Entomologia Experimentalis etApplicata 76: 241-245, 1995. 241 (~) 1995 Kluwer Academic Publishers. Printed in Belgium.

Laboratory rearing of the squirrel flea Ceratophyllus sciurorum sciurorum with notes on its biology

K. S. L a r s e n Danish Pest Infestation Laboratory, Skovbrynet 14, DK-2800 Lyngby, Denmark

Accepted: December 22, 1994

Key words: rearing, egg-laying, development period, Ceratophyllus sciurorum sciurorum

Abstract

A rearing method for the flea Ceratophyllus (Monopsyllus) sciurorum sciurorum, a pest on farmed mink in Europe, has been developed. The behaviour of egg-laying and the development period are described at 23 ~ and 80% r.h. The eggs of C. sciurorum sciurorum hatched after 4-7 days and cocoons were formed 10-12 days later. The development period from egg to newly emerged adult was 23-32 days. Females emerged, in general, earlier than males.

Introduct ion

The flea Ceratophyllus (Monopsyllus) sciurorum sci- urorum (Schrank) (Siphonaptera: Ceratophyllidae) is a pest of farmed mink (Mustela vison) in several European countries (e.g. Jurik & Kukla, 1974; de Jonge, 1979; Suciu et al., 1984; Larsen, 1992b, c). Heavy infestations of fleas on farmed mink may cause anaemia (Winding, 1970) or even death of the mink kits (de Jonge, 1979). The pelt may also be damaged when the mink reacts to fleas by scratching and bit- ing (Gorham et al., 1972; Wenzel & Berestov, 1986). Finally, fleas are considered as potential vectors of the Aleutian disease virus (Larsen, 1992d).

In spite of intensive control, flea infestations are common on some farms. A survey (Larsen, 1992a), found that more than 40% of farms were infested and a variety of insecticides failed to control flea popula- tions.

Apart from being a pest on farmed mink, C. s. sci- urorum is a common parasite on the red squirrel Sciu- rus vulgaris: it also frequently parasitises other Sciurus spp., glirids, martens, and many tree-inhabiting birds (Traub et al., 1983).

For a study of the biology of C. s. sciurorum and to test the efficacy of different insecticides for its control, it was necessary to rear the flea in the laboratory. This paper presents a method for rearing C. s. sciurorum on

the long-tailed field mouse (Apodemus sylvaticus) and the yellow-necked field mouse (Apodemusflavicollis) with notes on egg-laying and development.

Materials and methods

Field collection of fleas. Adults of C. s. sciurorum were obtained in March 1990 from a heavily infested mink farm near Herning, Central Jutland, Denmark. The fleas were collected from mink nests, nest boxes, the ground below the nest boxes, and from the inside and outside of the woodwork of the buildings contain- ing the cages. The fleas were caught using a pooter and transferred to glass vials containing strips of moistened filter paper to ensure high humidity during transporta- tion to the laboratory.

Flea rearing. In the laboratory, the collected fleas were anaesthetized using CO2, examined and sexed under a binocular microscope (30 • Only C. s. sciurorum was found. Forty female and ten male fleas were transferred to each laboratory-reared mouse, either A. sylvaticus or A. flavicollis, which were kept separately in glass cages (35 cm deep, 60 cm long, and 30 cm wide) with a metal mesh lid (9 mm sq.). Each cage housed a stainless steel mouse nest box (13.5 cm deep, 25 cm long, and 12.5 cm wide) (Fig. 1). The nest box had an entrance

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a

- - b

C

m d

e

Fig. 1. Mouse nest box. a = stainless steel box, b = straw material, c = nest box floor (metal grid), d = filter paper tray and e = metal grid protecting tray.

(diameter 5.8 cm) and four holes (diameter 1 cm) in the roof for ventilation. The floor of the nest box was elevated 1.5 cm above the underlying filter paper tray. A metal grid surrounded the filter paper tray to protect it from being destroyed by the mouse. The fleas and the mouse were put into the nest box, which was filled with barley straw. The straw was heated to 60 ~ before use to destroy predatory mites. Approximately 3.5 g dried bovine blood was placed in the filter paper tray to ensure adequate food for the flea larvae. The mice were given water and dry pelleted food (Altromin no 1324; Chr. Petersen A/S, Denmark) ad libitum. Having tested different environmental conditions in the room where the cultures were kept, 184- 1 ~ and 804-5% r.h. was found to create suitable rearing conditions for the fleas. Further, because the species of mice used are more active during the night (Gelmroth, 1969, 1970), a L 19:D5 hour photoperiod was used to encourage the mice to spend as much time as possible in the nest box with the fleas.

When fleas were needed for experiments, the mouse was removed from the nest box, and the con- tents of the box and filter paper tray were placed into plastic bags. To remove straw, uneaten food pellets and mouse faeces, the material from the bags was

sieved for 15 min in the sieving tower of a vibratory shaker (Retsch Laboratory Sieving Machine (VIBRO); Retsch, Germany). The sieving tower consisted of three sieves (mesh size 2.5 mm, 1.12 mm, and 0.5 mm respectively) and a bottom pan. The material from each of the sieves and the pan was collected in separate plas- tic bags. Still in the bags, the material was spread thinly on a white tray, so that all adult fleas could be seen and removed using a motor-driven pooter. Small amounts of the material from the pan were transferred to glass petri dishes and placed on a black background under a binocular microscope to collect larvae and eggs using a pooter. The adult fleas and sieved nest material were returned to the nest box along with the mouse.

Adult fleas, to supplement breeding colonies or to set up new ones, were reared from eggs and larvae. This method was also used, when adult fleas of known age were needed for experimental work. Flea eggs and larvae, collected from the sieved material as described above, were placed in 240 ml plastic cups containing larval food. These were then placed in an incubator at 23 4- 1 ~ and 80 4- 5% r.h. The larval food consisted of ground rabbit pellets, rat pellets, dried brewer's yeast, and dried bovine blood 50:50:40:10 by weight. This rearing medium was kept in a freezer at - 18 ~ before use to avoid contamination by mites and fungi.

Starting 7 days after collection of eggs and lar- vae, cocoons were obtained by sieving the larval rear- ing medium on the vibratory shaker. Sieves with a mesh size of 2.5 mm and 1.12 mm were used. The cocoons were retained only by the 1.12 mm sieve, but the 2.5 mm sieve is important for an efficient sepa- ration of the cocoons from the often lumpy rearing medium.

The rearing medium was sieved each week until the cocoon formation had ceased. Cocoons were placed in a thin layer on the screens (mesh size 1.2 mm) of the hatching funnels (Fig. 2). The adult fleas which emerged were collected daily by gently shaking the hatching funnels, so that the fleas passed through the screen into the glass vial.

When fleas are not required for experimental pur- poses, the breeding colonies can be left for long periods (up to 3 months) without sub-culturing. However, to keep down the number of mites, it is recommended that the nest box material is sieved every second or third week.

Egg-laying and development. For studying egg-laying behaviour, female fleas with enlarged abdomens were taken from sieved nest box material and placed sepa-

Fig. 2. Hatching funnel, a = plastic cup, b = mesh screen (glued to the cup), c = glass funnel (glued to the cup) and d = glass vial for collecting the emerged fleas. When containing cocoons, a lid is placed on top of the cup.

rately in glass vials with a small tuft of mink hair as a substrate. The number of eggs laid, their ability to hatch, as well as the number of faeces produced by the fleas, were recorded.

The duration of the developmental stages from egg to adult was determined by using eggs collected direct- ly from nests. To ensure that the eggs were newly laid, nests were sieved to clear old eggs, then sieved again within 24 h. Only healthy looking, single-laid eggs were used.

Five eggs were placed in each of the vials contain- ing a thin layer of larval medium, covering approxi- mately half the floor space of the vial, together with a small tuft of mink hair, as a microhabitat for the larvae. Hatching of eggs, cocoon formation and emer- gence of adult fleas, was recorded. The emergence of males and females were analyzed using student's t test (SAS Institute, 1989).

In both the egg-laying and the development stud- ies the vials were kept in darkness in incubators at 23 :i: 1 ~ and 80 + 5% r.h., and examined daily using a binocular microscope and cold light illumination. By keeping the juvenile fleas mainly in darkness, their natural environment was assumed to be mimicked as

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closely as possible. The larvae were observed to be strongly negatively phototactic, and were therefore kept without light.

Results

Egg-laying. In the study of the egg-laying behaviour only 25 of the 83 female fleas which appeared to be gravid actually produced eggs. All eggs were laid with- in 24 h after placing the fleas in the vials and a total of 89 eggs was produced. Each female laid from one to seven eggs (mean= 3.56). The eggs were found on the floor of the glass vials as well as in, or on, the tufts of hair. Although batches of up to five eggs were found, 74% of the eggs was laid singly. Also when select- ing eggs from sieved nest material for other research purposes, mainly single eggs are observed, however, batches of up to nine eggs have also been found.

It was also found that the fleas placed in the vials produced small blood spots at the time of oviposition or shortly after.

Development. The eggs laid in the vials started to hatch on the fourth day following oviposition and hatching continued over a 4-day period. The eggs collected from the nests were found to have a hatching period similar to that of the eggs laid in the vials (Table 1), but with a greater hatching success (75.0% against 31.5%).

Table 1 also presents the time from egg-laying until cocoon formation and emergence of adults, as well as the rate of survival of these stages. The total devel- opment period from egg to adult emerging from the cocoon varied between 23 and 32 days (Fig. 3). How- ever, female fleas were found to emerge significantly earlier (average 3 days) than males (P<0.0001, d f= 25, t = 4.97). The male: female ratio of the emerged fleas was found to be 1:1.25. At the temperature and humid- ity in the test mortality occurred only during the egg- and larval stages.

Discussion

For more than 3 years, the technique described has proved to be successful in producing a regular sup- ply of C. s. sciurorum in the laboratory. A. sylvaticus was easier to handle than A. flavicollis, which is highly active. The presence of baby mice in the nests made the removal of nesting material more labourious. There- fore male mice were preferentially used.

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Table 1. Length of time (mean 4- SE) required from oviposition to egg hatching, cocoon formation and emergence of adults as well as the rate of C. sciurorum sciurorum eggs developing into the different stages. Figures in brackets are ranges, a = eggs laid in glass vials (n = 89) and b = eggs selected from mouse nests (n =44). The fleas were kept at 23 ~ and 80% r.h.

Eggs Eggs Cocoon Emergence of adults b hatching '~ hatching b formationb 9 9 <Y o"

Days following 4.96 + 0.84 5.30 4- 0.68 16.00 4- 1.30 25.47 -I- 1.46 28.42 4- 1.62 oviposition (4-7) (4-7) (14-19) (23-28) (26-32)

Percentage 34.1 27.3 developed 31.5 75.0 61.3 into this stage 61.3

Fig. 3. The distribution of the developmental time for each sex of C. sciurorum sciurorum when kept at 23 ~ and 80% r.h.

The egg-laying behaviour of C. s. sciurorum is sim- ilar to that of Ceratophyllus styx, Ceratophyllus gal- linae (Humphries, 1963) and Ctenophthalmus nobilis (Cotton, 1970b). These three species all lay their eggs within 24 -30 h after being removed from the host.

The production of eggs can be related to the age of the female flea (Cooke, 1990; Dryden, 1989). In this study, the age of the oviposit ing female was unknown, and it remains uncertain as to whether 3-4 eggs per flea per day is a true average rate of egg-laying for this

species. The difference found in hatching success between

eggs collected from the nests and eggs from the fleas in the vials, may be explained in two ways. First, only healthy looking eggs were se lec ted from the sieved nest material. Secondly, these eggs were up to 24 h old

when placed at 23 ~ and 80% r.h. Both temperature and humidity have an effect on the hatching success of flea eggs (e.g. Silverman et al., 1981; Amin et al., 1993).

Defaecation at egg-laying is also known from other flea species, such as Orchopeas howardi (Ceratophyl- lus wickhami) (Sikes, 1931), C. gallinae and C. styx (Humphries, 1963) and Ct. nobilis (Cotton, 1970b). Humphries (1963) suggested that this acts as a readily available meal for the first larval instar. These blood spots are easily recognized on the straw from the rear- ing units and in the heavily infested nests of farmed mink.

As noted by Cotton (1970a), the time of emergence of the adult from the cocoon is partly dependent on mechanical disturbance. The time to emergence from the cocoon found in the present trial, should therefore be regarded as the minimum time at 23 ~ and 80% r.h., because the fleas were mechanical ly disturbed each day. The observed peaks of emergence between males and females has been demonstrated for a range of flea species (see Buxton, 1948; Sharif, 1948; Hudson & Prince, 1958; Vaughan & Coombs, 1979; Cooke, 1990; Amin et al., 1993).

Acknowledgments

I acknowledge the support of the Research Secretariat of the Danish Ministry of Agriculture, and in particu- lar, the staff of the Danish Pest Infestation Laboratory. Special thanks to Kristian Fordsmand for assistance at the rearing work, and to Karl-Martin Vagn Jensen, who prepared the illustrations. The author is also grate-

ful to F. C l a r k , M. T. G r e e n w o o d , J. B. J e s p e r s e n and

B. O. N i e l s e n fo r t he i r e n c o u r a g e m e n t .

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