CHAPTER 6: S haematobiuml S intercalatum (Lower Guinea) interactions

Origin of species

The Lower Guinea strain of S. intercalatum used in this study was isolated from 26 naturally infected Bulinus forskalii snails collected in Edea, Cameroon in 1998. It was maintained for 2 years in laboratory-bred B. wrighti and albino TO mice prior to use.

Eggs of S. haematobium were isolated from urine collected from the population of the village of Barombi Mbo, Cameroon in 2000. Laboratory-bred B. wrighti were exposed to the miracidia hatched from the eggs, and the isolate was passaged once in the laboratory through albino TO mice and B. wrighti prior to use in this study.

Experimental infections

The experimental design was aimed at determining the interactions between males and females of S. haematobium and S. intercalatum in sequential infections in hamsters with S. haematobium as the first infection and S. intercalatum as the second (Fig 2.9). The experiment extends the work carried out by Southgate et al. (1982) on the interactions between these two species in both simultaneous infections, and infections with S. intercalatum as the first infection, and S. haematobium as the second. Taking into account the differences in patency of the two species in hamsters (approximately 60 days for S. haematobium and approximately 45 days for S. intercalatum (Loker, 1983)) hamsters were exposed individually by the paddling technique (section 2.1.4) to 150 mixed-sex cercariae o f S. haematobium, and 4 weeks later to 150 mixed-sex cercariae of S. intercalatum (Zaire strain) so that S. haematobium would reach patency approximately 2 weeks earlier than S. intercalatum. It is assumed that S. haematobium will form homospecific pairs before S. intercalatum, and the usual male-bias of schistosome infections should result in an excess of S. haematobium males which may compete with S. intercalatum males for S. intercalatum females.

Fig 2.9 Schematic representation of the time-course of infections in chapter 6.

1 = infection with S.

!

Day 0 Day 28 Day 60 Day 73 Day 164-185

13-16 weeks

\ ^ CULL

Hamsters were killed and worms collected by perfusion and dissection of the hepatic portal vein and mesenteric venous systems of each infected hamster (section 2.1.5), 13-16 weeks post reinfection. Each pair of worms recovered, and any unpaired worms from the infected mice were segregated into individual containers and the male and female worms from each pair were separated. Females were taxonomically identified whilst still alive: male worms were stored in separate, labelled cryotubes in liquid nitrogen prior to identification.

Species identification

Females were identified by microscopic examination of the morphology of the intrauterine eggs: eggs of S. haematobium are oval at their anterior end and conical at the posterior end, tapering to a terminal posterior spine (Fig 1.7 c), whereas eggs of S. intercalatum are larger, with a more conical anterior end than eggs of S. haematobium (Fig

Since the taxonomic identity of the male worms could not be determined by morphology, individual males were examined for glucose-6-phosphate-dehydrogenase (G6PDH) activity using the isoelectric focusing technique (section 2.3.3.). Worms of the isolates of both species used in this study are monomorphic for G6PDH but differ in the pi value of the enzyme, producing an identifiable profile which enables the two species to be distinguished from one another (Fig 2.10)

1

2 3 4

5

6

7 8 9 10 11 12 13

A'

Fig. 2.10 G6PDH enzymes from extracts of adult male worms. Lanes 1, 2, 9-11 and 13, S.

2.2.5 CHAPTER 7: S. haematobiuml S. mansoni interactions

Origin of species

The isolate of S. haematobium used in this study is as described in section 2.2.4. The isolate of .S', mansoni used in this study is as described in section 2.2.2.

Experimental infections

The experimental design was aimed at determining the mating interactions which take place when a single sex of S. mansoni is introduced into a pre-established infection of mixed male and female S. haematobium. S. mansoni male and female cercariae were obtained from B. glabrata snails infected by a single miracidium. As male and female cercariae cannot be morphologically distinguished from one another, the sex of the S. mansoni infections from the snails was determined in two separate ways. Firstly, DNA was extracted and purified from cercariae shed from each unmiracidially infected snail (section 2.3.2), and tested for PCR amplification of the S. mansoni female-specific W1 marker. The W1 sequence yields a PCR amplification product of ~ 450bp with female S. mansoni DNA, together with a series of higher molecular weight products resulting from degenerate regions in the repeats. Under the PCR conditions below, male DNA produces a complex pattern of non-specific amplification products which contrasts significantly with that produced by female DNA (Fig 2.11).

-450bp

h

Fig 2.11. PCR amplification of female-specific marker W1 from cercariae shed from unimiracidially infected snails. Note the strong band of approximately ~450bp in the female lane. Lane M, size marker (Bioline “Hyperladder I”).

For the amplification of the W1 sequence, 25 ql PCR reactions were performed as below for each extraction and for two control samples (from a male and a female adult worm), using the primers 5’-GTGAAATTCTTCCTTCACAC and 5’-GACATTCAACTCAATGTTCG (Dias Neto gf a/., 1993).

Reagent Volume

DNA 0.5 pi of each extraction

dNTP 4pl (at 1.25mM)

1 OX PCR buffer 2.5pl

Tag DNA polymerase (Cambio) 0.25pl (at 5U/pl)

Primer 0.5pl of each at lOOpmol/pl

ddHzO 16.75pl

TOTAL 25pl

Samples were overlaid with mineral oil, as the PCR machine used (a Perkin-Elmer 480 Thermocycler) did not have a heated lid.

Amplification was performed in a Perkin-Elmer 480 Thermocycler programmed for: 94°C for 5 mins; 30 cycles of [ 94°C for 1 min, 50°C for 1 min and 72°C for 2 mins]; 72°C for 5 mins.

20pl of each sample was mixed with 3 pi of loading dye and run out on a 1% agarose gel containing ethidium bromide.

In addition to the W1 method of sex determination, controls for each snail were carried out by infecting one control mouse for each shedding snail and waiting approximately 35 days for the development of the adult schistosomes (which have morphologically distinct sexes) before culling the mice to reveal the schistosome sex.

Hamsters were exposed individually via the paddling technique (section 2.1.4) to 200 mixed male and female cercariae of S. haematobium, and then either to 100 male cercariae of S. mansoni (experiment #1) or to 100 female S. mansoni cercariae (experiment #2) 8

weeks later, so that worms of the initial S. haematobium infection would be ready to form pairs approximately 5 weeks before the S. mansoni worms were ready to do so (Fig 2.12)

Fig 2.12 Schematic representation of the time-course of infections in chapter 7.

t = infection with both sexes of S. haematobium cercariae j = infection with either female or male S. mansoni cercariae = onset of patency (and pairing) for S. haematobium = onset of patency (and pairing) for S. mansoni = recovery of heterospecific and/or homospecific worm pairs, plus unpaired worms

I

Day 0 Day 56 Day 60 Day 91 Day 140

•> _{7-8 weeks}

C U L L

The hamsters were culled, and worms collected by perfusion and

dissection of the hepatic portal vein and mesenteric venous systems of each infected mouse 7-8 weeks post- reinfection (section 2.1.5). Each pair of worms recovered, and any unpaired worms from the infected mice were segregated into individual containers and the male and female worms from each pair were separated. Mature females were taxonomically identified whilst still alive: immature females and male worms were stored in separate, labelled cryotubes in liquid nitrogen prior to identification.

Species identification

Gravid females were taxonomically identified by the microscopic examination of the morphology of intrauterine eggs: those of S. haematobium have a terminal spine (Fig 1.7 c), whilst S. mansoni eggs have a pronounced lateral spine (Fig 1.7 b).

Non-gravid females with a clearly discernible ovary were identified by the ovary postion which tends to be at/in the posterior third of the body in S. haematobium, but in the anterior half of S. mansoni females (Fig 2.13) (Cowper, 1971).

— oral sucker — - ventral sucker ovary

1

Fig. 2.13. Adult females of S. haematobium and S. mansoni (diagrammatic), showing the differing position of the ovary Adapted from Cowper (1971).

Immature females without a discernible ovary were identified by extracting and purifying DNA from them (section 2.3.1) and testing a sample of the DNA for PCR amplification of the S. mansoni-speciüc Sml07 marker as described in section 2.2.1. This marker produces a characteristic “ladder” pattern for S. mansoni, allowing S. mansoni to be distinguished from other species such as S. haematobium (Fig 2.14).

- 1 0 0 0 0 bp - 5 0 0 0 bp SOOObp -2 0 0 0 b p — 1500 bp — lOOObp — SOObp — eoobp — 200bp

Fig. 2.14. PCR amplification of marker Sm107 to distinguish between immature females of S.

mansoni (lanes 2-10) and S. haematobium (lanes 1, 11 and 12). Lane M, size marker (Bioline “Hyperladder I”).

Male worms were identified by examining them for glucose-6-phosphate-dehydrogenase (G6PDH) activity using the isoelectric focusing technique (section 2.3.3). S. haematobium and S. mansoni are both monomorphic for G6PDH but differ in the pi value of this enzyme, producing an identifiable profile which enables the two species to be distinguished from one another (Fig 2.15)

1 2 3 4 5 6 7 8

Fig. 2.15 G6PDH enzymes from extracts of adult male worms. Lanes 1-4, S. haematobium] lanes 5-8, S. mansoni.