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The in vitro development of the pentastomid Porocephalus crotali from the infective instar to the adult stage

The in vitro development of the pentastomid Porocephalus crotali from the infective, seventh (VII) instar, dissected from the tissues of rat intermediate hosts, to the adult male (X) and female (XI) instars, normally resident in the lung of rattlesnake definitive hosts, is described. The culture medium comprised washed human blood cells, resuspended in bovine serum (50[ratio ]50, v/v), with 20% minimum essential medium and antibiotics. Two batches of approximately 100 pentastomids, maintained at a density of 2 worms/ml, were cultured in 500 ml bottles in an atmosphere of 5% CO2 in air at 28°C. Culture bottles were rotated slowly on a Rollacell system and the medium was replenished every 2–3 days. Growth was monitored at various intervals by weighing worms, measuring cast cuticles with attached moulted hooks recovered from the medium, and by dissecting worms for measurements of various organ systems. Three moults separated the infective and adult male instar, whereas 4 moults were necessary in the case of females. In both cultures natural mortality was about 10% over a 160-day period, by which time 33–51% of females and 62–70% of males had reached the terminal instar. Some males attained full sexual maturity with seminal vesicles loaded with viable sperm: such males were indistinguishable from males recovered from naturally infected rattlesnakes. However, females never achieved full size, even after 200 days, and although copulation did not occur in vitro, some females became patent.