MATERIALS AND METHODS

2.1 MAINTENANCE OF LIVE MATERIAL 2.1.1 Overview

Schistosomes were maintained in the laboratory by repeated cycles (“passages”) of snail (intermediate host) and mouse/hamster (definitive host) infection. The length of time for which they can be maintained in laboratory passage is highly species, strain and even isolate dependent: S. mansoni, for example, can be maintained for many years whereas S. haematobium rarely survives beyond one or two laboratory passage cycles. Schistosomes were introduced into the laboratory by infected snails: either naturally infected, wild-caught snails, or wild-caught or laboratory-bred snails deliberately exposed to miracidia hatched fi'om eggs originating fi*om the livers, urine or faeces of naturally infected definitive hosts.

2.1.2 Snail infection procedure

In the laboratory, snails were infected by exposing them to miracidia hatched fi-om eggs fi"om the livers of infected laboratory rodents: albino TO outbred mice {Mus musculus) and golden hamsters {Mesocricetus auratus). Infected livers were removed from freshly sacrificed rodents (culled in a CO2 chamber) and forced through a 1mm metal mesh into a

beaker containing 0.85% saline. The liver suspension was allowed to sediment under gravity for 30 mins and the supernatant was then discarded. The sediment was re-suspended in saline and washed several times, and the supernatant discarded after each wash. After washing, the sediment containing schistosome eggs was resuspended in tank water (which had been biologically filtered by guppies to remove residual heavy metals) to initiate hatching, which is stimulated by a drop in osmolarity. The suspension was transferred to petri dishes and placed under a lamp for 30 mins to provide heat and light: both are pre­ requisites for miracidial hatching (Erasmus, 1972). After this incubation, the petri-dishes were examined using a binocular microscope: miracidia were clearly visible and could easily be removed using a pasteur pipette. Snails were infected by placing them individually in small pots containing filtered tank water and 5-7 miracidia. The snails were left overnight at 26°C, and then pooled and transferred to a plastic tray containing fi*esh filtered tank water. Snails were maintained at ambient temperatures of 26°C with a 12 hour light/dark cycle; high intensity illumination; and on a diet of dried lettuce and tropical fish

food flakes. Cercariae were shed from snails from approximately 28 days post infection onwards (depending on the schistosome species).

2.1.3 Obtaining cercariae from infected snails

Infected snails were transferred into small beakers of fresh filtered tank water and placed under strong illumination. All species of schistosome used in the mating experiments in this work have cercariae which emerge in response to the sunlight (as opposed to some species, such as those parasitic in rodents, which emerge during the night): strong illumination in the laboratory mimics the effect of the sun to stimulate cercarial emergence from the snails.

2.1.4 Procedure for infecting rodents

Water containing newly shed cercariae was transferred into a petri dish, and the cercariae observed under a binocular microscope where they were easily visible. The number of cercariae required to infect each rodent was removed using a pasteur pipette and transferred to a 500ml opaque plastic beaker containing filtered tank water (200ml for mouse infections, 300ml for hamster infections). The mouse or hamster was allowed to paddle in the infected water for approximately 30 minutes (the ‘partial immersion” technique of infection (Watson & Azim, 1949)), sufficient time for the cercariae to penetrate the skin.

2.1.5 Recovery of worms from infected animals by perfusion

After waiting for the full development of the adult worms, mice were culled using CO2, and

the worms recovered by the following perfusion technique:

1. Using a sharp pair of scissors, the area of skin covering the abdomen was cut away to leave the gut and liver clearly exposed

2. The ribcage was cut open to expose the heart

3. The rodent was pinned onto a vertical perfusion rack, and a 100 pm sieve positioned immediately beneath, to retain the perfused worms

4. The hepatic portal vein leading from the gut to the liver was cut

5. A 60ml syringe was filled with citrated saline (0.75% sodium citrate, 0.85% sodium chloride) and a large bore needle attached

6. The needle was inserted into the base of the heart the fluid injected with some force to push the worms out of the circulatory system onto the sieve

7. The abdominal cavity was washed with 0.85% saline and the washings allowed to drain through the sieve

8. The worms retained by the sieve were washed into a separate pot of saline

9. Any worms which had retained their attachment in the mesenteric veins were dissected out.

Worm pairs were quickly segregated after recovery as the constituent partners often separate after approximately 20 minutes, and may be confused with unpaired worms.

Care was taken to number each pair and each individual worm, prior to taxonomic identification.

2.2 ORIGIN OF SPECIES, EXPERIMENTAL INFECTIONS AND SPECIES IDENTIFICATION FOR THE MATING MODELS IN CHAPTERS 3-7

2.2.1 CHAPTER 3 : S. margrebowiei! S, mansoni interactions

Origin of species

The isolate of S. mansoni used in these experiments originated from Egypt and was introduced into the laboratory by Prof Mike Doenhoff (University of Bangor) where it was maintained for several years in Biomphalaria glabrata and albino TO mice.

The isolate of & margrebowiei used in these experiments originated from the Lochinvar National Park, Zambia and was isolated in 1991 by exposing laboratory-bred Bulinus wrighti to miracidia recovered from the freshly deposited faeces of Kobus Leche (lechwe). It was maintained in the laboratory in Bulinus natalensis and albino TO mice.

Experimental Infections

The experimental design was aimed at determining how male and female worms of both species interact in mixed infections in the vertebrate host, and whether a specific mate recognition system and mating competition exists between the two species. Mice were exposed individually by the paddling technique (section 2.1.4) to a fixed number of male and female cercariae of both species in a series of 5 experiments (Fig 2.1).

The experiments were of two types, the first of which (experiment #1) involved the simultaneous infection of mice with 150 mixed-sex cercariae of S. margrebowiei and 150 mixed-sex cercariae of S. mansoni, thus allowing the schistosomes total choice of mate from either species. The second type (experiments #2-5) involved the sequential infection of mice: firstly with 150 mixed-sex cercariae of S. mansoni, and two weeks later with 150 mixed-sex cercariae of S. margrebowiei (experiment #1) and vice versa (experiment #2), and repeated with a three-week interval between infections (experiments #3 and #4).

Fig 2.1 Schematic representation of the time-course of infections in chapter 3.

1 = infection with S. mansoni cercariae t infection with S. margrebowiei cercariae

^ = onset of patency (and pairing) for S. mansoni onset of patency (and pairing) for S.

margrebowiei = recovery of heterospecific and/or homospecific worm pairs, plus unpaired wonns Day 0

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Day 35 Day 49 Day 84 - 89

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