The Effects of Temperature and Humidity on the Survival and ...

Aust. J. Zool., 1988, 36, 649-59

The Effects of Temperature and Humidity on the Survival and Development of the European Rabbit Flea, Spilopsyllus cuniculi (Dale) B. D. CookeA and M. A . SkewesB Animal and Plant Control Commission, G.P.O.Box 1671, Adelaide, S.A. 5001, Australia. South Australian Department of Agriculture, Loxton Research Centre, Box 411, Loxton, S.A. 5333, Austra!ia.

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Abstract The development of rabbit fleas from eggs to adults is strongly influenced by both temperature and relative humidity. Normal development only occurs if the temperature is between 15 and 30°C and humidity lies between 70 and 95% RH. This is consistent with the ecology of flea larvae which develop in the rabbit's nest where young rabbits generally maintain a warm, humid environment. At 27°C new adult fleas emerge from pupae about 21 days after egg laying. If environmental air becomes too dry, the water content of the air, even in the rabbit nest, may not always be sufficient to maintain a sufficiently moist microclimate in the nest for the flea larvae. Adult fleas do not appear to have high tolerance to heat or desiccation and, in the more arid parts of Australia, if they left the rabbits during the summer the microclimate of burrows is likely to be too harsh for them to persist.

Introduction The distribution of the European rabbit flea, Spilopsyllus cuniculi (Dale), in Australia is limited to the more humid parts of the range of its specific host, the introduced European rabbit, Oryctolagus cuniculus. Generally the arid inland limit of the fleas falls between the 200 and 250 mm isohyets (King and Wheeler 1982; Cooke 1984; King et al. 1985). In some arid localities, fleas have temporarily colonised large areas but then have died out again; the disappearance of these populations occurred most commonly in drought years, particularly during the hotter summer months (Cooke 1984). Such losses could result from the direct effects of high temperature or low humidity on the adult fleas, but they could also be caused less directly-for example if the dry conditions inhibited the successful production of flea larvae. The consequences of low production of flea larvae would not become obvious until late in the spring or in early summer when there was little recruitment of young fleas to match the losses of the older ones and the size of the population would fall. Field observations have not led to clear definition of those factors which limit the distribution of the fleas. However, it is known that temperature within rabbit burrows varies seasonally and that it follows a more or less set pattern from year to year. On the other hand, humidity can vary more widely depending on rainfall. Consequently, the association between the loss of fleas and drought suggests that humidity, rather than temperature, might be the main limiting factor. To provide a better understanding of the limiting factors, experiments have been carried out on the effects of temperature and humidity on both juvenile and adult rabbit fleas. The results should enable us to determine the likely effects of burrow microclimates on the flea population and identify those life stages of the fleas which might be most vulnerable to low humidity or high temperatures.

B. D. Cooke and M. A. Skewes

Materials and Methods Collection and Maintenance of Eggs, Larvae, Pupae and Adult Fleas Fleas were maintained on a colony of breeding rabbits at the Northfield Laboratories, Adelaide, using the methods described by Sobey et al. (1974). However, when young rabbits were born, fleas were collected from the rabbits' nests or combed from the rabbits' ears and confined with two new-born rabbits in a paper lined container. The eggs laid by the fleas adhered to the paper and batches of 8-10 eggs were obtained by cutting tiny sections from the paper using a scalpel (Vaughan and Coombs 1979). The eggs were collected daily, so that their age was known to the nearest day. The paper sections with adhering eggs were placed in 10 mL glass vials with gauze caps. These vials also contained about 3 g of rearing medium which consisted of sterile sand, rabbit blood dried at 40°C and finely powdered, and pasteurised yeast. The eggs were examined daily to record hatching. The survival and development of larvae was also recorded by regular observation using a stereo-microscope (20X). Pupae used in experiments were collected from cultures of flea larvae which had been grown on the rearing medium at 25OC and 90% relative humidity (RH). These cultures were inspected daily and only freshly spun cocoons were collected, so that batches used in the experiments were of even age. In experiments with adult fleas, freshly emerged adults were used to ensure that the experimental fleas were of similar age and physiological condition. Temperature and Humidity Control Constant temperature cabinets were used to maintain eggs and immature stages of the fleas at specific temperatures. The temperatures used in the experiments were from 10 to 35'C, to + 1°C accuracy, which covers the range of temperatures which could possibly be encountered by developing fleas in rabbit burrows (Hall and Myers 1978). Humidity chambers consisted of plastic containers with tight-fitting lids and contained solutions of sulphuric acid which were adjusted to maintain in the chambers specific humidities ranging from 10% RH to saturation (Solomon 1952; Weast 1972). Experiments with Eggs and Larvae To observe the effect of different temperatures and humidities on the flea eggs, batches of eggs on paper substrate were first examined under the microscope (25 x), to ensure that they were fully turgid, then placed in vials and used in experiments as follows: Experiment I . To determine the effect of temperature on the survival and development of eggs and larvae, three vials each containing 10 freshly laid eggs were set up at 10, 15, 20, 25, 30 and 35°C. For this experiment all vials were kept at 90% RH. They were monitored regularly to record times taken to complete each growth stage. Experiment 2. To determine the effects of humidity on survival and development of the flea eggs and larvae, temperature was kept constant at 25OC and seven different levels of humidity were used ranging from 50% RH to saturation. A temperature of about 25°C was selected because this is about the temperature which flea larvae experience in the rabbits' nests. Experiment 3. Some attempt was also made to explore the interactions between temperature and humidity because temperature may have not only a direct effect on the survival of the larvae but also some indirect effect by influencing saturation deficit. For this work, a factorial experimental design was used which included 12 treatments based on three levels of temperature (15, 20 and 25"C), and four levels of humidity (50, 60, 70 and 80% RH). Three vials each containing 10 newly laid eggs were used as replicates within each treatment. Experiments with Fleas in the Cocoon Stage Experiment 4. To explore the effects of humidity on the development of fleas in the cocoon stage, batches of 10 1-day-old cocoons were placed into glass vials and one vial was placed at each of a range of humidities from 10 to 90% RH. This same simple experiment was later repeated using groups of 4-day-old cocoons. Daily inspection of the cocoons enabled emergence times to be recorded. The proportion of fleas which emerged from each batch was also noted. Experiments with Adult Fleas Experiment 5. The first experiment with adult fleas was designed to test the hypothesis that the microclimate of rabbit burrows, as recorded for arid sites in mid-summer (Hall and Myers 1978), might

Effects of Temperature and Humidity on S. cuniculi

cause high mortality among fleas which lived away from the rabbits. A factorial experimental design was used with three levels of temperature (25, 30 and 3S°C), and three levels of relative humidity (32, 52 and 95%). Within each treatment there were 10 glass vials each containing five newly emerged fleas. A small piece of rabbit fur was added to each tube to provide a suitable substrate and prevent the fleas from adhering to the glass. This was particularly important at the high humidities. The fleas were checked daily and the numbers of survivors recorded. From these data, mean survival times of the fleas in each tube were derived. No attempt was made to determine separately the survival rates of fleas of each sex.

Experiment 6. A second experiment with adult fleas was designed to compare the survival of fed and unfed fleas. The reason for the experiment was to test the hypothesis that fleas which obtained a blood meal from their host might better withstand desiccation. Again, a factorial experimental design was used with three levels of humidity and fleas at three levels of feeding: an unfed control group, fleas allowed to feed on a rabbit for 2 dyas, and others allowed to feed for 4 days. The humidity levels were 50, 80 and 95% RH. Six vials each containing five fleas were used in each treatment, and the survival time of the fleas in each vial was measured. Mean survival times calculated from these data were used in subsequent statistical analyses. Experiment 7. This experiment was designed to test the hypothesis that although fleas may lose water in dry conditions, they might be able to recoup water losses during periods of high humidity. For example, it is possible that the rabbit fleas might lose water during the day as the air within the rabbit burrows became warm and dry, but regain some water when the air became sufficiently cool at night and humidity was substantially increased. In the experiment fleas were held at constant temperature, 25OC, but subjected to low humidity, 50% RH, for 16 h then high humidity, 95% RH, for 8 h each day. Their mean survival time was compared with that of two other groups of fleas held constantly at high and low humidity respectively. For each treatment 10 glass vials each containing five fleas were used and once again mean survival time for the fleas in each treatment was estimated.

Results

Effects of Temperature on Development and Survival of Eggs and Larvae For each temperature level used in experiment 1, the percentage of eggs which finally developed into adult fleas is shown in Fig. 1. It is quite clear that high survival from eggs to adults occurred only between the temperatures of 20-25°C. The figure also shows the mean time to egg hatching, cocoon spinning and emergence of adults at each temperature level. At 10°C none of the eggs were able to hatch, although some well developed young could be seen through the shell membranes. At temperatures of 15-30°C the eggs hatched normally, although the time taken to complete hatching was shortened with increasing temperatures. At 35°C only about 40% of the eggs hatched and the newly emerged larvae died within a day. For the larval stages, it was found that the one flea which completed development at 15°C took almost 70 days; at 25°C the time from egg-laying to emergence of new adults was on average about 24 days and at 30°C the average time to emergence was only 16 days. Effects of Humidity on Development and Survival of Eggs and Larvae At 25°C the rate of development of fleas eggs at all humidity levels was rapid and most eggs hatched within 3 or 4 days (experiment 2). Even at the lower relative humidities of 50-70% when the eggs lost their normal turgid appearance and began to shrink from loss of water this did not affect the success of hatching (Fig. 2). The most obvious effect of humidity was on the survival of flea larvae. An analysis of variance of the data showed that the differences between survival of the flea larvae at different humidities was highly significant (PcO.001). Fig. 3 shows how survival changed with humidity. No flea larvae were able to survive long enough to complete their development when relative humidity was 70% or less. Survival was also reduced at very high humidities. Optimum survival was observed between 80 and 90% RH.


Temperature (C)

Fig. 1. Percentage survival and mean development times of eggs, larvae and pupae at different temperatures. All measurements were made at 90% RH.

Table 1. The influence of temperature and humidity on the time of hatching of flea eggs The variate is the number of eggs in each treatment which hatched within 3 days of laying. At that time all eggs which had not hatched still contained viable, developing embryos. There were three vials each containing 10 eggs in each treatment Relative humidity (%)

Treatment totals at: 15°C 20°C 25°C

Effects of Temperature and Humidity on S. cuniculi

Relative humidity

(%I

Fig. 2. The effect of humidity on the hatching of eggs and subsequent survival of flea larvae kept at 25°C.

Interaction between Temperature and Humidity Experiment 3, which considered the interaction between temperature and humidity, showed that there was a significant interaction between these factors in terms of the hatching of flea eggs (for temperature (T), F2,,, = 350.3, P < 0.001; humidity ( H ) , F3,24= 18.97, P < 0 . 0 5 ; and T x H , F6,,,= 1 3 . 2 , P e 0 . 0 1 ) (Table 1). Specifically, it was apparent that flea larvae hatched and survived very poorly at the lower temperatures unless humidity was high. Such an observation is quite unexpected because, as temperature falls, the saturation Table 2. The combined effects of temperature and humidity on the net production of flea larvae The variate is the number of flea larvae in each treatment 10 days after egg-laying. There were three vials each containing 10 eggs in each treatment Relative humidity (%)

Treatment totals at: 15°C 20°C 25°C


deficit also falls at any given level of relative humidity. As a consequence, flea eggs and larvae might be expected to tolerate low relative humidities if the temperature was low. However, low temperature also delays the rate of development of embryos and retards hatching (Fig. 1). Furthermore, at any given temperature, the level of humidity also influences the time taken for embryos to develop to hatching (Table 1). The net result of these interactions between temperature and humidity is that the waterloss which is incurred because of the slow development overrides any benefit gained from the lowered saturation deficit (Table 2). This means that humidity is the dominant factor (F3,24 = 18 4, P C 0 -01). Generally, hatching and subsequent survival of larvae is unsuccessful if the relative humidity is less than 70% RH. 100-

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Relative humidity (%) Fig. 3. Emergence of adult fleas from 1 and 4-day-old cocoons held at different humidities at 2 5 C . Table 3. The effect of humidity on the time of emergence of adult fleas from pupae

The data show the number of fleas which emerged by day 12 as a percentage of the total number to emerge at each humidity level. Total fleas in each treatment, 40 Relative humidity (%)

Emergence (%) At day 12 Total

Survival of Fleas in the Cocoon Stage at Different Humidities In experiment 4, batches of 1-day-old cocoons and Cday-old cocoons were kept at constant temperature but different humidities. The percentage of cocoons which yielded

Effects of Temperature and Humidity on S, cuniculi

adult fleas are given, for each specific humidity, in Fig. 3. With 1-day-old cocoons, at 60% RH or above the emergence of adult fleas was high, but below this level emergence was uniformly low. With 4-day-old cocoons a high proportion of fleas emerged at all humidity levels. Data collected on the emergence times of the adult fleas at each humidity level show quite clearly that the pupae held at low humidities emerged earlier than those held at high humidities (Table 3). The data show for each humidity level, the percentage of pupae which had emerged by day 12 after spinning cocoons. This observation cannot be explained simply by the fact that at low humidities the most slowly developing larvae were the most likely to die from desiccation. Such mortality might reduce the time of average emergence but it would not explain why emergence began a day or two earlier at low humidities than at high ones. The mean emergence time of fleas held at 10% RH was 11.2 days, whereas fleas emerged from pupae held at 90% RH at 14.0 days on average. Table 4. The survival of unfed, adult fleas at different temperatures and humidities Variate is In mean survival time (days) Relative humidity (%)

Treatment totals at:

25°C

30°C

35°C

Fig. 4. The relationship between In survival time of adult fleas (days) and saturation deficit at three different temperatures.

Saturation deficit (rnm Hg)

Survival of Adult Fleas at Different Temperatures and Humidities From experiment 5, clear evidence was gained that both temperature and humidity caused significant changes to the mean survival time of fleas. Bartlett's test showed that the variances within each treatment were heterogeneous (Xi= 27.7, PC 0.001) but transformation of these data using natural logarithms reduced this heterogeneity (Xi= 7.8, n.s.). An analysis of variance was carried out using In survival times (Table 4). The results show that both temperature and humidity had significant effects on survival (for temperature, F2,36 = 98.00,


PC0.001 ; for humidity, F2,36 = 52 00, P < 0.001). There was no significant interaction term, however, which implies that the effects of temperature and humidity are independent. This is surprising because temperature changes can affect the saturation deficit of the air even though relative humidity remains virtually constant. Nevertheless, even if the data are considered in relation to the calculated saturation deficit it remains clear that temperature still has a marked but independent effect on survival (Fig. 4). It can also be seen from Fig. 4 that, for any given temperature, the relationship between log-transformed survival times and the saturation deficit can be assumed to be linear. Table 5. Analysis of variance of the survival of fleas at different levels of feeding and humidity Variate is In mean survival time. There were six replicates in each treatment Level of feeding 0 days 2 days 4 days

Treatment means at: 50% RH 80% RH 1.10 1.29 1.24

1.02 1.90 2.05

95% RH 2.04 2.15 2.36

The Effect of Feeding on the Survival of Adults In experiment 6, the survival of fed fleas was examined in relation to that of unfed fleas held at 25"C, and 50, 80 and 95% RH. The results (Table 5) show that fleas which had been fed for 4 days and also those fed for 2 days survived significantly longer than unfed controls (F2,,,= 12.2, P