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First published online January 8, 2007
Journal of Experimental Biology 210, 261-268 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02631
Growing out of a caste - reproduction and the making of the queen mole-rat
1 Department of Biological Sciences, Vanderbilt University, Nashville, TN
37235, USA
2 Department of Neuroscience Graduate Program, Vanderbilt University,
Nashville, TN 37235, USA
* Author for correspondence (e-mail: ken.catania{at}vanderbilt.edu)
Accepted 7 November 2006
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Summary |
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Key words: eusocial, vertebra, lumbar, birth, bone
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). They are
eusocial mammals with separate castes and a social structure based on division
of labor (Jarvis, 1978;
Jarvis, 1981;
Burda, 1990;
Alexander et al., 1991). Each
colony contains a single, sexually active `queen' that breeds with 1-3 males
while other colony members help with pup rearing, colony maintenance, and
defense (Brett, 1991;
Jarvis, 1981;
Jarvis, 1991;
Lacey et al., 1991). When the
queen dies or is removed from the colony, subordinate females can undergo
rapid sexual maturation, and a violent competition often ensues until a victor
emerges to become the new queen (Clarke and
Faulkes, 1997). Mole-rats thus share a remarkable number of
features in common with eusocial insects and these findings have generated
considerable interest in both the evolutionary pressures resulting in
eusociality and the mechanisms that allow different castes to develop in such
disparate species (Thorne,
1997; Thorne et al.,
2003).
An important finding in this regard was the demonstration that queen
mole-rats and non-reproductives are dimorphic in their anatomy and thus
represent separate morphological as well as behavioral castes. O'Riain and
colleagues showed that established mole-rat queens have significantly longer
lumbar vertebrae for their body size compared with all other colony members
(Jarvis et al., 1991;
O'Riain et al., 2000). In
addition to confirming that physical castes exist in mole-rats, these findings
provided another obvious parallel between queens in insect and mole-rat
colonies; both have elongated abdomens to accommodate the reproductive tract
during gestation.
The findings raise a number of intriguing questions. For example, what triggers vertebral growth in formerly non-reproductive mole-rats that transforms them into the queen caste? Is this an irreversible step that will progress to completion once initiated? What is the time course for this transformation and how is it influenced by reproduction? What are the candidate physiological mechanisms that may contribute to this transformation? Here, we begin to address these questions by documenting changes in the morphology of newly established queens during their first 30 months of reproductive activity. We show that growth of the lumbar vertebrae is increased in new queen mole-rats during pregnancy but returns to normal rates during intervening periods without pregnancies. In addition, this growth is not uniform during the course of each pregnancy but instead appears to reach a maximum in the last few weeks of the 70-day gestation period. These results show a direct link between vertebral growth and pregnancy and suggest that hormones related to pregnancy may play the primary role in caste transformation.
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Materials and methods |
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Animal subjects
Fifteen naked mole-rats were used in this study. Procedures followed
National Institutes of Health guidelines and standards set by the Animal
Welfare Act and the Vanderbilt University Institutional Animal Care and Use
Committee. Twelve mole-rats (six females, six males) were isolated from a
single colony and pair-housed to form breeding pairs. Two females died and are
not included in this report. Of the remaining paired females, three became
reproductively active and were designated Breeder 02-02, Breeder 02-04 and
Breeder 02-05. The three breeders displayed swollen, perforate vaginas,
prominent teats and reproduced multiple times (
5 births). One pair-housed
female mole-rat's vagina remained imperforate and she failed to reproduce.
This animal served as a `Nonbreeder 02-13' control. These paired females were
closely observed during the study and underwent weekly radiographs. The paired
males, along with additional mole-rats from established colonies, were also
radiographed intermittently during the study for comparison to the pair-housed
females.
Radiography
Before radiography, each mole-rat was anesthetized with isoflurane in an
induction chamber. Anesthesia did not exceed 5 min. Mole-rats were placed in
an MX-20 specimen x-ray cabinet (Faxitron X-ray; Wheeling, IL, USA). X-rays
(34 kV at 0.3 µA for 80 s) were taken in the dorsal and sagittal planes at
a magnification of 1.5x (Fig.
1). Female mole-rats and their offspring showed no obvious effects
from the anesthesia and imaging. The breeding male (02-01) lost mass over the
course of the study, however breeding males typically lose body mass with
sustained breeding responsibilities
(Jarvis et al., 1991).
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).
L4 also corresponds to the vertebra examined in O'Riain et al.
(O'Riain et al., 2000),
identified as L5 (nomenclature variation is due to differing labeling schemes
- see Fig. 1B for our
labeling). ZA width measurement provides a control for general bone growth
(O'Riain et al., 2000) and is
not part of reproduction-induced morphological changes in queens. All
measurements were corrected for magnification of the radiograph images. Two
experimenters measured radiographs independently and inter-rater reliability
for this study was r=0.92.
Data analysis
To determine differences in L4 lengthening between animals, we plotted the
L4 length over time (Fig. 2).
We also used the ratio of L4 length divided by zygomatic arch width (L4/ZA) to
control for non-specific body growth (Fig.
3A). In addition, we compared our results to the L4 lengths and ZA
widths of nonbreeders and of breeding males reported in O'Riain et al.
(O'Riain et al., 2000) by
using data from their plots of L4 (called L5) and ZA in their
fig. 2. Mann-Whitney tests were
used to assess significance in all analyses.
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). Any time
period between pregnancies that was greater than 7 days was considered a
non-pregnancy period (see Table
2 for examples). Growth rate was calculated for pregnancy and
nonpregnancy time periods by dividing the change in L4 length (mm) by the time
period (weeks). For the linear regression graph
(Fig. 5D), the L4 length values
for each week during the pregnancy and non-pregnancy periods for the three new
queens were standardized to baseline before being averaged to illustrate
growth independent of initial differences in L4 length.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). Finally, we compare growth rates during pregnancies
and intervening periods without pregnancies
(Fig. 4).
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Changes in L4 length
Fig. 2 shows L4 length over
time for three female mole-rats (Breeders 02-02, 02-04, 02-05) as they
transition from virgins to multiparous breeders. The length of L4 in these new
queens was compared to L4 lengths of a nonbreeding female (02-13), established
female breeders (C1 and BG Queens) and male mole-rats (02-01 and 02-15). The
mole-rats initially had a wide range of L4 lengths and body masses, but clear
trends developed through the course of the study.
First, the new queens showed growth of their L4 vertebra over time (Fig. 2). Initially, the lengths of their L4 vertebrae were not significantly different from the other no-pregnancy animals (Nonbreeder 02-13 and Males 02-01, 02-15) (Mann-Whitney test; z=-1.091, P=0.275), but by the end of the study the L4 of the new breeders were significantly longer than the L4 of the no-pregnancy animals (Mann-Whitney test; z=-1.964, P=0.050, N=6).
Second, by the end of the study, the L4 vertebrae in the new queens had increased by an average of 34% (5.20±0.29 mm, mean ± s.e.m.), making them nearly as long as the L4 in established queens (5.66±0.18 mm). By contrast, L4 in the no-pregnancy mole-rats increased by an average of 7%, with an L4 mean length of 3.95±0.09 mm (details in Table 1). Thevertebral growth in the new queens did not level off, suggesting that L4 will continue to grow for some time in these mole-rats. However, L4 growth in older queens that had been breeding for several years (Fig. 2, Est. Breeders C1 and BG Queens) appeared minimal, suggesting that L4 (and the rest of the lumbar vertebrae) eventually stops growing in established queens.
Third, close examination of L4 length as a function of time during pregnancies revealed that growth of L4 in the new queens was most rapid in the second half of each pregnancy and reached a maximum just before parturition. This is shown in detail in Fig. 2B.
L4 length compared with body size
To control for nonspecific growth due to skeletal maturation, we used the
width across the ZA as an index of body size. O'Riain et al. demonstrated that
ZA width provides a convenient index of body size that can be used to identify
caste-specific differences in the relative length of the lumbar vertebrae
(O'Riain et al., 2000). Using
this measure as a comparison, we found that growth of L4 in the new queens was
not accounted for by changes in body size. A plot of the ratio of L4 length
divided by ZA width (L4/ZA) over time revealed the lumbar-specific growth in
nascent queens (Fig. 3). The
percentage change in L4 length from the beginning of the study to its
conclusion for Breeders 02-02, 02-04 and 02-05 was always greater than the
equivalent percent change for the ZA width
(Fig. 3B;
Table 1). However, this was not
true for nonbreeding mole-rats (02-15 and 02-13) or the breeding male
(02-01).
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).
Fig. 3 shows the progressive
increase in the L4/ZA index of new queens in our study as they join this
morphological class (note that Breeder 02-04 reached an L4/ZA index that
exceeded an established queen) whereas the non-breeding female and the males
showed no such trend. Although the difference in the L4/ZA index between the
paired females and males at the beginning of the study was not statistically
significant (Mann-Whitney test; z=-0.218, P=0.827), the
females that went on to breed successfully had significantly larger L4/ZA
indexes than the nonbreeders and breeding male by the end of the study
(Mann-Whitney test; z=-1.964, P=0.050, N=6). As
would be expected, the increased growth rate of L4 during late pregnancy (e.g.
increased growth close to parturition) was still apparent after controlling
for body size (Fig. 3).
To compare our results in breeding females with a larger control
population, we examined the L4 and ZA measurements of nonbreeding females and
breeding males in the previous study by O'Riain et al.
(O'Riain et al., 2000) (data
derived from their fig. 2). We
found that the L4/ZA index for our nonbreeding female and males was not
statistically different (0.210±0.003, mean ± s.e.m.) from the
L4/ZA index of the nonbreeders and breeding males in the O'Riain et al. study
(0.203±0.002, N=16) (Mann-Whitney test; z=-1.006,
P=0.314). However, our new queens showed a statistically significant
increase in lumbar-specific length over time when compared with the O'Riain
female nonbreeders (N=10). Initially, the L4/ZA values of our females
(02-02, 02-04, 02-05) were not different (0.205±0.009) from the O'Riain
non-breeding females (0.205±0.002, N=10) (Mann-Whitney test;
z=-0.676, P=0.499) but, by the end of the study, the new
queens had L4/ZA values (0.260±0.009) that were significantly larger
than those of the O'Riain nonbreeding females (Mann-Whitney test;
z=-2.535, P=0.011).
Although our analyses focus on L4 length as a representative vertebra for changes occurring throughout the lumbar spinal column, we did find subtle variations in the growth patterns within the lumbar region in the breeding females. This can be appreciated by examining the change that occurred over time in the vertebra to ZA index (L#/ZA) for each of the eight vertebra. Fig. 4 illustrates this as a percentage change of the L#/ZA index for each vertebra in each mole-rat over the duration of the study. Overall, lumbar growth seemed to peak around the L3 region. The right side of Fig. 4 shows the percentage change in this index over time for the entire lumbar spinal column [i.e. (L1-L8)/ZA], including intervertebral spaces. The (L1-L8)/ZA index for Breeders 02-02 and 02-05 increased by 15% whereas this index for Breeder 02-04 increased by 33%. The nonbreeding female showed little increase in this index, whereas the male breeder had no overall increase.
L4 growth rate compared during and between pregnancies
In the course of our experiment, there was a relatively long period during
which the breeding females ceased reproduction. There were no births and no
obvious signs of pregnancy. Mole-rat breeding is sensitive to environmental
variables, and we suspect that a 2.2°C drop in ambient temperature in our
animal facility during this time caused stress to the animals, which are
poikilothermic and derive body heat from their environment
(Jarvis and Bennett, 1993;
Woodley and Buffenstein,
2002). When the temperature was readjusted, the animals returned
to their regular breeding cycles. This unexpected change in environmental
conditions provided an unpredicted insight: that increased L4 growth in female
breeders was specific to pregnancy periods.
This difference in growth rate is most dramatic when comparing bone growth during pregnancies (0.020±0.012 mm week-1, N=18) to the bone growth during the long non-pregnancy period (0.002±0.001 mm week-1, N=3; see Fig. 2, approximately weeks 70-110). There is a 10-fold increase in growth rate during the pregnancy periods. This variation in growth rate is also apparent in the shorter non-pregnancy periods experienced by each animal (Table 2).These shorter periods of slower L4 growth (or L4 reduction possibly related to lactation - see Discussion) between pregnancies suggest that temperature per se was not the cause of the decreased L4 growth during the longer non-pregnancy period.
To illustrate these differences, we compared mean L4 growth rates for each new queen during all pregnancy periods and all non-pregnancy periods (Table 2). The difference in L4 growth rate between pregnancy and nonpregnancy periods is also illustrated graphically in Fig. 5. Fig. 5A-C shows mean pregnancy and between-pregnancy growth rates for each new queen. The growth rates are plotted over an arbitrary 10-week time scale. Fig. 5D combines the data from the three mole-rats, fitting regression lines to the mean pregnancy and non-pregnancy growth during a 10-week period. The values were standardized to baseline prior to averaging so that they could be combined. Note that both lines indicate an initial period of slow growth, which may reflect the mineral demands associated with lactation, as the early period of pregnancy (or post-parturition non-pregnancy) is generally when young from the previous litter are nursed. However, by week 5, more length was added to L4 during pregnancy than during nonpregnancy times, and this trend became more pronounced through the remaining five weeks up to parturition. The growth rates during the nonpregnancy periods were similar to the growth rates of the other, non-queen mole-rats in the study. The lengths of the nonpregnancy periods varied (resulting in varying N values for each time point) (see Table 3), but for comparison to the pregnancy periods, they are displayed to a maximum length of 10 weeks.
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Discussion |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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). We
found that pregnancy was the critical factor for upregulating lumbar-specific
bone growth in female mole-rats. During periods of pregnancy, nascent queens
experienced increased bone growth, but this growth tapered off between
pregnancies. The lumbar spinal column continued to grow at an increased rate
with each subsequent pregnancy, and after 136 weeks and 5-7 pregnancies, the
vertebrae in each new queen continued their disproportionate expansion. These
findings suggest that hormones associated with pregnancy play an important
role in the increased bone growth specific to the lumbar region.
Interestingly, one of the smallest new queens (02-04) at the beginning of
the study experienced the greatest amount of lumbar growth and grew the
longest over the monitored time period. Her final L4 length after seven
litters rivaled our most established queen (BG) after 12 litters. The rapid
growth of breeder 02-04 was reduced but not eliminated when normalized to
general skeletal growth. It is likely that breeder 02-04 was low on the
dominance hierarchy in the colony and had remained small as a result of her
worker status. Jarvis and colleagues have found that body size is labile and
correlated with social status in the colony
(Jarvis, 1981;
Jarvis et al., 1994). The
largest subordinate females in the colony tend to have a greater likelihood of
becoming the next queen if the colony is removed from reproductive
suppression. Without competition for queen status, as in our pair-housing
conditions, the size or former status of the female becomes unimportant,
providing a unique chance for a small female to become the breeder. This could
explain her rapid growth, both general and lumbar specific.
A pregnancy-induced mechanism for lumbar expansion, which accommodates a
larger reproductive tract, probably arose as an extension of the less dramatic
reproduction-related bone growth that prepares many mammals for lactation.
Investigators have shown increased bone growth or density changes during
pregnancy in rats, sheep, monkeys and humans, as an adaptation for
concentrating mineral reserves that can be used for fetal skeletal development
and milk production (Miller and Bowman,
1998; Miller and Bowman,
2004; Bowman and Miller,
1999). However, most gains are abruptly lost after parturition and
subsequent lactation (Bowman and Miller,
1999; Redd et al.,
1984).
A number of hormonal changes occurring during the reproductive cycle may
support such skeletal modifications. For example, sex steroids such as
estrogen, androgens and progesterone, which are up-regulated during pregnancy,
have been shown to contribute to bone formation and remodeling
(Syed and Khosla, 2005).
Mole-rat queens differ from other mammals in that a net gain in bone length is
achieved throughout each breeding cycle. In this sense, more general
pregnancy-related variations in bone density that prepare rodents for
lactation may have been an exaptation
(Gould and Vrba, 1982) for the
elongation of the lumbar region that accommodates large litters in
mole-rats.
In this regard, queen mole-rats are uniquely challenged. They have the sole
responsibility for reproduction in a colony that requires a large workforce
and there has clearly been selection for large litter sizes. Well-established
queens may have 15 or more pups in a litter (with a record of 28 recorded in
the wild; Table 4 shows the
number of pups born per litter for each of the breeding females in the current
study). Yet the queen's girth is limited by the size of the colony tunnels
(tunnel diameters average 35-45 mm)
(Jarvis, 1991), which she must
traverse to impose reproductive suppression on her colony members
(Jarvis, 1991;
Faulkes and Abbott, 1991;
Faulkes et al., 1991). The need
to interact with members of the colony prevents the queen from remaining
isolated in her nest chamber during the later stages of pregnancy. Female
mole-rats had to adapt to these constraints, and selection seems to have
favored increased lumbar length as a mechanism for achieving large litter
sizes without overly increasing girth. This scenario is supported by the
observation that litter size increases over time in new queens
(O'Riain et al., 2000;
Jarvis and Bennett, 1991). The
alternative, that mole-rats make wider tunnels to accommodate the pregnant
queen, would be metabolically exorbitant.
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Acknowledgments |
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References |
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