Targeting of host cell lineages by vertically transmitted, feminising microsporidia

International Journal for Parasitology 36 (2006) 749–756 www.elsevier.com/locate/ijpara

Targeting of host cell lineages by vertically transmitted, feminising microsporidia Robert T. Weedall a, Michael Robinson b, Judith E. Smith a, Alison M. Dunn a,* a

Faculty of Biological Sciences, Institute for Integrative and Comparative Biology, University of Leeds, Miall Building, Clarendon Way, Leeds LS2 9JT, UK b Faculty of Arts, Computing, Engineering and Science, Sheffield Hallam University, Sheffield, UK Received 16 November 2005; received in revised form 24 January 2006; accepted 26 February 2006

Abstract Feminising microsporidian parasites are transmitted vertically from generation to generation of their crustacean hosts. Little is known about the mechanisms underpinning vertical transmission, in particular, parasite transmission to the host gonad during host development. Here, we investigate the burden and distribution of two species of vertically transmitted, feminising microsporidia (Dictyocoela duebenum and Nosema granulosis) during early embryogenesis (zygote to eight-cells) of the Gammarus duebeni host. Parasite burden differs between the two parasites with N. granulosis being higher by a factor of 10. Whilst D. duebenum replicates during the first few host cell divisions, there is no increase in N. granulosis burden. Only merogonic parasite stages were observed in the host embryo. Distribution of both parasites was non-random from the two-cell embryo stage, indicating biased parasite segregation at host cell division. Dictyocoela duebenum burden was low in the germline and somatic gonad progenitor cells but was highest in the ectoderm precursors, leading us to propose that the parasite targets these cells and then secondarily infects the gonad later in host development. Targeting by N. granulosis was less specific although there was a persistent bias in parasite distribution throughout host cell divisions. Parasite burden was highest in the ectoderm precursors as well as the germline progenitors leading us to suggest that, in addition to using the ectodermal route, N. granulosis may also target germline directly. Biased segregation will be adaptive for these parasites as it is likely to lead to efficient transmission and feminisation whilst minimising virulence in the host. q 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. Keywords: Vertical transmission; Embryogenesis; Sex ratio distorter; Microsporidia; Gammarus duebeni; Dictyocoela duebenum; Nosema granulosis

1. Introduction The Microspora are a diverse phylum of endosymbiotic eukaryotic parasites that are most closely related to the fungi (Hirt et al., 1999; Baldauf, 2003) and infect a wide range of invertebrate and vertebrate hosts (Wittner and Weiss, 1999). Microsporidia exhibit diverse life cycles (Dunn and Smith, 2001) and commonly make use of vertical transmission. Vertical transmission occurs in all the major lineages of the phylum suggesting that it may have arisen multiple times or that it may be an ancestral strategy (Terry et al., 2004). For some microsporidia, vertical transmission is supplementary to the main horizontal route whilst for others it is the major transmission route (Dunn and Smith, 2001). Vertical transmission is unusual amongst eukaryotic parasites and selection is likely to favour the evolution of complex strategies to ensure * Corresponding author. Tel.: C44 113 3432856; fax: C44 113 3432835. E-mail address: [email protected] (A.M. Dunn).

that the parasite reaches target host tissues. However, little is known about the evolution of vertical transmission in the microsporidia nor have the mechanisms of vertical transmission been fully described. Here, we investigate patterns of within host transmission during early host development by two phyogenetically distant species of microsporidian parasite: Nosema granulosis and Dictyocoela duebenum (Terry et al., 2004). For microsporidia of invertebrates, the most common route of vertical transmission is transovarial transmission; parasites are passed from mother to offspring via the ova. Because of the small size of sperm, male hosts rarely transmit the infection and so transmission is mostly maternal (Dunn et al., 2001). Many species of microsporidia enhance their transmission and spread by manipulating host reproduction towards overproduction of female offspring. Microsporidia have been shown to achieve this by distorting host sex ratio through male killing (Lucarotti and Andreadis, 1995) and feminisation (Terry et al., 2004). This goes hand in hand with other

0020-7519/$30.00 q 2006 Australian Society for Parasitology Inc. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.ijpara.2006.02.020

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biological adaptations. Whilst horizontally transmitted parasites are often present in high burden with associated high levels of pathogenicity (e.g. Lange et al., 1996), vertically transmitted parasites are dependent on host reproduction for their transmission and have been shown to exhibit lower virulence (e.g. Agnew and Koella, 1999). Vertically transmitted parasites are subject to conflicting selective pressures (Dunn et al., 1998). Because transmission is linked to reproduction, a high parasite burden will be selected if it increases the opportunities for successful transmission to the gametes and so to the next generation of hosts. However, a high parasite load is likely to lead to increased pathogenicity which may reduce host survival or reproduction and hence transmission of the parasite to the next host generation. We predict that these conflicting selective pressures will lead to the evolution of targeting of host cell lineages by parasites during host development. The amphipod crustacean Gammarus duebeni is host to three species of vertically transmitted, feminising microsporidia (Terry et al., 2004) of which N. granulosis is best studied. Previous studies have shown that the life cycle of N. granulosis is tightly coupled to host reproduction. The parasite primarily infects the gonadal tissue of the adult female host. Sporogony occurs in the follicle cells in synchrony with host vitellogenesis and infection of the oocytes occurs during primary vitellogenesis (Terry et al., 1997). The pattern of parasite distribution in host embryos is less well understood. Vertical transmission to future generations depends on successful transmission to the gonad of the developing host. We can envisage two extreme alternative strategies for a vertically transmitted parasite to target the gonad. On the one hand, high replication and burden in all lineages may ensure infection of key tissues but is likely to impose a high cost on host fitness and consequently on future parasite transmission. Alternatively, targeting of particular lineages may reduce the metabolic burden imposed by the parasite whilst still ensuring future transmission. We have shown that, during early embryogenesis, N. granulosis is present in low burden only as merogonic stages (Dunn et al., 1998). Distribution patterns indicated that the parasites showed biased segregation into host lineages during embryonic cell divisions (Dunn et al., 1998), although we were unable to determine which cell lineages were preferentially infected. However, recent descriptions of amphipod embryonic cleavage and cell fates (Wolff and Scholtz, 2002; Gerberding et al., 2002a,b) have enabled us to investigate parasite distribution in relation to embryo architecture. Here, we use empirical observations and theoretical modelling to examine parasite burden and distribution in host cells during early embryogenesis for two species of vertically transmitted microsporidia infecting G. duebeni; D. duebenum and N. granulosis. 2. Materials and methods 2.1. Parasite distribution and burden Ten D. duebenum-infected and 10 N. granulosis-infected G. duebeni females were allowed to form precopula pairs with

males and were observed daily. Gammarus duebeni embryos develop in the mother’s marsupium until hatching. At each cell stage from the zygote to eight-cell, up to five embryos were taken from each mother and stained with DAPI (a DNA fluorescent dye) and examined under a Zeiss Axioplan fluorescence microscope. The diplokaryotic nuclei of microsporidia could be seen in the cytoplasm of the host cells. Host cell type was identified on the basis of size and location according to Wolff and Scholtz (2002). The smallest quadrant of the four-cell stage contains the blastomere A. From the dorsal aspect the other cells are identified in a clockwise direction as B, C and D. Each of these blastomeres will divide asymmetrically to give one macromere ventrally and one micromere dorsally. These daughter cells occupy the same position as the mother cell of the four-cell stage. Parasite burden per cell was recorded. Parasite burden was measured for 84 D. duebenum-and 156 N. granulosis-infected embryos from the zygote to the eight-cell stage. Early embryogenesis has recently been described in the amphipods Orchestia cavimana and Parhyale hawaiiensis and an early restriction of cell fates has been identified (Wolff and Scholtz, 2002; Gerberding et al., 2002a,b). The embryos show a stereotyped pattern of development with total cleavage. The macromeres that come from blastomeres B, D and C of the four-cell stage form the ectoderm of the morphological right side, left side and the posterior ectoderm. The micromeres b and d that derive from these blastomeres will form the mesoderm of the left and right sides whilst micromere c will form the endoderm (gut and epithelia). Blastomere A will produce macromere A that will form the visceral mesoderm and micromere a that will form the primordial germline. 2.2. Parasitism and host development Host development time was measured for uninfected embryos and for embryos infected with D. duebenum and N. granulosis. Embryos were collected from the brood pouches of 27 mothers of known infection status and the timing of division observed under a dissecting microscope at 15 8C. 2.3. Statistical methods Initial analyses indicated a difference in parasite burden for the different cell types for both parasites. However, the embryonic cells differ in volume, therefore, we examined parasite distribution correcting for relative cell volume. To estimate relative cell volumes, we dissected 10 embryos and squashed each individual cell beneath a coverslip. The relative cell volume is then proportional to r2 where r is the radius of the squashed cell. Parasite count data at the four- and eight-cell stage were square root transformed and analysed using ANOVA unless otherwise specified. Post-hoc tests (Tukey tests and model simplification) were used to look for homogenous subsets of cells. Data for the probability of infection of each cell type were not corrected for cell volume and were analysed as binary response variables (Crawley, 1993).

R.T. Weedall et al. / International Journal for Parasitology 36 (2006) 749–756

2.4. Theoretical modelling Although the fate of the individual cells at the two-cell stage has been described (Wolff and Scholtz, 2002; Gerberding et al., 2002a,b), it was not possible to distinguish the cell types in the current study. Therefore, parasite distribution at the two-cell stage was considered separately, according to a binomial model, initially similar to that used by Dunn et al. (1995). An individual parasite in the zygote is considered to have a probability P of moving into the ‘correct’ cell and a probability 1Kp of ending up in the ‘wrong’ lineage. Thus, PZ0.5 represents an unbiased distribution and PO0.5 represents the parasite targeting the correct lineage. Assuming that there is no parasite replication at this stage and that the observed numbers of parasites in the ‘right’ and ‘wrong’ lineages at the two-cell stage are nR and nW, respectively, we can say that for an individual embryo, the probability of observing precisely this distribution is given by
  nKnR n pðx Z nR Þ Z ; pnR 1Kp nR where x is the theoretical number of parasites in the right lineage and nZnRCnW is the total number of parasites. To test our hypothesis, however, we consider the probability that x lies further from the expected value pn. In other words, the probability of obtaining the observed distribution according to this hypothesis is given by ! nR X n i nKi pðx% nR Þ Z if nR ! pn p 1Kp i iZ0 ! n X n i nKi if nR O pn: pðxR nR Þ Z p 1Kp i iZnR The special case of nRZpn is considered below. However, a complication to this model arises, since at the two-cell stage, it is not possible to determine which cell is which. If we consider the observed data for the two-cell embryos, and let A and B represent the numbers of parasites in the two-cells, such that ARB this may represent either nR Z A;

nW Z B

or nW Z A;

nR Z B:

We must therefore, consider the two possibilities: that the right lineage has either the maximum number of parasites A or the minimum number BZnKA. This means that when AOpn, we include both tails of the binomial distribution, calculating the probability that this distribution could arise according to our hypothesis as

In the rare case of AZpn, we simply observe that the probability of getting a parasite count at the expected result, or further from it, is unity. Thus, the probability p(H) of obtaining the observed result according to this hypothesis is given by ! 8 A X n i nKi > > > p 1Kp > > > i > iZ0 < pðHÞ Z 1 > ! ! > nKA n > X > n i n i nKi X nKi > > p 1Kp p 1Kp C > : i i iZ0 iZA

pðx% A OR x% nKAÞ Z pðx% AÞ:

A! pn A Z pn AO pn:

2.4.1. Normal approximation to the binomial distribution In the case of N. granulosis, the total parasite numbers at the two-cell stage are always above 900 and we therefore, use the standard Normal approximation to estimate the binomial probabilities, using a mean of pn and a variance of p(1Kp)n. 2.4.2. Varying p We first considered the case of unbiased segregation into daughter cells, i.e. PZ0.5. Second, we considered that the probability was determined according to the cell volume with the larger cell being the target lineage, i.e. PZ0.517. Finally, we looked at a range of probabilities from PZ0.5 upwards, to determine the targeting probability that would best fit the data. 3. Results 3.1. The effect of parasitism on embryonic division Parasitism had no effect on embryonic division (Table 1). The mean time taken for the G. duebeni embryos to develop from two to four-cells was 170.1 min (SE 4.24) and there was no significant difference in the development time of uninfected, D. duebenum-infected or N. granulosis-infected embryos (F2,17Z0.609, PO0.05). Similarly, parasitism had no effect on the time taken to progress from four to eight-cells (mean timeZ167.0 min, SE 2.82, F2,26Z1.02, PO0.05) or on the time taken to develop from eight to 16 cells (mean timeZ 155.9 min, SE 3.33, F2,21Z0.01, PO0.05. 3.2. Parasite burden Total parasite burden was much higher for N. granulosis than for D. duebenum (F1,225Z726, P!0.01) and the parasites

Table 1 The mean times in min (SE) for uninfected, Nosema granulosis-infected and Dictyocoela duebenum-infected embryos to develop from 2 to 4, 4 to 8 and 8 to 16 cells

pðxR A OR x% nKAÞ Z pðxR AÞ C ðx% nKAÞ: However, if A!pn, then necessarily B!pn also, and so the cases where x%B are a subset of x%A and the probability that this could arise if our hypothesis is true is given by

751

2–4 cells 4–8 cells 8–16 cells

Uninfected

Nosema granulosis infected

Dictyocoela duebenum infected

178.1 (15.4) 171.6 (4.9) 155.9 (6.19)

157.5 (0.0) 169.3 (6.3) 155.1 (4.25)

166.1 (4.75) 162.5 (3.9) 156.5 (6.6)

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R.T. Weedall et al. / International Journal for Parasitology 36 (2006) 749–756 Table 2 Number of embryos at which the binomial distribution hypothesis is rejected or accepted for D. duebenum distribution in the two-cell embryo

Parasite Number

160 140 120

Number of embryos

100

60 40 20 0 Zygote

2 Cells

4 Cells

8 Cells

Host embryonic stage 3000

Parasite Number

Hypothesis rejected

Hypothesis accepted

At 0.1% level

At 5% level

80

2500 2000 1500 1000 500 0 Zygote

2 Cells

4 Cells

8 Cells

Host embryonic stage Fig. 1. (a) Mean parasite burdenGSE of Dictyocoela duebenum during early host embryogenesis. (b) Mean parasite burdenGSE of Nosema granulosis during early host embryogenesis.

also differed in their pattern of replication during early host embryogenesis (Fig. 1). Dictyocoela duebenum burden increased significantly during host development (F3,144Z 4.05, P!0.01). Inspection of Fig. 1(a) indicates that parasite burden increased during the first two embryonic cell divisions but did not change during the four- to eight-cell stage and model simplification supports this conclusion (simplification of the model to combine the four- and eight-cell stages lead to an insignificant increase in deviance, F1,141Z0.001, PO0.05). In contrast, there was no significant increase in N. granulosis burden from host zygote to the eight-cell stage during host development (F3,80Z2.4, PO0.05, Fig. 1(b)). 3.3. Parasite distribution during early host embryogenesis 3.3.1. Dictyocoela duebenum Parasite distribution for D. duebenum was uneven in the two-cell embryo. At the two-cell stage, choosing a targeting probability of PZ0.5 (unbiased segregation) or PZ0.517 (probability based on cell volume) yielded the same results, in terms of the number of embryos for which the binomial hypothesis could be accepted or rejected. Out of the 42 embryos, which had non-zero parasite loads, 25 showed that the hypothesis could be rejected at the 5% level and of these 21 could be rejected at the 0.1% level (Table 2). The targeting probability that gave the fewest number of embryos where we could reject the hypothesis was PZ0.75

PZ0.5 and PZ0.517 Total parasite load!9 Total parasite loadO9 All embryos PZ0.75 Total parasite load!9 Total parasite loadO9 All embryos

At 5% level

Total

0

1

11

12

21

24

6

30

21

25

17

42

0

0

12

12

17

19

11

30

17

19

23

42

(noticeably higher than for N. granulosis), but even in this case the hypothesis could be rejected at the 5% level in 19 out of 42 cases, with 17 of these rejected at the 0.1% level. Closer examination of the results reveals a noticeable difference between low and high parasite burdens; where the parasite burden is low, the binomial hypothesis is far more likely to be accepted than the cases where the parasite burden is high. This is not surprising, given the nature of the binomial distribution; indeed for nZ8 and PZ0.5 for example, only a distribution where no parasites ended up in one of the cells would produce a result where we would reject the hypothesis. If we exclude the 12 embryos where the total parasite burden was less than nine, therefore, we see our binomial hypothesis accepted in far fewer cases. With PZ0.5 or PZ0.517, the hypothesis is rejected at the 5% level in 24 out of 30 cases, with 21 of these rejected at the 0.1% level. Even for the optimum targeting probability of PZ0.75, the hypothesis is rejected in 19 cases at 5%, with 17 of these rejected at 0.1%. Table 3 shows the value of p(H) for each individual embryo. In the four-cell embryo, parasite burden differed significantly between the cell types (F3,139Z20.73, P!0.01) with cell type accounting for 44% of the total deviance in parasite number. Parasite burden also differed between families (F9,139Z6.29, P!0.01), however, the family ! cell type interaction was not significant (F27,139Z0.65, PO0.05), indicating that patterns of between-cell distribution did not differ between families. Parasite burden was lowest in blastomere A (Fig. 2(a), Tukey test, P!0.05). This pattern also occurred when we compared the presence/absence of parasites in each cell type. The probability of infection differed significantly between cell type (X32 Z 33:4, P!0.01) and this could be attributed to a lower probability of infection in blastomere A compared with blastomeres B, C and D (combining the factor cell type to 2 levels; ABC; and D caused no significant increase in the deviance of the data from the model, X22 Z 4:5, PO0.05). In most embryos, blastomeres B, C and D were infected (probability of infectionZ0.98G

R.T. Weedall et al. / International Journal for Parasitology 36 (2006) 749–756

Min

Max

0 0 0 0 1 1 0 0 0 0 0 0 9 7 11 1 10 1 27 4 9 44 34 24 3 28 43 41 56 31 19 82 53 92 38 88 98 16 38 100 8 176

1 1 1 1 1 1 2 2 2 2 4 8 17 28 26 40 39 49 33 63 74 46 76 94 119 98 97 103 93 127 148 96 137 101 168 125 124 243 236 174 311 331

Total embryo parasite load 1 1 1 1 2 2 2 2 2 2 4 8 26 35 37 41 49 50 60 67 83 90 110 118 122 126 140 144 149 158 167 178 190 193 206 213 222 259 274 274 319 507

p(H) (3 d.p.) PZ0.5

PZ0.517

PZ0.5216

1.000 1.000 1.000 1.000 1.000 1.000 0.500 0.500 0.500 0.500 0.125 0.008 0.169 0.001 0.020 0.000 0.000 0.000 0.519 0.000 0.000 0.916 0.000 0.000 0.000 0.000 0.000 0.000 0.003 0.000 0.000 0.330 0.000 0.565 0.000 0.013 0.093 0.000 0.000 0.000 0.000 0.000

1.000 1.000 1.000 1.000 0.733 0.733 0.501 0.501 0.501 0.501 0.126 0.008 0.175 0.001 0.023 0.000 0.000 0.000 0.533 0.000 0.000 0.496 0.000 0.000 0.000 0.000 0.000 0.000 0.006 0.000 0.000 0.379 0.000 0.607 0.000 0.026 0.135 0.000 0.000 0.000 0.000 0.000

1.000 1.000 1.000 1.000 0.438 0.438 0.625 0.625 0.625 0.625 0.320 0.100 0.180 0.322 0.309 0.000 0.288 0.000 0.001 0.000 0.001 0.000 0.095 0.143 0.000 0.272 0.074 0.192 0.000 0.068 0.000 0.000 0.200 0.000 0.016 0.000 0.000 0.000 0.000 0.000 0.000 0.000

Bold italics indicate the cases where the hypothesis is not rejected at the 5% level.

0.01) where as the probability of infection of blastomere A was only 0.66G0.09. The data for parasite burden in the eight-cell embryos were overdispersed with the micromeres being uninfected in many embryos, whereas the macromeres contained parasites in the majority of embryos (Fig. 2(b)). Transformation of the data for parasite burden in eight-cell embryos did not normalise these data and so we analysed the data specifying a binomial error structure. Parasite burden differed significantly between the cell types (X72 Z 424, P!0.01). Parasite burden also differed between families (X72 Z 96, P!0.01) and there was a 2 Z 76, P!0.01). significant family ! cell type interaction (X49

Z

Parasite Number

Observed parasite numbers in the two-cells

(a) 35 30

Z

25

Z

20 15 10

Y

5 0 A

B

C

D

Host Cell (b) 40 35

Parasite Number

Table 3 Probability p(H) that the observed distribution of D. duebenum in the two-cell embryos is consistent with the binomial distribution hypothesis

753

X

X

30 25

X

20 15 10

Y

5

Z

Z

Z

a

b

c

Y

0 A

B

C

D

d

Host Cell Fig. 2. (a) Dictyocoela duebenum distribution in the four-cell host embryo. A total of 38 embryos were screened. MeanGSE parasite number in each cell type is plotted. To take into account differences in the volume of the blastomeres, parasite burden has been corrected for host cell volume and expressed as (parasite number/microlitre). Post-hoc (Tukey tests) showed that parasite burden was lowest in cell A (homogenous subsets labelled Y and Z). (b) Dictyocoela duebenum distribution in the eight-cell host embryo. A total of 39 embryos were screened. To take into account differences in the volume of the host cells, parasite burden is corrected for host cell volume and expressed as (parasite number/microlitre). MeanGSE parasite number in each cell type is plotted. Cells in homogenous subsets (revealed in post-hoc tests) share the same letter label.

Simplification of the model indicated that there were four homogenous groups; macromeres BCD (highest burden); macromere A and micromere d; and micromeres abc (lowest burden); model simplification caused no significant increase in the deviance of the data from the model, X52 Z 7:48, PO0.05. A further analysis was carried out to compare the proportion of each cell type infected. These results were broadly in accord with the non-parametric tests on parasite burden. The probability of a cell being infected was affected by family (X72 Z 19:6, P!0.01) and by cell type (X72 Z 108:4, P!0.01) with cell type explaining 29% of the deviance. There was no significant family ! cell interaction. Simplification of the model indicated that there were four homogenous groups: macromere A, probability of infectionZ0.74G0.07; macromeres B, C and D, probability of infectionZ0.97G0.02; micromere a, probability of infectionZ0.23G0.05; micromeres b, c and d probability of infectionZ0.61G0.05; model simplification caused no significant increase in the deviance of the data from the model, X42 Z 0:4, PO0.05). Macromeres were more likely to be infected than micromeres and this can be attributed to differences in cell volume (presence/absence data were not corrected for cell volume). The lower frequency of

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R.T. Weedall et al. / International Journal for Parasitology 36 (2006) 749–756

Table 4 Number of embryos at which the binomial distribution hypothesis is rejected or accepted for N. granulosis distribution in the two-cell embryo Number of embryos

PZ0.5 PZ0.517 PZ0.522

Hypothesis rejected

Hypothesis accepted

At 0.1% level

At 5% level

At 5% level

14 12 11

19 14 12

2 7 9

Total

21 21 21

infection in macromere A and micromere a follows from the distribution observed at the four-cell stage. 3.3.2. Nosema granulosis For N. granulosis, parasite distribution was also biased in the two-cell embryo. At the two-cell stage, assuming that the targeting probability PZ0.5 (i.e. unbiased segregation), we would clearly expect an even distribution of parasites between the cells. In fact, the distribution is highly uneven and the hypothesis is rejected at the 5% level in 19 out of the 21 embryos considered. It is a measure of how uneven the distribution is that in 14 of these cases, we can reject the hypothesis even at the 0.1% level (Table 4). We then considered distribution taking account of differences in cell volume. Assuming that the targeting probability is proportional to the cell volumes, i.e. that PZ0.517, increases the number of embryos where our hypothesis could be accepted but it is still rejected at the 5% level in 14 out of 21 embryos, with 12 of these rejected at the 0.1% level. The targeting probability which yields the best result is close to that suggested by the cell volumes, at PZ0.522 (noticeably lower than for D. duebenum) but even in this case the hypothesis is rejected at 5% confidence levels in 12 out of 21 embryos. All but one of these can also be rejected at the 1% level. Table 5 shows the value of p(H) for each individual embryo. At the four-cell stage, all cells were infected and there was no significant difference in parasite burden between cell types (Fig. 3(a), F3,70Z2.48, PZ0.07). Parasite burden also differed between families (F9,79Z10.32, P!0.05). All cells were infected in the eight-cell stage and parasite burden was significantly affected by cell type (F7,104Z6.19, P!0.01) and post-hoc (Tukey) tests showed that the cells could be grouped into three homogeneous subsets (Fig. 3(b)). Parasite burden differed between families (F6,104Z13.42, P!0.01), however, the family x cell type interaction was not significant (F42,104Z1.00, PO0.05), indicating that patterns of betweencell distribution did not differ between families. 4. Discussion Our results show different patterns of replication and distribution in the two vertically transmitted microsporidia. Parasite numbers are higher in N. granulosis by a factor of 10 and, whilst D. duebenum does replicate during early host embryogenesis, there is no increase in N. granulosis burden. In Section 1, we proposed two alternative strategies for a

Table 5 Probability p(H) that the observed distribution of N. granulosis in the two-cell embryos is consistent with the binomial distribution hypothesis Observed parasite numbers in the two-cells Min

Max

155 376 583 476 645 743 904 924 819 1301 1324 1415 1108 1302 1231 1031 1644 1588 1697 1523 1440

754 787 655 785 958 863 980 1039 1312 1504 1517 1459 1817 1712 2088 2331 1765 1913 1884 2072 2493

Total embryo parasite load 909 1163 1238 1261 1603 1606 1884 1963 2131 2805 2841 2874 2925 3014 3319 3362 3409 3501 3581 3595 3933

p(H) (3 d.p.) PZ0.5

PZ0.517

PZ0.5216

0.000 0.000 0.044 0.000 0.000 0.003 0.084 0.010 0.000 0.000 0.000 0.422 0.000 0.000 0.000 0.000 0.040 0.000 0.002 0.000 0.000

0.000 0.000 0.208 0.000 0.000 0.055 0.405 0.145 0.000 0.023 0.038 0.160 0.000 0.000 0.000 0.000 0.477 0.000 0.144 0.000 0.000

0.000 0.000 0.309 0.000 0.000 0.108 0.460 0.255 0.000 0.063 0.097 0.070 0.000 0.000 0.000 0.000 0.332 0.002 0.300 0.000 0.000

Bold italics indicate the cases where the hypothesis is not rejected at the 5% level.

vertically transmitted parasite to target the gonad; high replication and burden in all lineages versus targeting of particular lineages. Our data indicate that, for both parasites, targeting of host cell lineages begins at the earliest stages of embryogenesis. From the two-cell stage, our data indicate biased parasite segregation in both D. duebenum and N. granulosis even when we control for differences in cell volume. Therefore, we conclude that biased segregation and targeting of host lineages have evolved in diverse branches of the microsporidia. Dictyocoela duebenum distribution is non-random at the four- and eight-cell stages. At the four-cell stage, whilst, in the majority of embryos, blastomeres B, C and D contain parasites, blastomere A is infected in only 66% of embryos and parasite numbers are significantly lower in this cell. The observed pattern at four-cells leads to a biased distribution in the eightcell embryo with most macromeres infected. The micromeres, in particular micromere a, were less likely to be infected and, even when correcting for cell volume, parasite number was lower in these cells than in the macromeres. The blastomere A of the four-cell stage is believed to be the progenitor of the primordial germ cells in the amphipods O. cavimana (Wolff and Scholtz, 2002) and P. hawaiensis (Gerberding et al., 2002a,b; Extavour, 2005). Blastomere A is also reported to proliferate the naupliar mesoderm and parts of the endoderm in O. cavimana (Wolff and Scholtz, 2002) and the entire visceral mesoderm in P. hawaiensis (Gerberding et al., 2002a,b). Thus, at the four-cell stage, the parasite was in the germline precursor cells in only 66% of embryos, at the eight-cell stage the parasite

R.T. Weedall et al. / International Journal for Parasitology 36 (2006) 749–756

Parasite Number

450 400 350 300 250 200 150 100 50 0 A

B

C

D

Host Cell 450

Z

Parasite Number

400 350 300

Y X

250

Z X

Z X

Y X

Y X

Y

Y

b

c

200 150 100 50 0 A

B

C

D

a

d

Host Cell Fig. 3. (a) Nosema granulosis distribution in the four-cell host embryo. A total of 20 embryos were screened. MeanGSE parasite number in each cell type is plotted. To take into account differences in the volume of the blastomeres, parasite burden has been corrected for host cell volume and expressed as (parasite number/microlitre). (b) Nosema granulosis distribution in the eightcell host embryo. A total of 40 embryos were screened. MeanGSE parasite number in each cell type is plotted. To take into account differences in the volume of the host cells, parasite burden has been corrected for host cell volume and expressed as (parasite number/microlitre). Post-hoc (Tukey) tests showed that the cells could be grouped into three homogeneous subsets, these are labelled X Y and Z.

was in the germline precursor cells in only 23% of embryos. Theoretical models predict that a feminising parasite cannot be sustained in the population when transmission to future generations falls below 50% (Hatcher and Dunn, 1995). The observed distribution therefore, leads us to conclude that D. duebenum cannot achieve successful vertical transmission solely through its presence in the germline precursor cells. In addition, the parasite does not appear to target the somatic gonad as we find low parasite burden in the progenitor cells. The highest rate of infection and highest parasite burden was found in macromeres B, C and D, which are precursors of the ectoderm leading us to propose that the parasite targets these cells and then secondarily infects the gonad later in host development. In N. granulosis, the targeting of cell lineages is less specific. This may reflect the higher overall burden and hence less strong selection for targeting. Nonetheless, there is evidence of persistent bias in the distribution of parasites throughout host cell divisions. By the eight-cell stage, parasite burden was highest in cells, which are progenitors of the ectoderm (macromeres C and D) and the germline (micromere a). These data lead us to suggest that in addition to using the

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ectodermal route, which might lead to infection of the somatic gonad, N. granulosis may also target germline directly. Over representation in the ectoderm is in accord with previous studies that report N. granulosis to be localised in the subcuticular cells of juvenile G. duebeni but absent from the muscle, nerve and gut tissue (Terry et al., 1997). However, presence in germline precursors is surprising given that in the adult the parasite is located in the somatic gonad (follicle cells) and is only transmitted to oocytes at vitellogenesis (Terry et al., 1997). Only merogonic parasite stages were observed and these have no mechanism for between-cell transmission. The observed distributions appear to reflect biased segregation of the parasites at host cell division and raise the question of the mechanism by which these parasites target cells at host mitosis. Previous work has shown close association of N. granulosis with host astral microtubules (Terry et al., 1997) and this may provide a mechanism for targeted parasite segregation. We saw no evidence of parasite mortality during early embryogenesis. However, we cannot exclude the possibility that selective replication or mortality of parasites may occur later in development as tissues differentiate. The distribution of parasites observed in the current study differed from that observed for vertically transmitted Nosema locustae (Raina et al., 1995). Vertical infection was lethal in 70–90% of locust hosts (Raina et al., 1995), whereas N. granulosis and D. duebenum cause little pathogenicity with the majority of hosts going on to transmit the parasite vertically to future generations (Terry et al., 1998; Ironside et al., 2003). Nosema locustae was found to undergo sporulation in the yolk of the host embryo with spore germination leading to infection of the midgut epithelium. In contrast only merogonic stages of the D. duebenum and N. granulosis parasite are present during host development and this may reflect strong selection for reduced pathogenicity. Infection of the germline by N. granulosis may ensure infection of target tissues early in development. In contrast, secondary infection of the gonad requires that both N. granulosis and D. duebenum undergo spore development in the ectoderm and subsequent infection of the gonad. Parasite distribution and burden are also likely to be important for feminisation of the host. Studies of the reproductive parasite Wolbachia have found that expression of the parasite may be density dependent. Wolbachia dosage is positively correlated with parthenogenesis induction in Muscidifurax uniraptor (Zchori-Fein et al., 2000). Reduced Wolbachia density under elevated temperatures has been shown to reduce male killing in Drosophila (Hurst et al., 2000) as well as the feminisation efficiency of Wolbachia that infect woodlice (Porcellionides pruinosus, Rigaud et al., 1997). Similarly, low temperatures are associated with a reduced parasite burden for both N. granulosis and D. duebenum (Dunn et al., in press) and reduced feminisation by N. granulosis (Kelly et al., 2002). Nosema granulosis has been shown to induce feminisation in G. duebeni by preventing differentiation of the androgenic gland and the production of androgenic gland hormone which controls male sexual differentiation (Rodgers-Gray et al., 2004). Our data do not

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support parasite targeting of androgenic gland precursor cells. The gland probably develops from the mesodermal cell lineages, which proliferate from micromeres b and d (Wolff and Scholtz, 2002; Gerberding et al., 2002a,b) and parasite numbers are low in these cells for both species of feminiser. Sexual differentiation in Crustacea occurs relatively late in development. For G. duebeni, it takes place from moults two to four of the juvenile (Charniaux-Cotton and Payen, 1985) and future work should track parasite distribution during this period of androgenic gland differentiation. Acknowledgements RTW was supported by a White Rose studentship. We thank Angela Douglas, Ian Hope, Mel Hatcher and Beth McClymont for helpful discussions and Rupert Quinnell and Bill Kunin for statistical advice. References Agnew, P., Koella, J.C., 1999. Life history interactions with environmental conditions in a host–parasite relationship and the parasite’s mode of transmission. Evol. Ecol. 13, 67–89. Baldauf, S.L., 2003. The deep roots of Eukaryotes. Science 300, 1703–1706. Charniaux-Cotton, H., Payen, G., 1985. Sexual differentiation. In: Bliss, D.E., Mantel, L.H. (Eds.), Integument, Pigments and Hormonal Processes. Academic Press, London, pp. 217–299. Crawley, M.J., 1993. GLIM for Ecologists. Blackwell Science, Oxford. Dunn, A.M., Smith, J.E., 2001. Microsporidian life cycles and diversity: the relationship between virulence and transmission. Microb. Infect. 3, 381–388. Dunn, A.M., Hatcher, M.J., Terry, R.S., Tofts, C., 1995. Evolutionary ecology of vertically transmitted parasites: transovarial transmission of a microsporidian sex ratio distorter in Gammarus duebeni. Parasitology 111, S91–S109. Dunn, A.M., Terry, R.S., Taneyhill, D.E., 1998. Within-host transmission strategies of transovarial, feminizing parasites of Gammarus duebeni. Parasitology 117, 21–30. Dunn, A.M., Terry, R.S., Smith, J.E., 2001. Transovarial transmission in the microsporidia. Adv. Parasitol. 48, 57–100. Dunn, A.M., Hogg, J., Hatcher, M.J., in press. Transmission and burden and the impact of temperature on two species of vertically transmitted microsporidia. Int. J. Parasitol. Extavour, C.G., 2005. The fate of isolated blastomeres with respect to germ cell formation in the amphipod crustacean Parhyale hawaiensis. Dev. Biol. 277, 387–402. Gerberding, M., Browne, W., Lall, S., Patel, N., 2002a. Separation of the germ line at the eight-cell stage—The invariant cell lineage of the amphipod Parhyale hawaiensis. Dev. Biol. 247, 522–523. Gerberding, M., Browne, W.E., Patel, N.H., 2002b. Cell lineage analysis of the amphipod crustacean Parhyale hawaiensis reveals an early restriction of cell fates. Development 129, 5789–5801.

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