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Impact of a selfish B chromosome on chromatindynamics and nuclear organization in Nasonia

Megan M. Swim, Kelsey E. Kaeding and Patrick M. Ferree*W. M. Keck Science Department of Claremont McKenna, Pitzer and Scripps Colleges, 925 N. Mills Avenue, Claremont, CA 91711, USA

*Author for correspondence (pferree@kecksci.claremont.edu)

Accepted 10 July 2012Journal of Cell Science 125, 5241–5249� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.113423

SummaryB chromosomes are centric chromosomal fragments present in thousands of eukaryotic genomes. Because most B chromosomes arenon-essential, they can be lost without consequence. In order to persist, however, some B chromosomes can impose strong forms of

intra-genomic conflict. An extreme case is the paternal sex ratio (PSR) B chromosome in the jewel wasp Nasonia vitripennis.Transmitted solely via the sperm, PSR ‘imprints’ the paternal chromatin so that it is destroyed during the first mitosis of the embryo.Owing to the haplo-diploid reproduction of N. vitripennis, PSR-induced loss of the paternal chromatin converts embryos that should

become females into PSR-transmitting males. This conversion is key to the persistence of PSR, although the underlying mechanisms arelargely unexplored. We assessed how PSR affects the paternal chromatin and then investigated how PSR is transmitted efficiently at thecellular level. We found that PSR does not affect progression of the paternal chromatin through the cell cycle but, instead, alters its

normal Histone H3 phosphorylation and loading of the Condensin complex. PSR localizes to the outer periphery of the paternal nucleus,a position that we propose is crucial for it to escape from the defective paternal set. In sperm, PSR consistently localizes to the extremeanterior tip of the elongated nucleus, while the normal wasp chromosomes localize broadly across the nucleus. Thus, PSR may alter or

bypass normal nuclear organizational processes to achieve its position. These findings provide new insights into how selfish geneticelements can impact chromatin-based processes for their survival.

Key words: Nasonia vitripennis, Chromosome segregation, Early insect development, Genome conflict, Selfish DNA

IntroductionSince their discovery, B chromosomes have persisted as

intriguing components of many eukaryotic genomes. Found inover two thousand plant and animal species so far (Palestis et al.,2004), B chromosomes are diminutive, extra chromosomes thatare distinct from the normal chromosomes. Because they are not

a part of the wild-type genome, B chromosomes are, in mostcases, not essential for the fitness of the organism (Camacho et al.,2000; Jones et al., 2008). Many B chromosomes contain large

amounts of highly repetitive, non-coding DNAs, includingsatellite repeats, transposable elements, and degenerate rDNAgenes (Jones, 1995; Jones et al., 2008). This quality owes to the

likelihood that B chromosomes derive from the centric andpericentric regions of the normal chromosomes, where thesesequences are normally located (Jones, 1995; Perrot-Minnot and

Werren, 2001; Jones et al., 2008). Largely free from functionalconstraints, B chromosomes can change rapidly in their sequencecomposition over short evolutionary periods, and they have beenimplicated in major genome events such as the origin of sex

chromosomes (Gamero et al., 2002). Thus, B chromosomes areregarded as both genome parasites and potentially importantfacilitators of genome evolution.

A fundamental question is how B chromosomes persist despitethe fact that they are largely non-essential. This question isespecially compelling given that many B chromosomes are

known to be unstable during meiosis or mitosis (Jones, 1995).Some B chromosomes counter their instability through meioticand mitotic drive mechanisms (Kayano, 1957; Frost, 1969;

Gonzalez-Sanchez et al., 2003). For example, one B chromosome

in the plant Lilium callosum positions itself on one side of themetaphase plate during female meiosis so that it can

preferentially segregate into the meiotic product that willbecome the gamete (Kayano, 1957; Jones et al., 2008). B

chromosomes in plants also can utilize non-disjunction duringmitosis in order to accumulate in the generative (pollen) products

and not the vegetative ones (Jones et al., 2008). Such drive

mechanisms normally are effective only when aspects of thechromosome segregating system (e.g. the microtubule-based

spindle apparatus) are intrinsically asymmetrical and can betaken advantage of, as is the case in many plants.

An alternative, and more extreme, mode of B chromosomepropagation is exemplified by paternal sex ratio (PSR), a B

chromosome found in the jewel wasp Nasonia vitripennis. PSR istransmitted to new wasp progeny through the sperm (Nur et al.,

1988). In the presence of PSR, the paternal chromatin becomeshyper-condensed and fails to resolve into distinct chromosomes

during the first mitotic division of the embryo (Werren et al.,

1987; Reed and Werren, 1995). In insects, the paternal andmaternal chromosome sets remain physically separated during

the first mitosis by an incompletely broken down layer of nuclearenvelope surrounding each set (Callaini and Riparbelli, 1996;

Kawamura, 2001). This unique property gives rise to a bipartitespindle apparatus known as the ‘gonomeric’ spindle (Kawamura,

2001). Because the two chromosome sets do not mix during this

first mitotic division, the maternally derived chromosomes areunaffected by the PSR-altered paternal chromatin and segregate

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normally to form two haploid nuclei (Reed and Werren, 1995).

The paternal chromatin never resolves into distinct chromosomes

or segregates, and is eventually lost. However, PSR is somehow

not affected and escapes this destruction (Reed and Werren,

1995). N. vitripennis and all other insects belonging to the insect

order Hymenoptera (i.e. ants, bees and wasps) exhibit a

specialized mode of reproduction known as haplo-diploidy, in

which females develop as diploids from fertilized eggs while

males arise as haploids from unfertilized eggs. By completely

destroying the paternal chromatin, PSR effectively converts

fertilized N. vitripennis eggs that should become females into

PSR-transmitting males (Werren et al., 1987). Owing to its

extreme effect on the paternal half of the wasp genome, PSR is

considered to be the most extreme selfish genetic element known

in nature (Werren and Stouthamer, 2003). PSR was discovered in

N. vitripennis, but subsequently, similar B chromosomes have

been found in distantly related wasps belonging to the genus

Trichogramma (Stouthamer et al., 2001).

A critical aspect of PSR propagation is the ability of PSR to alter

the paternal chromatin so that it fails to resolve into distinct

chromosomes and segregate. However, little is known about the

underlying mechanism. One possibility is that PSR specifically

disrupts some aspect of chromosome condensation. Alternatively,

PSR may block paternal chromosome condensation secondarily by

affecting one of several developmental steps preceding the first

embryonic mitosis. During spermiogenesis, the spherical nucleus of

the post-meiotic spermatid becomes highly elongated into a

‘needle’ shape (Tokuyasu, 1974). Subsequently, the sperm

chromatin is packaged into an extraordinarily condensed state

with paternal histone-like proteins known as protamines (Balhorn,

2007; Rathke et al., 2007). Once the sperm nucleus enters the egg

cytoplasm, its nuclear envelope dissolves, the protamines are

removed, and the paternal DNA is repackaged with conventional

histones (Poccia and Collas, 1996). These events are believed to be

essential for the paternal chromatin to undergo replication

synchronously with the maternal chromatin so that the two nuclei

can enter together into the first mitosis (Poccia and Collas, 1996).

PSR could inhibit one or more of these important processes, thus

altering normal cell cycle progression and, ultimately, blocking

paternal chromosome resolution. Interestingly, PSR somehow

evades the same fate that it imposes on the paternal set in order

to be transmitted, despite the fact that both entities consist of

chromatin and reside closely together in the sperm nucleus.

Another important aspect of PSR propagation is the extremely

high transmission rate of this chromosome from father to

offspring. Previous studies showed that PSR is transmitted to

progeny at frequencies ranging from 94% to 100% (Beukeboom

and Werren, 1993b). These numbers suggest either that PSR

segregates stably during cell division in the male germ line or,

alternatively, that PSR is propagated through previously

undetected drive mechanisms. Perhaps the most critical period

for PSR transmission is during the first mitotic division of the

embryo. In order to be transmitted, PSR must first dissociate

from the paternal chromatin. This goal is particularly noteworthy

because the paternal set remains as a three-dimensional mass of

chromatin (Werren et al., 1987; Reed and Werren, 1995). How

PSR is capable of avoiding entanglement with the paternal

chromatin mass (PCM) is currently unknown. Once free, PSR

must then associate with the maternal chromosomes. A possible

scenario is that PSR, unlike the paternal chromatin, resolves into

distinct sister chromatids that segregate with the maternal

chromatids during the first mitotic division.

In this study we utilized cytological methods to address if PSR

affects the ability of the paternal chromatin to progress through

several key developmental stages leading up to the first mitotic

division. Additionally, we performed the first-ever visual analysis

of PSR during early development to address how it becomes

transmitted at the cellular level. Our experiments suggest that

PSR does not prevent the paternal chromatin from undergoing

replication during S phase or from entering into mitosis, but,

instead, alters phosphorylation of Histone H3 and proper loading

of the chromosome condensation machinery. Additionally, we

found that PSR maintains a position at the periphery of the

paternal nucleus, which we propose is important for its escape

from the paternal chromatin. PSR also localizes at the anterior-

most region of the elongated nucleus of all sperm, in striking

contrast to the normal chromosomes, which are positioned

broadly across the sperm nucleus. Thus, PSR may manipulate or

bypass normal nuclear organization in the wasp sperm, thus

adding a new dimension to its selfish nature.

ResultsPSR disrupts paternal Histone H3 phosphorylation andCondensin loading but not cell cycle progression

We first analyzed young N. vitripennis embryos produced from

crosses between wild-type females and males carrying PSR. As

expected, these crosses yielded broods of all male progeny that

were nearly identical in size to broods from control crosses that

produced both sexes (t50.08, P50.94, d.f.521; Table 1).

Examination of 0–1.5-hour embryos revealed previously

described nuclear defects that occurred during the first mitotic

division of the embryo (Werren et al., 1987; Reed and Werren,

1995). In PSR-positive embryos, the maternal and paternal nuclei

became juxtaposed next to one another in a manner that was

indistinguishable from control (wild-type) embryos (Fig. 1A).

Additionally, both nuclei exhibited similar chromatin densities at

this stage (Fig. 1A). During prometaphase, both chromatin sets

underwent condensation but the paternal chromatin became

abnormally denser than the maternal chromatin (Fig. 1A).

Moreover, in contrast to the maternal chromatin, which

resolved into individual chromosomes at metaphase, the

paternal chromatin remained unresolved throughout the first

mitotic division (Fig. 1A). During anaphase and telophase the

maternal chromatids segregated normally into daughter nuclei,

Table 1. Brood size and sex of progeny produced from N. vitripennis crosses

CrossNumber of male progeny

(mean 6 s.e.m.)Number of female progeny

(mean 6 s.e.m.)Brood size

(mean 6 s.e.m.)

AsymC6AsymC(n512)

10.662.4 41.963.2 52.564.2

AsymC6AsymC-PSR(n511)

53.065.2 0.260.1 53.265.1

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while the paternal chromatin, now considered the PCM, remained

at the metaphase plate (Fig. 1A). The PCM persisted near the

plasma membrane in older embryos that had undergone several

rounds of mitosis (Fig. 1B), but eventually disappeared in later

stages (not shown).

We addressed the possibility that PSR might induce formation

of the PCM secondarily by preventing the paternal chromatin

from progressing normally through the first cell cycle. In order to

investigate this possibility, we examined the localization of

proliferating cell nuclear antigen (PCNA) in young PSR-positive

embryos. PCNA is a highly conserved component of the

replication complex and has served as a marker for

incompletely replicated DNA (Yamaguchi et al., 1991;

Easwaran et al., 2007; Landmann et al., 2009). We observed an

equal intensity of PCNA on both the maternal and paternal

chromatin in both wild-type embryos (not shown) and PSR-

positive embryos undergoing their first round of replication

(Fig. 2A). Additionally, PCNA disappeared synchronously from

both sets at the onset of mitosis (Fig. 2A). These observations

suggest that the PSR-altered paternal chromatin undergoes DNA

replication properly during the first mitotic division.

Although we detected no defects in replication of the paternal

set preceding the first mitotic division, we did find that the PCM

fails to become PCNA-positive during entry into the second

round of DNA synthesis (Fig. 2A). Additionally, we detected

high levels of broken DNA within the PCM during this stage that

resemble levels present in yolk nuclei that are undergoing

apoptosis (Fig. 2B). These findings suggest that the PCM may

accrue chromatin defects during the first division. Nevertheless,

proper replication of the paternal set before the first mitosis

implies that the processes leading up to S phase, such as the

removal of protamines from the paternal DNA and its

repackaging with maternal histones, also occur normally.

We next addressed if the paternal chromatin is capable ofentering properly into mitosis. To do this, we stained embryoswith antibodies that recognize phosphorylated serine 10 of

Histone H3 (PH3S10), a well-established chromatin marker ofmitosis (Wei et al., 1999; Hsu et al., 2000; Tram and Sullivan,2002). In these embryos, both the paternal and maternalchromatin became PH3S10 positive in a synchronous manner

(Fig. 3A), demonstrating that the paternal nucleus is capable ofentering normally into mitosis. However, the paternal chromatinbecame more enriched for PH3S10 relative to the maternal

chromatin during metaphase (Fig. 3A). Additionally, the PCMabnormally retained this strong PH3S10 signal as the daughternuclei derived from the maternal chromatin set appropriately lost

this mark upon exit from mitosis (Fig. 3A). In later stageembryos the PCM retained PH3S10 inappropriately duringinterphase when the other nuclei were devoid of this mark

(Fig. 3B). These observations suggest that the presence of PSRleads to the abnormal retention of PH3S10 on the paternalchromatin and its failure to exit properly from mitosis.

The PH3S10 mark is functionally associated with the propercondensation of chromatin into chromosomes at the onset of mitosis(Koshland and Strunnikov, 1996; Strahl and Allis, 2000).

Fig. 1. PSR induces loss of the paternal genome during the first

embryonic mitosis. (A) In both control (top row) and PSR-positive (bottom

row) embryos, the maternal and paternal nuclei become juxtaposed normally.

However, in PSR-positive embryos, the paternal chromatin (white arrows)

becomes hyper-condensed at prometaphase and never resolves into distinct

chromosomes, as it does in control embryos. (B) Control and PSR-positive

embryos during later cleavage divisions. The paternal chromatin mass (white

arrow) can be seen at this stage but disappears during later stages (not shown).

DNA is blue in all panels. Scale bar: 10 mm (A) and 40 mm (B).Fig. 2. In the presence of PSR, the paternal chromatin undergoes the first but

not subsequent rounds of DNA replication, and incurs DNA damage during

the first mitosis. (A) Both maternal and paternal chromatin become enriched for

PCNA (green) during the first round of DNA replication (top row), and lose this

mark upon entry into the first mitosis (middle row). White arrows indicate the

paternal chromatin mass. Maternally derived nuclei become enriched for PCNA

upon entry into the second round of replication, while the paternal chromatin mass

shows no PCNA localization (bottom row). DNA is blue. Scale bar: 5 mm.

(B) The paternal chromatin mass (white arrow) becomes positive for TUNEL

(red) during the first mitosis (top row). This pattern resembles yolk nuclei (white

arrows in the bottom panel), which are present in the middle of later stage embryo

and become TUNEL positive as they become degraded by apoptosis. DNA is

blue. Scale bar: 8 mm in the top row and 50 mm in the bottom panel.

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Specifically, this mark is though to be necessary for the loading of

Condensins to chromatin (Ono et al., 2003; Hirano, 2005; Cobbe et al.,

2006). At least two forms of the Condensin complex, each defined by

different subsets of associating proteins, appear to play distinct but

interrelated roles in the conversion of chromatin into distinct

chromosomes (Hirota et al., 2004; Cobbe et al., 2006). At the core

of these complexes are two proteins, SMC2 and SMC4 (Hirano,

2005; Cobbe et al., 2006). We reasoned that PSR-induced hyper-

condensation of the paternal chromatin may involve abnormal

Condensin localization as a result of inappropriate PH3S10

persistence. To test this idea, we stained wild-type and PSR-

positive embryos with an antibody raised against Drosophila

melanogaster SMC2 (Cobbe et al., 2006). This antibody strongly

labeled metaphase chromosomes but not interphase chromatin in

older wild-type embryos (Fig. 4), patterns that reflect normal

Condensin localization in D. melanogaster (Cobbe et al., 2006).

However, in PSR-positive embryos, SMC2 localized at a much higher

level to the PCM than to the condensed maternal chromosomes

(Fig. 4). This pattern mirrors the bright staining of DNA and high,

persisting levels of PH3S10 that we observed on the paternal

chromatin. Thus, PCM formation likely involves overloading of

Condensins, in addition to abnormal phosphorylation of Histone H3.

PSR exhibits unique placement in the sperm-derived

nucleus

We sought to determine when and how PSR dissociates from the

PCM to join the maternal chromosomes for transmission. To do

this, we designed fluorescently labeled DNA probes against two

previously identified satellite DNA repeats present on the PSR

chromosome (see Materials and Methods) (Eickbush et al.,1992). When hybridized to meiotic metaphase chromosomes,

these probes highlighted two small blocks of satellite DNA, one

on each end of PSR, thereby delineating its entire chromosomal

length (Fig. 5A). The fact that these two satellite blocks arelocated on the chromosome ends supports the idea that PSR

consists largely of non-coding repetitive sequences because these

sequences are normally located in the pericentric regions and not

within the chromosome arms in many higher eukaryotes.Furthermore, this finding is consistent with the idea that PSR

derives evolutionarily from the pericentric region of a normal

chromosome (Perrot-Minnot and Werren, 2001).

We also generated probes that specifically recognize two

different normal chromosomes. One of these probes is directed

against a satellite DNA repeat located in the rDNA spacer region(Stage and Eickbush, 2010). This probe highlights a single locus

that overlaps with a chromosomal constriction that we interpret to

be the Nuclear Organizer Region (NOR; Fig. 5A). This probe did

not, however, localize to PSR, suggesting that PSR, unlike manyother B chromosomes (Camacho et al., 2000; van Vugt et al.,

2005), does not contain rDNA. To confirm this conclusion, we

performed hybridizations with a combination of three probes to

the highly conserved 18S region of the rDNA locus (seeMaterials and Methods). Like the rDNA spacer probe, these

18S probes localized to a single locus on a N. vitripennis

chromosome but not to PSR (not shown). In addition to these

rDNA probes, we generated a probe that recognizes a singlesatellite block on a normal wasp chromosome that is distinct from

the one containing the NOR (Fig. 5A).

Using the PSR-specific probes, we analyzed PSR dynamicsduring the first embryonic mitosis (Fig. 5B). As expected, we

observed a single PSR-specific signal in only one of the two

juxtaposed nuclei of post-fertilization embryos (Fig. 5B). It is

highly likely that the PSR-positive nucleus is the paternally derivedone because in many embryos this nucleus often appeared

elongated, as does the sperm nucleus just after fertilization. A

single PSR-specific signal was observed within the paternal

chromatin before entry into the first mitosis and during the firstmetaphase (Fig. 5B). However, during anaphase and telophase, a

single PSR-positive signal was associated with each of the two

Fig. 3. PSR causes the paternal chromatin to abnormally retain

phosphorylation of Histone H3 (PH3) at serine residue 10 upon exit from

the first mitosis. (A) Neither set of chromatin exhibits the PH3S10 mark

(green) before entry into the first mitosis (top row). However, both sets are

PH3S10 positive during the first metaphase (middle row). Maternally derived

daughter nuclei lose PH3S10 while the paternal chromatin mass abnormally

retains this mark (bottom row). (B) A later stage cleavage embryo in

interphase; the paternal chromatin mass retains the PH3S10 mark while the

maternally derived nuclei do not. White arrows indicate the paternal

chromatin. DNA is blue. Scale bar: 12 mm.

Fig. 4. The paternal chromatin mass exhibits abnormally high levels of the

core Condensin protein, SMC2. An anti-SMC2 antibody (green) strongly

labels metaphase chromosomes of a wild-type embryo during a later cleavage

division (top row). The paternal chromatin is highly enriched with SMC2 (white

arrow in the bottom row) compared to the maternally derived chromosomes.

Arrowhead, maternal chromosomes. DNA is blue. Scale bar: 8 mm.

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maternally derived daughter chromatid sets undergoing segregation

but not with the lagging PCM (Fig. 5B). These observations

demonstrate that PSR escapes the defective paternal chromatin and

associates with the viable maternal chromosomes during anaphase

of the first mitotic division. The fact that PSR can effectively

segregate with the maternal chromosomes at this time demonstrates

that the centromeres of the PSR sister chromatids are functional. It

is also likely that the centromeres of the paternal chromatin are

functional because the lagging paternal chromatin often appeared

oblong as if under tension, with pointed ends stretching toward the

two maternally derived nuclei (Fig. 1A, Fig. 5C). We also noticed

that the PCM occasionally segregated with one of the two daughter

chromatid sets derived from the maternal nucleus (Fig. 5C). This

co-segregation has the potential of destroying the associated

chromatid set. However, embryos in which this occurs are likely

to survive due to the presence of the second, free daughter set.

PSR was found to localize to the periphery of the paternal

nucleus in all young embryos analyzed between fertilization and

the first prophase (n519/19; Fig. 5B). Additionally, PSR often

was present in a position immediately adjacent to the maternal

chromosomes (Fig. 5B). In several cases we observed PSR

residing in a condensed form just outside and separate from the

uncondensed paternal chromatin during metaphase (Fig. 5C).

The rDNA locus also was found at the nuclear periphery,

suggesting that localization of PSR at the nuclear periphery is a

general characteristic of pericentric chromosomal regions in N.

vitripennis (Fig. 5C). Nevertheless, we speculate that this

peripheral position allows PSR to effectively escape the PCM

for association with the maternally derived chromosomes.

We analyzed the nuclear position of PSR in developing and

mature sperm (Fig. 6). PSR was present near the anterior tip of

the elongated nucleus in all of the mature sperm examined

(Fig. 6A,D). This position is at the end of the sperm that contains

the acrosome vesicle and first enters the egg during fertilization.

In contrast to PSR, two different wasp chromosomes localized

broadly across the elongated sperm nucleus (Fig. 6B–D). Earlier,

during the process of spermatid nuclear elongation, PSR

Fig. 6. Unlike the normal wasp chromosomes, PSR localizes to a defined

region in the sperm nucleus and is stably segregated in the testis. (A) An

elongated sperm nucleus with PSR (red) at the anterior-most tip (top panel),

and multiple sperm nuclei with PSR at this anterior position (red arrows in the

bottom panel). (B,C) Elongated sperm nuclei hybridized with probes that

mark two different normal chromosomes. (D) A schematic showing the

localization frequency of PSR and the two autosomes within quadrants of the

elongated sperm nucleus. Numbers are percentage localization for each

quadrant. For PSR, the anterior-most quadrant is divided into the first 10th

and second 15th percentiles. (E) Localization patterns of PSR (red, with red

arrow) and the rDNA locus (green, with green arrow) during the early stages

of sperm nucleus elongation. (F) Localization of PSR (red) and the rDNA

locus (green) in the germ line and somatic cells of a whole-mount testis is

shown in the large panel. The top right panel depicts a single, pre-meiotic cell

at the anterior of the testis (white arrow in large panel). The middle right

panel shows a somatic cell in the mid testis (yellow arrow) that is polyploid,

as indicated by its large nucleus and the expanded PSR and rDNA satellite

signals. The bottom right panel shows a post-meiotic cyst of interconnected

spermatid cells in the posterior region of the testis (white outline). DNA is

blue in all panels. Scale bar: 5 mm (A), 3 mm (C), 3 mm (E) and 100 mm (F).

Fig. 5. PSR localizes at the periphery of the paternal nucleus and migrates

from the paternal to maternal chromatin during the first anaphase.

(A) Meiotic chromosome spreads from male testes hybridized with different probes

that recognize specific satellite DNA repeats on PSR and the normal wasp

chromosomes. The red arrow in the first panel indicates PSR; the probes recognize

two satellite blocks, one on each end of the PSR chromosome. The white arrow

indicates the normal chromosome carrying the rDNA locus. The green arrow in the

second panel indicates a single satellite block that overlaps with the nucleolar

organizer region (NOR) seen in the first panel (white arrow). The green arrow in

the third panel indicates a single satellite block on a different normal wasp

chromosome. (B) Different stages of the first mitosis in PSR-positive embryos are

shown. White arrows indicate the paternal chromatin. PSR is red. (C) The first

panel depicts the paternal chromatin mass (white arrow) co-segregating with a

maternally derived daughter nucleus following the first mitosis. The second panel

shows metaphase of the first mitosis; PSR resides just outside the paternal nucleus

(white arrow). The third panel shows prophase of the first mitosis; both PSR (red)

and the rDNA locus (green) associate with the periphery of the nuclear envelope.

DNA is blue. The rDNA locus is shown in green in A and C.

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localized to the edge of the nucleus that forms one of the twoelongating nucleus ends (Fig. 6E), showing that PSR is at theposition that will become the anterior nucleus tip from the

beginning of elongation. In contrast, the rDNA locus was presentin different regions within the elongating spermatid nucleus(Fig. 6E). This disparity in localization patterns between PSR and

the normal chromosomes suggests the intriguing possibility thatPSR may alter or bypass normal nuclear organization duringsperm nucleus elongation to obtain its peripheral position at the

tip of the sperm nucleus.

PSR is stably transmitted during meiosis and mitosis

Previous genetic studies suggested that PSR transmission doesnot involve meiotic or mitotic drive mechanisms (Beukeboomand Werren, 1993a). To cytologically confirm this conclusion,

we visualized PSR in whole testes. The majority of germ cells inthe testis are cysts of mitotically dividing (pre-meiotic)spermatocytes and post-meiotic spermatids. We observed a

single PSR signal within each nucleus of these cysts (Fig. 6F).We also found exceptionally large clusters of PSR signal withinthe giant nuclei of somatic cells (Fig. 6F). We interpret these

PSR clusters to be multiple chromosome copies due to thepolyploid nature of these and other somatic cells in N. vitripennis

(Rasch et al., 1977). A single region of PSR signal was detectedin nearly all post-meiotic spermatids (n5206/210; Fig. 6F) and

mature sperm (n5146/149). Similar patterns were found for thechromosome carrying the rDNA locus (Fig. 6F). Theseobservations demonstrate that PSR is stably transmitted during

the mitotic and meiotic divisions in the male germ line, a findingthat is consistent with the idea that PSR transmission does notinvolve pre- or post-meiotic drive.

DiscussionA fascinating aspect of B chromosomes is how they can persistwithin a given eukaryotic genome without contributing to thefitness of the organism. The B chromosome, PSR, exhibits a uniquemeans of achieving this goal – it takes advantage of the haplo-

diploid reproduction of N. vitripennis, completely destroying thepaternal nuclear material of this organism so that all embryosfertilized by PSR-bearing sperm, which should become diploid

females, are transformed into haploid, PSR-transmitting males.Previous studies demonstrated that, in the presence of PSR, thepaternal chromatin does not properly condense into chromosomes

during the first mitotic division and is, therefore, renderedincapable of segregating with the maternal chromosomes (Nuret al., 1988; Reed and Werren, 1995). A fundamental question is

specifically how PSR induces this effect on the paternal half of thegenome while, at the same time, escaping this fate and becomingtransmitted to new progeny at near perfect frequencies.

The chromatin basis of paternal genome loss causedby PSR

Our results suggest that PSR does not destroy the paternalchromatin indirectly by altering its progression into the firstmitotic division. Specifically, we found that both maternal and

paternal sets synchronously undergo DNA replication and entryinto mitosis while in the presence of PSR. Instead, ourexperiments suggest that the process of chromosome

condensation is specifically affected. We found that SMC2, aprimary Condensin component, appears highly enriched on thepaternal chromatin relative to the maternal chromosomes during

the first mitotic division. Additionally, Histone H3 of the paternalchromatin remains phosphorylated at Serine residue 10 long after

the maternal chromosomes have exited from the first mitoticdivision. In fact, the PH3S10 mark persists on the paternalchromatin after multiple mitotic divisions, suggesting that thePCM fails to exit properly from mitosis. Our experiments do not

allow us to conclude if persistent PH3S10 leads directly toCondensin overloading. However, this hypothesis is consistentwith the likelihood that the PH3S10 mark is necessary for proper

Condensin loading to chromatin (Ajiro et al., 1996a; Ajiro et al.,1996b; Van Hooser et al., 1998). We speculate that PSR does notdirectly cause abnormal Histone H3 phosphorylation because the

paternal set is not PH3 positive before entry into the first mitosis.Instead, PSR may induce other, more upstream ‘epigenetic’marks that ultimately affect proper Histone H3 phosphorylation.

Previous groups have speculated that PSR ‘imprints’ the paternal

chromatin some time during spermatogenesis (Werren andStouthamer, 2003). During later stages of this developmentalperiod, histones are largely stripped away from the paternal DNA

before it is packaged with protamines. However, a fraction ofhistones are known to remain within the sperm chromatin in anumber of organisms, including Drosophila (Gatewood et al.,

1987; Dorus et al., 2006; Govin et al., 2007). In wasps, PSR couldmodify any remaining histones during sperm development in a waythat specifically affects their phosphorylation and ability to

facilitate chromatin condensation following fertilization.Alternatively, PSR could directly alter methylation of thepaternal DNA in the testis. Recent experiments have uncoveredsubstantial Cytosine methylation at a number of genes in N.

vitripennis (Park et al., 2011). In other organisms, DNAmethylation is closely linked with proper Histone H3 methylation(Jackson et al., 2002). Such marks, if altered, could conceivably

affect downstream chromatin processes such as chromosomecondensation. It is intriguing to speculate that PSR, while thoughtto be largely heterochromatic and gene-poor, encodes a DNA- or

Histone-modifying enzyme that is expressed in the testis and iscapable of modifying the paternal chromatin but not its ownchromatin. This scenario is plausible, first, because largelyheterochromatic chromosomes, such as the Y and 4th

chromosomes in D. melanogaster, are known to contain protein-coding genes (Adams et al., 2000). Second, a number of chromatinremodeling enzymes are known to act on distinct types of

chromatin. For example, the Histone methyltransferase 1 (HMT1)in human, the G9a Histone H3 methyltransferase in mouse, and theHistone H4 acetyltransferase MOF in Drosophila are associated

primarily with euchromatin (Akhtar and Becker, 2000; Wang et al.,2001; Tachibana et al., 2005; Kleefstra et al., 2006). A PSR-linkedenzyme that acts solely on euchromatin would be expected to affect

the normal wasp chromosomes but not PSR. Alternatively, thepresence of PSR could cause the mis-regulation of a euchromatin-modifying enzyme encoded in the wasp genome. Testing thesehypotheses will be greatly facilitated by genome-wide studies of

epigenetic marks and gene expression during spermatogenesis andearly embryogenesis in PSR-carrying wasps.

The cellular transmission of PSR

Our cytological analyses of PSR during early development aresummarized in Fig. 7. These experiments revealed that PSR

escapes the PCM and associates with the viable maternalchromosomes during anaphase of the first mitotic division. Thisfinding is not unexpected because association of PSR with the

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maternal chromosomes during the later mitotic divisions would

likely result in mosaic animals. However, what remains to be

determined is precisely how PSR utilizes the gonomeric spindle

for segregation. It is well established that, in addition to the

centrosomes – the primary organizers of the spindle apparatus –

chromatin also can play an active role in microtubule

organization. A primary example is the centrosome-less meiotic

spindle apparatus in the D. melanogaster oocyte, in which the

chromosomes alone nucleate the microtubule-based spindle

apparatus (Matthies et al., 1996). Additionally, in N. vitripennis

embryos in which the centrosomes have been inactivated by

the male-killing bacterium Arsenophonus nasoniae, the

chromosomes alone are capable of nucleating rudimentary

spindles (Ferree et al., 2008). An interesting question is

whether the PCM, along with the centrosomes, can support the

formation of its half of the gonomeric spindle. Future attempts to

answer this question will require careful analyses of the spindle

apparatus in young, PSR-positive wasp embryos.

We found that PSR localizes to the outer surface of the paternal

nucleus in all young embryos examined. This pattern is likely to be

highly important for the successful transmission of PSR. For

example, an alternative finding, such as the presence of PSR within

the center of the paternal nucleus, would likely result in the

entanglement of PSR with the unresolved paternal chromatin. We

never observed PSR to remain with the PCM following anaphase

of the first division, demonstrating the proficiency of this

chromosome in escaping the PCM. The localization of PSR to

the nuclear periphery likely stems from the general tendency of

certain centric and pericentric regions to associate with the inner

nuclear envelope (Rae and Franke, 1972; Marshall et al., 1996;

King et al., 2008). In support of this idea, the pericentric region of

the autosome containing the rDNA locus also was found to

associate with the nuclear periphery in N. vitripennis. A large

portion of the euchromatic arms of the autosomes likely extends

into the interior of the nucleus where the entangled chromatin mass

forms. PSR, in contrast, contains little or no euchromatin. Thus, its

entire contents reside at the nuclear periphery where it can resolve

freely from the paternal chromatin.

The fact that a single PSR chromosome resides within the

nucleus of each germ cell and mature sperm demonstrates that

PSR stably segregates during cell division in the testis. These

observations are consistent with the idea that PSR transmission

does not involve meiotic or post-meiotic drive mechanisms. The

fact that PSR stably segregates in the male germ line stands in

contrast to many other B chromosomes, which are highly

unstable and prone to loss during cell division (Jones, 1991).

Some of these B chromosomes have evolved ways of amplifying

or segregating asymmetrically, including directed non-

disjunction, in order to counter their instability (Jones, 1991).

A more universal problem of B chromosomes is that, unlike the

normal chromosomes, they lack pairing partners during meiosis.

PSR may be transmitted stably in the male germ line because

meiosis in this sex occurs as a modified mitotic division due to

the haploid state of males (Whiting, 1968). This idea predicts that

PSR should not be stably transmitted during female meiosis,

which involves homolog pairing (Beukeboom and Werren,

1993b). Support for this prediction stems from the unstable

nature of several PSR derivatives produced from genome

fragmentation or mutagenesis (Beukeboom and Werren, 1993a;

Perrot-Minnot and Werren, 2001). These chromosomes were

successfully obtained in females due to their reduced ability to

destroy the paternal chromatin. However, the instability of these

chromosomes to persist in the female germ line may have been

caused either by damage incurred to the centromeres or other

chromosomal regions that are important for their stability or by a

lack of a pairing partner in this tissue.

PSR disobeys normal patterns of nuclear organization

One of our most striking findings is that PSR localizes at the

anterior tip of the elongated sperm nucleus in all analyzed sperm.

This pattern is in contrast to the normal chromosomes, which

localize broadly across the nucleus. An important question

stemming from this observation is how PSR is able to achieve

such a high level of localization to the anterior tip of the sperm

nucleus. One possibility is that this region is where any extra

chromatin that arises in the genome is placed. An alternative and

more intriguing possibility is that PSR localizes to the sperm

nucleus tip in an active manner. How might this occur?

Sperm nuclear elongation likely involves the coordination of

extra-nuclear events, such as formation of a microtubule bundle

on one side of the nucleus that generates the elongating force

(Fawcett et al., 1971), with intra-nuclear events that are less

clearly understood. Early studies in birds documented the

presence of dense foci of DNA, presumed to be regions of

heterochromatin, at the position of elongation initiation in the

nuclei of post-meiotic spermatids (Dressler and Schmid, 1976).

In mice, foci of heterochromatin were observed in a position

beneath the nuclear envelope that eventually becomes the

anterior tip of the sperm nucleus (Czaker, 1987). Although the

mechanisms are unknown, these observations suggest that

heterochromatin plays a role in determining the initial site of

sperm nucleus elongation. In light of this possibility, we propose

Fig. 7. A schematic summary of PSR nuclear dynamics between spermiogenesis and the first embryonic mitosis in N. vitripennis. Blue and green represent

the paternal and maternal chromatin, respectively. Red symbolizes PSR. During spermiogenesis, PSR resides in the position that will become the anterior tip of the

elongated sperm nucleus. Following fertilization, the two nuclei become juxtaposed and undergo replication synchronously. During this time, PSR resides at the

periphery of the paternal nucleus, often adjacent to the maternal nucleus. Both sets enter mitosis together. However, during metaphase, the paternal chromatin fails

to condense and resolve properly while PSR resolves properly. PSR escapes the paternal chromatin and segregates with the normal maternally derived

chromosomes during anaphase. During interphase following the first mitosis, one copy of PSR is present in each daughter haploid nucleus. The defective paternal

chromatin is unused and eventually becomes lost.

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that PSR, being largely heterochromatic, may seed nucleus

elongation preferentially over the pericentric regions of the

autosomes.

A second important question is whether there is any functional

relevance of PSR localization to the anterior tip of the sperm

nucleus. A similar pattern of B chromosome localization also was

discovered in the sperm of maize (Rusche et al., 1997). It was

proposed that the localization of a B chromosome to the sperm

nucleus tip confers a fertilization advantage to the subset of

sperm that carry this chromosome (Rusche et al., 1997), although

no compelling mechanistic explanations were offered (Jones

et al., 2008). In N. vitripennis, all sperm from PSR-positive males

contain a single copy of this B chromosome, arguing against the

need for a fertilization advantage in this organism. Alternatively,

it is possible that placement at the tip of the sperm nucleus may

confer some epigenetic advantage to the PSR chromosome. Such

a hypothesis has been proposed for the sex chromosomes, which

localize invariably to discrete regions within the sperm of several

mammals (Greaves et al., 2003; Foster et al., 2005).

PSR-like B chromosomes in other hymenopteran insects

The paternal transmission of PSR is possible due to the haplo-

diploid reproduction of N. vitripennis. Because males normally arise

as haploids from unfertilized eggs, the destruction of the paternal

half of the genome by PSR successfully converts what should

become diploid females into transmitting haploid males. It has been

suggested that haplo-diploid organisms in general may be prone to

sex ratio distortion by similar, paternally transmitted B

chromosomes (Werren and Stouthamer, 2003). This hypothesis is

supported by the previous discovery of another paternally

transmitted B chromosome in a different hymenopteran insect,

Trichogramma kaykai (Stouthamer et al., 2001). It is highly likely

that this B chromosome in T. kaykai and PSR in N.vitripennis arose

through independent evolutionary events due to their large sequence

differences (Eickbush et al., 1992; McAllister, 1995; van Vugt et al.,

2005) and the distant relatedness of these two insect species. An

intriguing question is whether these B chromosomes are transmitted

through similar mechanisms. This idea appears to be supported at

the morphological level – the B chromosome in T. kaykai causes the

paternal genome to undergo hyper-condensation and chromosome

resolution failure in a manner that is very similar to PCM formation

in N. vitripennis (van Vugt et al., 2003). Further studies in N.

vitripennis and T. kaykai will be important for exploring if PCM

formation and B chromosome transmission involve similar

chromatin-based mechanisms, and more broadly, how such

systems of genome conflict can evolve at the molecular level.

Materials and MethodsWasp lines and crosses

Experimental crosses involved AsymC (wild-type, Wolbachia-uninfected) virginfemales with either AsymC males or AsymC males carrying PSR (AsymC-PSR).Wasps were allowed to mate en mass overnight with 50% honey in water.Subsequently, mated females were separated and individually allowed to depositembryos into fresh blowfly hosts.

Embryo collection and fixation

Embryos were removed from host pupae and fixed in a solution of 5 ml heptane,1.5 ml 16PTX (16phosphate-buffered saline with 0.5% Triton X-100) and 600 ml37% formaldehyde (final concentration of 4% in the aqueous layer) for26 minutes. Fixed embryos were removed, placed on a small piece of Whatmanpaper, allowed to air-dry for ,1 minute, and adhered to double-sided adhesivetape on the surface of a 22 mm Petri plate. Approximately 2 ml of 16 PTX wasimmediately placed into the dish to rehydrate the embryos. A 28-gaugehypodermic needle was used to remove embryos from their vitelline

membranes. Embryos were transferred to a 0.6 ml microfuge tube, washed threetimes with 16 PTX and treated with RnaseA for 1–2 hours at 37 C beforefluorescence in situ hybridization or antibody staining.

Fluorescence in situ hybridization

The following sequences were used for fluorescence in situ hybridization probes:59-TCAAAAGTCTTGACTTTGGCTTACACGCTT-39 and 59-TATAATCAAA-AGTCTCGACTTATGATTGGA-39 are regions of two unique satellite DNArepeats on PSR (Eickbush et al., 1992); 59-TTTTTAAGACGTTTTATTGT-TACTGGTAGT-39 is a region of a satellite DNA repeat that is found on a singleautosome (Eickbush et al., 1992); and 59-GTATGTGTTCATATGATTTTGG-CAATTATA-39 is a 240-bp spacer repeat of the rDNA locus (Stage and Eickbush,2010). Additionally, three sequences corresponding to regions of the 28S rDNAgene also were used for probes: 59-TGGTTGCAAAGCTGAAACTTA-AAGGAATT-39, 59-ATTTAGAGGAAGTAAAAGTCGTAACAAGG-39, 59-AT-GTTTTCATTAATCAAGAACGAAAGTTAG-39. All of these sequences werechemically synthesized (IDT, Inc., USA) and 59-end-labeled with either Cy3 orCy5 for detection. Mapping the hybridization sites of the synthesized probes wasperformed on meiotic chromosomes from testis tissues. Whole testes weredissected in 16 PBS and fixed in 4% paraformaldehyde and 50% acetic acid for4 minutes. Immediately following fixation, testes were squashed, immersed intoliquid nitrogen for 5 minutes, dehydrated in 100% ethanol for 10 minutes, and air-dried for at least 1 hour. 125 ng of each probe was diluted in 20 ml of 1.16hybridization buffer, which was placed onto the tissue and slide and covered with acoverslip. Samples were incubated overnight at 30 C, washed three times for20 minutes each wash in 0.16SSC buffer, and mounted in Vectashield mountingmedium with DAPI (Vector Labs, Inc., USA). Probe hybridizations to embryoswere carried out as described in (Ferree and Barbash, 2009).

Immunostaining and TUNEL

Primary antibodies were incubated with fixed embryos overnight at 4 C for thefollowing dilutions: rabbit anti-PH3 (Santa Cruz Biotech, USA) at 1:200; rabbitanti-PCNA (a gift from Paul Fisher, Stony Brook University) at 1:500; rabbit anti-SMC2 (a gift from Margarete Heck, University of Edinburgh) at 1:200. TUNELreactions were performed on whole embryos using the In Situ Cell Death Detectionkit, TMR red (Roche, Inc., USA) according to the manufacturer’s instructions fortissues in liquid medium.

Microscopy

Images of chromosomes were captured on an Olympus IX81 epifluorescencemicroscope and ImagePro 6.3 imaging software. Images of whole mount tissues(testes and embryos) were taken with a Zeiss DMIRB confocal microscope.Constant gain and pinhole settings were used to collect all images within a givenexperiment. All images were subsequently processed with Adobe Photoshop CS5version 12.

AcknowledgementsWe thank John Werren for the wasp lines, Margarete Heck for theanti-SMC2 antibody, and Paul Fischer for the anti-PCNA antibody.We also thank John Werren, Richard Stouthamer, and Emily Wileyfor helpful discussions. Finally, we thank the Harvey Mudd BiologyDepartment for the use of their confocal microscope.

FundingWe would like to thank the Kenneth T. and Eileen L. NorrisFoundation of Scripps College for a summer research award given toM.S.

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