Systemic activity of the avermectins against the cat flea (Siphonaptera: Pulicidae)

Ivermectin has potent systemic activity against numerous species of nematodes and arthropods, but there are some important species in these two groups, such as the cat flea, Ctenocephalides felis (Bouché), that appear to be refractory to it. In an effort to determine if the lack of systemic activity against C. felis is specific to ivermectin, or if it is a class-wide phenomenon, 20 avermectin derivatives were tested in an artificial membrane flea feeding system at concentrations of 20, 10, and 1 microg/ml. Results showed that ivermectin had LC90 and LC50 values against fleas of 19.1 and 9.9 microg/ml, respectively. Only four of the other 19 compounds evaluated possessed both LC90 and LC50 values more potent than ivermectin and even then the advantage was modest. Among those four compounds was a two-fold increase in potency relative to ivermectin when the LC90 values were considered (range, 9.2-10.3 microg/ml) and a two- to eight-fold increase when the LC50 values were examined (range, 1.23-5.26 microg/ml). Neither the possession nor the number of oleandrosyl sugars on the macrocyclic backbone were relevant for additional flea activity because among these four compounds were two disaccharides, a monosaccharide and an aglycone. Also, bond disposition between C-22 and 23 did not contribute to increase in activity because these molecules comprise members with either single or double bonds. One of these avermectin analogs was scaled-up and tested subcutaneously in a dog at >100 times the commercial ivermectin dosage and zero efficacy was observed against the flea. We conclude that even the best in vitro avermectin does not have the in vivo potential to become a commercial oral or subcutaneous flea treatment for companion animals.     

Spectrophotometric method of quantifying adult cat flea (Siphonaptera: Pulicidae) feces.

Cat flea, Ctenocephalides felis felis (Bouché), feces are an essential part of larval nutrition under natural conditions. The mass values of adult flea feces can be measured by dissolving samples of flea feces in Drabkin's reagent, filtering, centrifuging, and measuring absorbance spectrophotometrically at 540 nm. Either flea feces or air-dried host blood can be used to generate the standard curve used to convert absorbance (optical density) values into mass values. Debris collected from flea-infested house cats averaged 23.02 mg debris per cat per h with an average of 10.41 mg flea feces per cat per h. Flea feces deposited in the environment serve as potential larval food. Adult flea feces comprised an average of 44.28% of the debris deposited from infested domestic house cats in this study.     

Blood consumption by the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae)

The volume of blood consumed by actively reproducing female cat fleas, Ctenocephalides felis (Bouché), while feeding in confinement feeding chambers on cats for 3 h, was investigated using two radionuclide blood tags (51Cr-erythrocyte and 125I-albumin) and the gravimetric method. Female fleas consumed an average of 0.110 ml (+/- 0.026 ml) of blood per 100 females in 3 h as determined using the dual radionuclide system. The single 51Cr-erythrocyte tag overestimated blood consumption by 11.3% and the single 125I-albumin tag underestimated blood consumption by 6.4%. The gravimetric method underestimated blood consumption by 72.2% compared with the dual radionuclide value. Investigations of blood consumption of nonconfined female fleas were conducted with cats housed in metabolic cages and restricted from grooming. These investigations were conducted using the single 51Cr-erythrocyte tag, and the data obtained were corrected to compensate for using a single erythrocyte tag. Female cat fleas consumed an average of 13.6 microliters (+/- 2.7 microliters) of blood per day, which was equivalent to 15.15 times their body weight. In an additional study, direct exposure to 40 kHz ultrasound did not reduce blood consumption by the fleas.     

Chorion formation and ultrastructure of the egg of the cat flea (Siphonaptera: Pulicidae)

Oocyte development in adult female cat fleas, Ctenocephalides felis (Bouché), was studied by light and electron microscopy to determine the formation and ultrastructural morphology of the eggshell. As oocytes develop, somatic follicle cells from the lining of the ovariole migrate around the oocytes. The follicle cells produce electron-dense granules that form the vitelline membrane around the developing oocyte. Subsequently, electron-lucent granules containing an electron-dense core (precursors of the chorion) are produced from the rough endoplasmic reticulum that appear as dilated and clear linear clefts in the cytoplasm of the follicle cells. Exocytosis and coalescence of the granules around the oocyte as the follicle cells disintegrate give rise to the chorion. The chorion was found to consist of 4 distinct layers. The external surface of the egg shell consists of a particulate layer approximately 760 nm thick, composed of an electron-lucent layer of widely dispersed granules. Embedded in this layer are electron-dense spheres that project above the surface of this granular layer. Beneath this outermost layer is a band of electron-dense material, consisting of densely packed granules and is half as thick as the outer particulate layer. The 3rd layer consists of relatively thick, weakly laminated chorion, with a felt-like appearance due to a meshwork of microfibrils. Projections of this network of microfibrils form pillars that attach this layer to a thin relatively compact 4th or basal layer. The pillars and the air-filled cavities lying between the 3rd and 4th chorionic layers constitute the chorionic meshwork known as the palisades or trabecular layer that form the major respiratory organ of the eggshell. The trabecular layer is connected to the external environment by means of the lateral and anterior aeroplyes. The vitelline membrane lies between the chorion and oocyte and is a granular, uniform, moderately electron-dense layer measuring approximately 260 nm thick. The micropyle at the posterior of the flea egg consists of a rosette of 50-80 apertures and possesses an internal electron-dense plug between the chorion and the vitelline membrane. An aeropyle at the anterior end of the egg consists of a rosette of 40-50 apertures. An inconspicuous aeropyle appears as a cluster of hexagonal or polygonal-shaped plaques on the lateral surface of the chorion. Each plaque contains 3-8 pores.     

Diel patterns of cat flea (Siphonaptera: Pulicidae) egg and fecal deposition.

The quantity of eggs and feces deposited by replicate populations of cat fleas, Ctenocephalides felis (Bouché), from domestic house cats. Felis catus, were recorded at 1-h intervals over a 24-h period, in a photoperiod of 12:12 (L:D) (photophase, 0600-1800 hours [EST]). Egg production averaged 23.96 +/- 0.83 eggs per female per day and was greatest from 2400 to 0300 hours and lowest from 0700 to 0900 hours and from 1700 to 1900 hours. Daily fecal deposition was 0.77 +/- 0.03 mg feces per flea and did not vary significantly among hours. Flea eggs and adult feces were dropped continuously from infested cats, but significantly more eggs were shed at times of day when cats normally rest. More feces are concentrated in the small areas where cats rest than over the large areas they roam. Consequently, eggs and feces would not be deposited uniformly throughout the hosts home range, resulting in a clumped distribution of larval development sites at host resting areas.     

Development of an assay for the screening of compounds against larvae of the cat flea (Siphonaptera: Pulicidae)

Larvae of the cat flea, Ctenocephalides felis (Bouché), are the target of numerous growth regulators. This study explores the development of an assay that tests the susceptibility of cat flea larvae to a wide range of compounds. Different rearing media and containment units were tested that would facilitate optimization. Larvae of various ages were compared, and 7-d-old larvae were found to be optimal because they were the most uniform in size and age and exhibited a need to feed. The assay could be used to distinguish insecticides from growth inhibitors. The insecticides chlorpyrifos and carbaryl caused 100% larval mortality in 24 h at 10 ppm, and cythioate and fipronil killed the larvae at concentrations of > or = 100 ppm within 24 h. The insect growth regulators methoprene and pyriproxifen caused molt delay at concentrations of 100 ppm and bioallethrin delayed molt at 1,000 ppm. This assay can be used to identify compounds that are specific to cat flea larvae that may be useful in the control of cat flea infestations.     

Off-host observations of mating and postmating behaviors in the cat flea (Siphonaptera: Pulicidae)

A blood meal was necessary for a male cat flea, Ctenocephalides felis (Bouché), to display the mating attempts to an unfed or fed female. More mating pairs resulted when both sexes were fed. Copulation occurred when fed fleas were placed on surfaces with temperatures from 34 to 42 degrees C. This article not only describes the mating and postmating behaviors of cat fleas, but also compares them with those of other fleas. The sequence of mating behavior began when a male approached a female ventrally, and the male's antennae and claspers became erect to attach to the abdomen of the female. Clasper attachment lasted until copulation ended, whereas the male retrieved his antennae immediately after genitalia linkage. The male generally grasped the female's tarsi with his claws during mating. The length of the mating interval terminated by the male ranged from 25 to 110 min and was significantly longer than that terminated by the female (averaging 12.11 min). After the mating pair separated, the male displayed a series of postmating behaviors discussed herein. This article documents grasping and postmating behaviors of the male cat flea.     

Effects of juvenile hormone on eggs and adults of the cat flea (Siphonaptera: Pulicidae)

Juvenile hormone III plays a major role in regulating feeding and reproduction in the adult cat flea, Ctenocephalides felis (Bouché). Both blood consumption and egg production increased in a dose-dependent manner up to a maximum at 1,250 ppm when fleas were continuously exposed to concentrations up to 12,500 ppm juvenile hormone. Histological studies demonstrated that juvenile hormone III also stimulated cellular differentiation of salivary gland epithelia, midgut epithelia, and fat body cells, enhancing the ability of the adult flea to digest blood and synthesize vitellogenins for the maturing oocytes. In unfed fleas, exposure of adults to concentrations of > or = 1,000 ppm juvenile hormone III applied to filter paper resulted in membrane lysis and destruction of salivary gland and midgut epithelial cells, fat body cells, and ovarian tissue. Unlike juvenile hormone mimics, which have potent ovicidal effects in fleas, juvenile hormone had little effect in preventing egg hatch; 58% of the eggs laid by fleas treated with 12,500 ppm juvenile hormone III hatched, and a concentration of 30,000 ppm was required to reduce hatch to 2% in untreated eggs exposed to treated filter paper for 2 h. Compared with the juvenile hormone mimic pyriproxyfen, juvenile hormone III was less toxic to fed adult fleas. However, at a concentration of 12,500 ppm, juvenile hormone killed approximately 45% of the adults and caused autolysis and yolk resorption in the developing oocytes. Thus, at high concentrations, juvenile hormone appears to have a pharmacological effect on fleas, which is highly unusual in insects.     

Studies on the growth of Bartonella henselae in the cat flea (Siphonaptera: Pulicidae)

Two out of three pools of cat fleas, Ctenocephalides felis (Bouche), that were fed Bartonella henselae-positive cat blood for 3 d and then bovine blood for 3 d, were polymerase chain reaction (PCR) positive for B. henselae. In a second experiment, three cats were inoculated with a streptomycin-resistant strain of B. henselae. After the cats were inoculated, caged cat fleas were fed on the cats during three different periods, and then pooled and transferred to noninfected recipient cats. In the first trial, the bacteria in the flea feces were below level of detection when the fleas were transferred from the infected cats to the recipient cat. After the fleas had fed on the recipient cat for 6 d, a bacteria level of 4.00 x 10(3) CFU/ mg was detected in the flea feces. Subsequently, the bacteria level increased for 4 d and then declined. In another experiment, the bacteria level in the flea feces was 1.80 x 10(3) CFU/mg at 2 h after collection and 3.33 x 10(2) CFU/mg at 72 h after collection. These data indicated that this strain of B. henselae can persist in flea feces in the environment for at least 3 d, and that B. henselae can multiply in the cat flea.     

Activity of insecticides against the preemerged adult cat flea in the cocoon (Siphonaptera: Pulicidae)

The activity of insecticides against the adult cat flea, Ctenocephalides felis (Bouché), inside the cocoon (preemerged) was determined by spraying the cocoons directly and observing subsequent emergence of adults over the next 14 d. Direct sprays of 0.5% trimethacarb wettable powder (WP) and chlorpyrifos emulsifiable concentrate (EC) and WP provided greater than 70% kill. The order of activity against the preemerged adult exposed to direct sprays was organophosphates greater than carbamates greater than pyrethroids. In general, pyrethroids and proprietary aerosols failed to kill preemerged adults. Even though most sprays did not kill preemerged adults, they stimulated adult emergence from cocoons. Preemerged adults placed in the nap of carpet were not killed by chlorpyrifos, diazinon, or propetamphos sprays; the carpet protected them effectively.     

Ovicidal and larvicidal modes of action of fenoxycarb against the cat flea (Siphonaptera: Pulicidae)

The mode of action of the insect growth regulator, fenoxycarb, on embryogenesis of cat flea eggs, Ctenocephalides felis (Bouché), was evaluated biologically and morphologically. Newly oviposited flea eggs were aged for 0, 3, 6, 12, 24, 48, or 72 h and exposed to fenoxycarb (1.1 micrograms/cm2) on filter paper disks for 0 (60 s), 2, 4, 8, 12, or 24 h at 23 +/- 1 degree C and 70 +/- 3% RH. Following exposure, samples of flea eggs were processed for microscopic examination and seeded onto carpet swatches containing flea-rearing medium in order to assess egg hatch, and larval, pupal, and adult development. Fenoxycarb exhibited embryocidal activity against eggs in early blastoderm formation, blastokinesis, and advanced larval development up to hatching. The ovicidal effect of fenoxycarb was not restricted to any specific developmental stage of embryogenesis, and no significant relationship was found between duration of exposure and lethal or inhibitory effects. Indeed, exposure for as little as 60 s produced a lethal inhibition of embryonic development. Conventional microscopy and ultrastructural observations of flea eggs exposed to fenoxycarb showed extensive cellular and tissue damage of the developing embryos, including membrane lysis, burst cells, pycnotic nuclei with coalesced and clumped heterochromatin, swollen and ruptured mitochondria, cellular autolysis, and collapse and discoloration of the eggshell. Fenoxycarb also exhibited larvicidal activity against newly emerged and 24-h-old flea larvae. The midguts of larvae exposed to fenoxybarb appeared disrupted and distorted with large gaps. These data document the ovicidal and larvicidal modes of action of fenoxycarb against the cat flea.     

Distribution of cat fleas (Siphonaptera: Pulicidae) on the cat

A total of 3,382 cat fleas, Ctenocephalidesfelis (Bouche), was taken from 164 of the 200 stray cats examined. It was observed that cat fleas preferred specific areas on the cat. A significantly higher mean number of fleas was found on the area of head plus neck than on the ventral part of the body. More specifically, the mean number of fleas was highest on both of the neck and dorsal areas. However, in terms of the density of fleas, the neck had more fleas than the dorsal area did. The fewest fleas were found infesting the legs and tail. Distribution of fleas on the cat may well be explained by the various grooming patterns of the cat, and the knowledge of flea distribution may be valuable for application of on-animal flea control procedures.     

Effects of multiple mating on female reproductive output in the cat flea (Siphonaptera: Pulicidae)

Multiple mating behavior of female cat fleas, Ctenocephalides felis (Bouché), was confirmed in this study, and its effects on fecundity and fertility were investigated as well. The number of fertile eggs produced by mated females was close to nil within 7 d after removal of males, but it was resumed when females were exposed to males again on day 7. Multiple-mated females displayed significantly higher fecundity (400.3 eggs per female) and fertility (182.8 viable eggs per female) than single-mated females (61.7 and 19.0, respectively) in the 24-d period, suggesting that multiple mating by females is an advantageous strategy for cat fleas. The duration of first mating averaged 63.1 min. The high ratio (55.56%) and short duration (34.0 min) of impotent mating suggested that cryptic female choice may be involved during copulation.     

Topical application as a method for comparing the effectiveness of insecticides against cat flea (Siphonaptera: Pulicidae)

A topical (micro-droplet) bioassay is described for the cat flea, Ctenocephalides felis (Bouche). Key parameters are the short carbon dioxide anesthetization and the use of 0.1 ml of acetone per flea. The method was used to compare the effectiveness of 13 insecticides. LD95 values in nanograms per flea were nitenpyram 0.68, fipronil 0.69, deltamethrin 0.70, imidacloprid 0.81, cypermethrin 5.4, fenthion 8.0, diazinon 12, permethrin 19, malathion 29, bendiocarb 170, DDT 710, propoxur 1,300, carbaryl >10,000. Experiments repeated 2-5 yr later, with five of the chemicals, in a different facility, showed only small shifts in potency (0.38- to twofold of the original LD50 values).     

Respiratory gas exchange in the flea Xenopsylla conformis (Siphonaptera: Pulicidae)

Respiratory gas exchange was measured in various developmental stages of the flea Xenopsylla conformis mycerini (Rothschild, 1904) originating from the central Negev in Israel. Gas exchange in fleas was measured using a flow-through respirometry system that monitored CO2 emission. Lowest metabolic rates were found in the cocooned stage, which included prepupae, early stage pupae, late stage pupae, and preemerged adults. Newly emerged adults and feeding larvae had metabolic rates 2.5-3.0 times greater than those for the cocooned stages. Highest rates of gas exchange were found in feeding fleas. The low metabolic rates of the cocooned stages is thought to contribute to their ability to survive for longer periods than free-living larvae and adults.     

Effect of diet composition on weight gain, sperm transfer, and insemination in the cat flea (Siphonaptera: Pulicidae)

Weight gain by adult cat fleas, Ctenocephalidesfelis (Bouché), was influenced primarily by the concentrations of protein and sodium chloride in the feeding solution. After 48 h of feeding, fleas fed whole blood weighed almost twice as much as fleas fed plasma or hemolyzed blood and 1.25 times as much as fleas fed 0.15 M sodium chloride. When fleas were fed sodium chloride solutions ranging from 0 to 0.5 M, weight gain was greatest on the 0.15- or 0.2-M solutions. Weight gain decreased significantly when fleas were fed plasma, hemolyzed blood or 0.3 or 0.5 M sodium chloride in place of whole blood, but improved when plasma was diluted 100% and when hemolyzed blood was diluted 10% with distilled water. Adenosine-5'-triphosphate did not appear to stimulate weight gain in cat fleas; weight gain was unchanged in fleas fed hemolyzed blood or 0.15 M sodium chloride to which 0.005 M ATP was added. Insemination did not occur in starved fleas or those fed protein-free diets. When fleas were starved or fed distilled water, sodium chloride, or other salt solutions, sperm was transferred from the testes to the vas deferens in 91-94% of males, but no females were inseminated. In contrast, when fleas were fed whole blood, hemolyzed blood, plasma, or bovine serum albumin (3.5 or 7.0 g/deciliter) dissolved in 0.15 M saline, 80, 80, 10, and 10% of the females were inseminated, respectively.     

Development of a larval bioassay for susceptibility of cat fleas (Siphonaptera: Pulicidae) to imidacloprid

Strategies for controlling cat fleas, Ctenocephalidesfelisfelis (Bouché), have undergone dramatic changes in the past 5 yr. With the advent of on-animal treatments with residual activity the potential for the development of insecticide resistance increases. A larval bioassay was developed to determine the baseline susceptibility of field-collected strains of cat fleas to imidacloprid. All four laboratory strains tested showed a similar level of susceptibility to imidacloprid. Advantages of this bioassay are that smaller numbers of fleas are required because flea eggs are collected for the test. Insect growth regulators and other novel insecticides can also be evaluated. Using a discriminating dose, the detection of reduced susceptibility in field strains can be determined with as few as 40 eggs.