). There is no significant difference between the mean temperatures sought by brooding and non-brooding females. This confirms what the field study revealed: that female P. scaber seek a higher temperature range than male. This may be a seasonal occurance to insure that brooding females will ex¬ perience temperatures which may be optimal to development of the embryos. To investigate the role of temperature in the rate of devel- opment of the young, I followed the in vivo development of eight- een broods. I chose and characterized seven stages of develop¬ ment which I could easily identify, counting emergence as stage "7", and selected eighteen females whose broods ranged through the first six stages. These brooding females were kept in containers of moist soil and vegetation, nine at 15°C and nine at 20°C. Every other day a few embryos were removed from each brood and staged. This technique required restraining the mother under filtered sea water in a tray and lifting a brood plate to allow a few embryos to float out. This method usually did not damage the brood pouch and the females recovered immediately. The average rate of development for each brood was calculated as the number of stages through which it developed, divided by the number of days it took to reach the last observed stage. The mean Behavioral and Physiological Adaptations of Female P. scaber rate of development was the average of the individual rates at each temperature. The mean rate at 20°C was .16 stages per day, twice as great as the mean rate at 15°C which was .23 stages per day, a significant difference (p.0l, student's t-test). Verhoeff (1920) reported durations of 19, 72, and 102 days in P. scaber, from the time of the first visible swelling of the brood pouch until the young emerged. He found that the variation in brooding duration in P. scaber may br greater than in other species of Oniscoidea. He also noted that the duration of individual stages was dependant on the weather, however, he did not report temperature data. At the stage which I called "l", the brood pouch was easily perceptible. Even allowing for the elapse of several days before "I", and for the possibility that my manipulations may have initi- ated early emergence of the young, my brood durations - about 25 to 28 days at 20'C - were shorter than Verhoeff's shortest. The in¬ creased rate of development associated with a warmer microhabitat is a likely factor in the temperature response shown by female P. scaber at this time of year. As an advantage, the shorter brooding duration decreases the length of a period during which the female is most vulnerable to mechanical injury and parasitism, both noted by verhoeff (1920). I observed several cases of damaged brood plates as well as the presence of worms within some brood pouches. Presumably there is increased desication stress associated with a warmer microhabitat. That the female seeks warmer temperatures may indicate that the brood poch envirorment provides a controlled Behavioral and Physiological Adaptations of Female P.scaber humidity to protect the young. To test the effects of desication stress on females and their broods, in comparison with males, the animals were placed in a constant environment of 50 percent humidity at 23 -25 C. Initially I used fourteen animals from each class and recorded the time of death for each. Afterward I found that differ- ences in weight as small as two milligrams could be masking any ef- fect of the animals sex on its survival time. I selected only the seven pairs in which the weight difference was less than one milli- gram. The mean survival time for the group of three brooding and four non-brooding females was 15.2 hours, siggnificantly longer than the mean survival time for the seven males, 11.1 hours (pé.Ol1, student's t-test). There is evidently some sex-related character- istic which makes it possible for females to resist desication stress longer than males. I have noticed a general difference between the shapes of each sex: males tend to be longer and narrow¬ er than females and brooding females of the same weight. At the time of her death, the brood of each female was still alive. This is additional evidence that the marsupium is a pro- tective environment. It is interesting that the mother is sacri- ficed while the brood is preserved, since the embryos desicate with- in a few hours if they remain in stress conditions. It seems more advantageous to the species that the mother sacrifice her brood and take advantage of the fluid in the marsupium,if she could possibly live to produce another brood. This may indicate that there is no link between the internal fluid system of the mother and the brood pouch fluid. Behavioral and Physiological Adaptations of Female P. scaber Summary Field studies have revealed that female P. scaber seek warmer temperatures than male. A laboratory experiment which allowed ani- mals to seek temperatures in a gradient confirmed the field obser¬ vations, yielding mean temperatures of 15.lC and from 18 to 20'C sought by males and females respectively. The experiment indicated no difference between the temperature responses of brooding and non-brooding females; the response does not seem to occur in the individual with the onset of the brooding period, but appears to be a characteristic of the sex. The positive response to warmer temperatures which has been shown by females may be a seasonal oc¬ curance which insures that brooding females will experience temp¬ eratures which may be optimum for embryonic development. It has been shown that increasing the temperature of the microhabitat from 15 to 20 C did indeed double the rate of embryonic development. Rate of development clearly may play an important role in the temp¬ erature response. In conjunction with seeking a warmer habitat, there is some evidence that females are able to withstand the effects of desication stress for a longer period than males. That the brood within the marsupium survives a period of desication which has killed the mother provides striking evidence of the protective qualities of the brood pouch. 0 Behavioral and Physiological Adaptations of Female P. scaber Acknowledgements I would like to thank Robin Burnett and Nat Howe for their assistence and for their encouragement. Behavioral and Physiological Adaptations of Female P. scaber References Bedford, Franklin T. Climates in Miniature. London: Faber and Faber Limited, 1955, p. 87. Brereton, J. LeGay. 1957. The distribution of woodland isopods. Oikos 8(1): 85-106. Cloudsley-Thompson, J.L. 1952. Studies in diurnal rhythms. II. Changes in the physiological responses of the woodlouse Oniscus asellus to environmental stimuli. J. Exp. Biol. 29(2): 295-303. Gunn, D.L. 1937. Humidity reactions of the woodlouse, Forcellio sca- ber (Latrielle). J. Exp. Biol. 11(2): 178-186. Heeley, W. 1911. Observations on the life histories of some terres- trial isopods. Proc. Zool. Soc. London. 111: 79-119. Paris, Oscar H. 1963. The ecology of Armadillidium vulgare (Isopoda: Oniscoidea) in California grassland: food, enemies, and weather. Ecol. Monogr. 33(1): 1-22. Verhoeff, Karl W. 1920. On the larvae, the brood sac, and the brood of the Oniscoidea (transletion by D.P. Abbott, May 1973), Zool. Anz. 51: 168-189. 3 U ++ 2 100 60- c5 40 20 Behavioral and PhysiologicalAdaptations ot Female scaber 100 80 1415 1819 1611 1213 TEMPERATURE 9C FIG. MEAN 14111 2122 21130 MAXIMUM TEMPERATURE PC FIG.2 t H Behavioral ard Physiolagical Adaptations ot Female E scaber 100- 80- 60 1111 MINIMUM TEMPERATURE SC rG5 TEMPERATURE GRADIENT APPARATUS 2 110 2 30c M M Pl H -INDIVIDUAL Ve RUNWAYS E P D GL THOT WATER BATH 7IRON BASE UNED WITH ALUMINUM FOIL FIG.4 ICE BATH .9 TABLE 1 INITIAL STAGE TEME 20 20 15 15 20 DAY 1 10 14 16 18 11 19 11 29 11 12 .9 1.3 1.5 75 20 111/3 33 43 DEVELOPMENTAL STAGES INACTIVE 3 1.5-1.8 DISTANCES IN MM 20 20 15 15 | 15 20 15 15 15 5 154 1 13 25 112 112 25 MOTILE IF OUTSIDE POUCH 20 12 15 /2 FIG. 5 15 Captions for Graphs Fig.1 - The felationship between the percentage of female P. scaber in field samples and the mean temperature at sampling locations. Mean percentage and standard deviation are indicated. Fig. 2 - The relationship between the percentage of female P. scaber in field samples and the maximum temper- ature at sampling locations. Mean percentage and standard deviation are indicated. Fig. 3 - The relationship between the percentage of female P. scaber in field samples and the minimum temperature at sampling locations. Mean percentage and standard deviation are indicated. e Captions for Tables Table 1 - The development of eighteen individual broods through an eighteen day period. Nine of the brooding females were kept at 20 C and nine were kept at 15 C. The av¬ erage rate of development in stages per day is indicated for each brood.