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Embryonic Staging System for the Black Mastiff Bat Molossus rufus (Molossidae) Correlated With Structure-Function Relationships in the Adult.

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THE ANATOMICAL RECORD 292:155–168 (2009)
Embryonic Staging System for the Black
Mastiff Bat, Molossus rufus (Molossidae),
Correlated With Structure-Function
Relationships in the Adult
MARK J. NOLTE,1,2 DORIT HOCKMAN,3 CHRIS J. CRETEKOS,1
RICHARD R. BEHRINGER,1* AND JOHN J. RASWEILER IV4
1
Department of Genetics, University of Texas M. D. Anderson Cancer Center,
Houston, Texas
2
Genes and Development Graduate Program, University of Texas Graduate
School of Biomedical Sciences at Houston, Texas
3
Department of Molecular and Cell Biology, University of Cape Town,
Cape Town, South Africa
4
Department of Obstetrics and Gynecology, State University of New York
Downstate Medical Center, Brooklyn, New York
ABSTRACT
An embryonic staging system for Molossus rufus (also widely known
as Molossus ater) was devised using 17 reference specimens obtained during the postimplantation period of pregnancy from wild-caught, captivebred females. This was done in part by comparing the embryos to a developmental staging system that had been created for another, relatively
unrelated bat, Carollia perspicillata (family Phyllostomidae). Particular
attention was paid to the development of species-specific features, such as
wing and ear morphology, and these are discussed in light of the adaptive
significance of these structures in the adult. M. rufus can be maintained
and bred in captivity and is relatively abundant in the wild. This embryonic staging system will facilitate further developmental studies of M.
rufus, a model species for one of the largest and most successful chiropteran families, the Molossidae. Anat Rec, 292:155–168, 2009. Ó 2008
Wiley-Liss, Inc.
Key words: bat; Chiroptera; Molossus rufus; Molossus ater;
embryogenesis; embryonic staging system
The bats (order Chiroptera) are one of the most successful mammalian groups. A recent compilation indicates that there are at least 1,116 species of bats, comThe first two authors contributed equally to this work.
Chris J. Cretekos is currently affiliated with Department of
Biological Sciences, Idaho State University, Pocatello, Idaho
83209.
Grant sponsor: National Institutes of Health; Grant numbers: HD17739RR05396 (JJR), CA09299 HD07325 (CJC),
HD07325 (MJN); Grant sponsor: National Science FoundationIBN 0220458 (RRB); Grant sponsors: Ben F. Love Endowment (RRB), Society for Integrative and Comparative
Research (Grants-in-Aid-of-Research Award) (DH), National
Research Foundation of South Africa.
Ó 2008 WILEY-LISS, INC.
prising a little more than 20% of all living mammalian
species (Simmons, 2005; Wilson and Reeder, 2005), and
they are exceedingly abundant mammals in absolute
*Correspondence to: Richard R. Behringer, Department of
Genetics, University of Texas M. D. Anderson Cancer Center,
1515 Holcombe Blvd., Houston, TX 77030. Fax: 713-834-6339.
E-mail: rrb@mdanderson.org
Received 29 February 2008; Accepted 9 September 2008
DOI 10.1002/ar.20835
Published online 16 December 2008 in Wiley InterScience (www.
interscience.wiley.com).
156
NOLTE ET AL.
numbers, particularly in tropical regions of the world.
Bats also exhibit remarkable diversity with respect to
habits, form, and function. This can be attributed to the
innovations of powered flight and, in the majority of species, echolocation. These have permitted many bats to
prosper and evolve in a niche, the night sky, which,
among mammals, has been exploited to a limited extent
only by those that glide. Bats now perform a variety of
ecologically important functions within this niche,
including pollination, seed dispersal, and predation (particularly of insects).
To fully understand the evolution of bats, knowledge
of their ecology, phylogeny, and ontogeny is needed. The
study of bat ecology and species-specific characteristics
allows us to postulate what selective pressures may
have encouraged the development and diversification of
these adaptations. An analysis of the embryonic and
postnatal growth of specialized structures (such as bat
wings), and comparisons with the formation of homologous structures in model organisms, provides an anatomical basis to begin understanding the genetic modifications that have played a role in their evolution.
Previous studies have described early oviductal and/or
uterine development of the embryo in many bat species
(Rasweiler, 1993; Badwaik and Rasweiler, 2000), and the
postnatal development of a variety of anatomical features (Pedersen, 1995; Reep and Bhatnagar, 2000; Vater,
2000; Phillips, 2000; Adams and Thibault, 2000; Hermanson, 2000; Elangovan et al., 2007). Relatively few
studies have examined development during the postimplantation period, and most of these have not included a
precise temporal framework (instead using embryo
crown-rump length or weight as indicators of relative
age). These have focused on the brain (Misek, 1989,
1990; Pirlot and Bernier, 1974, 1991; Reep and Bhatnagar, 2000), pituitary gland and vomeronasal organ (Reep
and Bhatnagar, 2000), and aspects of limb development
and skeletogenesis (Adams, 1992a,b; Wyant and Adams,
2007).
Recently, we used conceptuses collected after timed,
captive matings to create a detailed embryo staging system for the short-tailed fruit bat, Carollia perspicillata
(family Phyllostomidae) (Cretekos et al., 2005). Subsequently, this system has been used as a framework for
detailed descriptions of the embryonic development of a
pteropodid bat (Rousettus amplexicaudatus; Giannini
et al., 2006) and a vespertilionid bat (Pipistrellus abramus; Tokita, 2006). Staging systems had previously been
devised for another pteropodid (Syconycteris australis;
Lawrence, 1991) and the vespertilionid Myotis lucifugus
(Adams, 1992a).
Here, we add to current knowledge of bat embryonic
development through a description of this process in the
black mastiff bat, Molossus rufus (family Molossidae),
correlated with some major structure-function relationships in the adult. The molossids are generally known
as fast flying, acrobatic insectivores, although many (e.g.
M. rufus) are also highly adept at nonvolant, quadrupedal movement. It seems reasonable to expect that the
morphological characteristics that facilitate the distinctive locomotor activities of this group would be reflected
in their patterns of embryonic development.
Although the subject of this study has also been
widely referred to in previous studies (including the collection of the actual embryos) as Molossus ater, Dolan
(1989) made the case that this species was actually first
identified and described by Geoffroy Saint-Hilaire
(1805a,b) as M. rufus. According to the rules of zoological nomenclature (International Code of Zoological Nomenclature, Fourth Edition, 1999), the latter name
should therefore take precedence.
M. rufus is found widely in the neotropics, from northern Mexico to northern Argentina (Dolan, 1989). These
animals are relatively large (20–40 g) compared to most
other insectivorous bats (Rasweiler, 1990a), and the
adults have wings with a high aspect ratio and wingloading (Fenton et al., 1998). These long, narrow wings
combined with low frequency echolocation calls (20–30
kHz) allow M. rufus to specialize in open-air foraging
with fast, sometimes erratic flight patterns, and an
apparent preference for the consumption of hard-shelled
insect prey, for example, beetles (Freeman, 1981a,b;
Fenton et al., 1998).
The available evidence indicates that the Trinidadian
population of M. rufus that provided breeding stock for
this study is reproductively synchronized, and that some
of the females may carry two pregnancies per year in
quick succession (Rasweiler, 1988; Badwaik et al., 1998).
The reproductive biology of M. rufus has also been
extensively studied under controlled conditions in captivity (Rasweiler, 1987, 1988, 1990a,b, 1991a,b, 1992,
1993; Badwaik et al., 1998), revealing this species to be
an interesting model for studies of the biology of menstruation, trophoblastic growth and differentiation, and
placental morphogenesis. During its menstrual cycle, M.
rufus also exhibits, within its uterus, the most pronounced example of physiological (nonpathological)
angiogenesis thus far observed in any adult mammal
(Rasweiler, 1991a). Our description of the postimplantation embryology in M. rufus further advances the utility
of this bat species for developmental studies.
MATERIALS AND METHODS
Source of Animals and Species Identification
Over the course of 23 years, more than 2000 M. rufus
were collected on the West Indian island of Trinidad by
one of the authors (JJR). These were captured with mist
nets or live traps, as they exited from their diurnal
roosts to forage at dusk or dawn, or when they returned
and attempted to re-enter these roosts. The nets or traps
were carefully positioned to catch most of the bats as
they dropped from their exit holes and initially began to
fly off. Many additional M. rufus were observed within
their roosts. The roosts were found under roofs or in the
attics of buildings at 48 sites scattered over a wide area
of central Trinidad, roughly outlined by the towns and
villages of Arima, Valencia, Sangre Grande, Nestor,
Mamon, Mundo Nuevo, Tabaquite, Gran Couva, Freeport, Chaguanas, Caroni, and Arouca.
On the basis of their larger adult size and pelage coloration (always a lustrous jet black), these bats were
readily distinguished from the two smaller species of
Molossus that also live on Trinidad—Molossus sinaloae
trinitatus and Molossus molossus (Carter et al., 1981).
Recently, however, a small number of exceptional M.
rufus (about six individuals) exhibiting their russet (reddish) color phase were observed by the authors intermingled with a larger number of black animals in one
additional roost. The great preponderance of black indi-
MOLOSSID BAT EMBRYONIC DEVELOPMENT
viduals in this heavily-sampled population raises a serious question as to whether M. rufus is really a more
appropriate species name for these bats than M. ater.
Captive Maintenance
Some of the M. rufus (adult females and a smaller
number of males in breeding condition) were retained,
transported to New York and maintained at Cornell
University Medical College with methods described elsewhere in detail (Rasweiler 1987, 1988). The bats were
kept in a warm room (29–318C) with a controlled light
cycle (12 hr light: 12 hr dark). Initially, they were
housed in sexually-segregated groups in Jewell-type animal cages (45.7 cm deep 3 48.3 cm wide 3 35.6 cm
high). A portion of each cage (15.2 cm wide) on the right
side was almost completely enclosed, except for a small
(7.6 cm wide 3 5.1 cm high) access hole at floor level, to
provide a darkened retreat.
Each day, without exception, the bats were fed mealworms (the larvae of Tenebrio molitor) ad libitum in
shallow pans. Initially, the mealworms were fattened for
2–3 weeks on a calcium-supplemented medium and then
sprayed with a liquid multivitamin supplement (diluted
1:1 with water) immediately prior to being offered to the
bats. Later in the studies, the mealworms were sprayed
with the diluted vitamin mix, dusted directly with calcium carbonate, and then presented to the bats. Water
was also continuously provided via plastic chick fountains,
consisting of a reservoir bottle and a water trough.
157
did not exhibit a limited period of estrus, it proved
impossible to correlate the first appearance of spermatozoa in aspirates with ovulation and fertilization. Most
females exhibited an extended period of sperm-positive
aspirates, however, and the time elapsed from its onset
was generally correlated with the stage of embryonic development found in pregnant animals. Records of the
days on which sperm-positive aspirates were obtained
from many of the bats have been published elsewhere
(Rasweiler, 1988, 1990b).
Measurements of the Uterus and Embryo
M. rufus was found to ovulate only from the right
ovary and to carry conceptuses only in the right oviduct
and uterine horn. On dissection, the greatest diameter
of the right uterine horn was measured. In the initial
studies (Rasweiler, 1990b), embryos at the limb bud
stage or later were removed from the uterus, weighed,
fixed in Zenker’s fluid for 16–20 hr, and measured. The
embryos were then washed overnight in running tap
water and dehydrated through graded alcohols to 70%
ethanol for storage. When necessary, mercuric chloride
precipitated on the embryos was removed by treatment
with Lugol’s solution, followed by a 5% solution of sodium thiosulfate to remove the iodine. In later studies,
initial fixation of the embryos was done while they were
still in utero. Both embryonic weight and crown-rump
length of each embryo were then recorded postfixation.
These measurements have been recorded in Rasweiler
(1990b) and Table 1 of this article.
Observations of Locomotor Activity
M. rufus is specialized for a way of life that is significantly different in a number of respects from those of
many other bat species. Observations were made by one
of the authors (JJR) on M. rufus resting or moving about
in their natural, diurnal roosts in 48 buildings on Trinidad. This was accomplished by peering into attics or
subroof crawl spaces through access holes (‘‘manholes’’)
from the human living quarters with the assistance of a
flashlight, or by actually entering the roosts (which generally disturbed the bats). Observations were also frequently made on M. rufus when they left or re-entered
their roosts at dusk or dawn, while actively foraging in
flight for insects (which occurs while there is substantial
daylight), and in captive breeding colonies maintained
for about 5 years at Cornell. In both the field and captivity, bats occasionally fell to the ground or floor, and
attempted to escape by crawling or running.
Timing of Embryonic Development
Following their transport to New York, the bats were
initially housed in sexually-segregated groups for 2–7
months. Single males were then placed with groups of
females to permit breeding activity.
Histological studies provided evidence that many of
the M. rufus became polyestrous when maintained in
sexual isolation in captivity. These studies also revealed
that the species is a spontaneous ovulator with a functional luteal phase to its cycle. The cycle is terminated
by true menstruation (Rasweiler, 1988, 1991a).
Following the introduction of males, vaginal aspirates
were obtained from every female each morning and
examined for spermatozoa (Rasweiler, 1987). As M. rufus
RESULTS
Characteristics of Locomotor Activity
Extensive observations were made on locomotor activity by M. rufus during the course of both field work (48
building roosts) and captive experimentation with the
animals. Like other molossid bats, M. rufus possesses
long, narrow wings and compact craniofacial features
(Fig. 1A-D). These are thought to be adaptations, in
part, for their rapid mode of flight (Vaughan, 1966; Freeman, 1981a). When foraging, M. rufus is also capable of
sudden, seemingly erratic, changes in direction, which
are presumably used in part to catch insects. M. rufus
forages twice per night, at dusk and dawn. At both
times, the animals are flying outside their roosts when
there is significant daylight, and they are therefore at
risk of predation by raptors. On one occasion, during the
course of field work on Trinidad, M. rufus leaving a roost
to forage at dusk used sudden, evasive flight maneuvers
to successfully avoid being captured by a pair of bat falcons (Falco rufigularis) (Badwaik and Rasweiler, 2000).
Chase et al. (1991) have also observed bat falcons and
owls hunting the closely-related M. molossus.
Within their natural roosts (or wire cages in captivity), M. rufus were observed to walk or scamper quadrupedally across flat, inclined, or vertical surfaces with
great agility. This ability may help them to avoid predation by barn owls (Tyto alba), and possibly other predators (e.g. snakes), that also sometimes occupy the same
attic roosts (JJR unpubl. observ.; Esberard and Vrcibradic, 2007). M. rufus were never seen to take to flight
within their natural roosts (even when disturbed by the
entrance of a human observer) and have generally
158
NOLTE ET AL.
TABLE 1. Summary of M. rufus embryonic development
Day female
was killedc
Uterus
diameter
(mm)
Crown-rump
length (mm)
Mass
(gm)
13
MA25C
b
40
5.0
5.0
0.03
14
MA24Cb
MA41
MA66
MA29
MA74Bb
29
38
10
40
41
8.0
9.5
8.0
10.0
10.5
7.0
6.4
5.8
7.2
9.0
0.04
0.04
0.03
0.09
0.07
16
MA15
MA92Bb
44
36
12.0
10.0
9.7
10.0
0.13
0.08
17
MA53
34
11.0
11.4
0.17
18
MA22
MA25
MA44
MA83Bb
MA16
MA21
62
66
40
43
50
54
13.0
12.0
12.1
12.5
13.5
14.5
13.5
12.9
12.1
12.0
16.7
18.5
0.33
0.26
0.20
0.39
0.70
0.77
21
MA10b
60
19.0
19.0
0.45
22
b
70
19.0
28.0
2.80
Stage
15
20
Reference
specimensa
MA78
Key features*
Hindlimb buds form; forelimb AER; 3
pharyngeal arches.
Forelimb longer than wide; hindlimb
AER; nasal pits; cervical flexure;
propatagium primordium.
Hand and footplate; plagiopatagium
primordium; auditory hillocks;
premaxillary centers.
Pinnae and antitragii present; vibrissae
condensations; uropatagium
primordium; fore- and hindlimb digital
condensations.
Prominent snout; cervical flexure absent;
eyelids; fore- and hindlimb interdigit
tissue receding.
Mouth open and thumb free; forelimbs
cross at midline; crescent-shaped
pinnae; eyes begin to close; thumb and
hindlimb claw primordia.
Eyes closed; pinnae extend toward facial
midline; salient crus helix; knee
flexure; mouth obscured by forelimb;
digits II-IV juxtaposed/chiropatagium
narrow.
Autopod folded onto zeugopod; mouth
open and tongue visible.
Eyes begin to open; teeth formed;
antitragii broad and erect.
*Italicized text indicates unique features of M. rufus when compared to other bat species that were staged according to the
C. perspicillata staging system (Cretekos et al., 2005; Giannini et al., 2006; Tokita, 2006).
a
Measurements reported in Rasweiler, 1990b.
b
Measurements not included in Rasweiler, 1990b; C-R length and weight recorded postfixation.
c
Counted from the beginning of a prolonged period of sperm-positive vaginal aspirates.
AER, apical ectodermal ridge.
proved to be incapable of flight in captivity (e.g. when
escaping during their routine care or handling, and
dropping to the floor of a relatively large animal room).
As feeding time approaches in the wild, the bats can
often be heard crawling across ceiling panels and out
into roof overhangs to exit, usually from holes along the
edges of the roofs.
On exiting from such holes, M. rufus do not immediately
take to active flight. Rather, they first drop several meters
to pick up lift and momentum before flying off. This was
observed many hundreds of times in the course of catching
the bats. When individuals occasionally dropped to the
ground during collecting activities in the field, they could
scamper there but generally could not directly take flight.
Rather, they would have to first ascend to an elevated
position by crawling and then drop to take flight.
Courtship behavior was also frequently observed during periods when M. rufus were being cared for or
worked with in the captive breeding colonies established
in New York. This was possible, because the animals
were sometimes active in the open portions of their
cages during daylight hours. Courtship activity typically
involved extensive movement and interactions of the
animals on all fours. The animals possess a cutaneous
throat or gular gland, which is only active in adult
males. Males were sometimes noted crawling about their
cages to mark cage surfaces or females with the secre-
tion of their gular glands. Females also sometimes
exhibited intense interest in the gular glands of adult
males and would approach the males on all fours, in an
effort to nuzzle the glands. On occasion, this led the
females to crawl under the males, into the normal dorsum-to-venter mating position, and coitus eventually
took place (Rasweiler, 1987, 1992). Finally, if two adult
breeding males were placed together in a cage, fighting
would often erupt. This too was conducted on all fours.
Thus, for numerous reasons, quadrupedal movement
is very important to these animals, and they are highly
adept at it.
Timing of Embryonic Stages
It has been well-documented elsewhere that M. rufus
does not exhibit a limited period of estrus (Rasweiler,
1987, 1988, 1990b). Instead, most females usually exhibit intermittent brief periods of sperm-positive vaginal
aspirates, as well as one or more longer periods of such
aspirates. Although clear evidence was obtained that
mating activity was not always closely correlated with
ovulation, the possibility that spermatozoa may also
sometimes be stored for prolonged periods in the female
tract of M. rufus remains to be explored.
The time elapsed from the onset of the first prolonged
period of sperm-positive aspirates was usually correlated
MOLOSSID BAT EMBRYONIC DEVELOPMENT
159
distinct and set in relief compared to the rest of the cranium. The oral groove appears as a slit on the anterior
border of the first, or mandibular, arch and separates it
into an elongated distal mandibular component and a
shorter proximal maxillary component (Fig. 5A). In ventral view, the oral groove is visible as a horizontal slit.
The second, or hyoid, arch is asymmetrically shaped,
with a broader proximal and narrower distal component.
The third, or glossopharyngeal, arch is tucked underneath the second arch and protrudes toward the anterior
end of the embryo (Fig. 5A). Also, the otic vesicle is
apparent dorsal to the hyoid arch (Fig. 5A). Just anterior to the mandibular arch the lens vesicle is recognized
as a whitish opaque condensation. The heart protrudes
ventrally from the main body of the embryo and contacts
the future viscerocranium (Fig. 2A). Additionally, the
dorsal convexity of the embryo causes the tip of the curling tail to nearly touch the head (Fig. 2A).
Fig. 1. Gross morphology of adult M. rufus. (A) Frontal view of
head. (B) Lateral view of head. (C) Dorsal view showing extended
wing. (D) Dorsal view showing wings retracted for quadrupedal movement; scale bar 5 1 cm.
with the stage of embryonic development found in pregnant animals (Table 1), although there were some exceptional cases. This general pattern is best appreciated,
however, when data for younger and more advanced conceptuses are also examined (see Rasweiler, 1988, 1990b).
Because embryonic development was not closely correlated with the onset of a prolonged period of sperm-positive vaginal aspirates, it was impossible to determine
the duration of each embryonic stage.
Stages of Embryonic Development
One hundred-thirty-eight M. rufus embryos were generated from captive matings. Sixty-five embryos were
collated into nine developmental stages beginning at
stage 13 and ending with stage 22; we did not have a
specimen for stage 19 (Figs. 2, 3). Between two and six
embryos were observed for stages 13 to 20. For stages
21 and 22, we had 10 and 29 embryos, respectively. The
17 embryos used as reference specimens are listed in Table 1. Seventy-three embryos were earlier or later in development than these numbered stages (Rasweiler, 1988,
1990b). Some embryos of M. rufus may be slightly more
or less mature than those of the reference specimens. In
such cases, it would be appropriate to label such embryos
as ‘‘early’’ or ‘‘late’’ stage embryos (e.g. 13E or 13L).
Stage 13.
The reference specimen for this stage
possesses 33 somite pairs, and late stage 13 embryos are
presumed to have up to 36 somite pairs (see description
for stage 14 below). Additionally, this stage is hallmarked by the presence of both fore- and hindlimb buds
(Figs. 2A–D, 4A, B). The forelimb buds possess an apical
ectodermal ridge (AER), which likely becomes more visible by late stage (Fig. 4A). Three pharyngeal arches are
Stage 14.
An early stage 14 reference specimen
possessed 37 somite pairs. The late stage 14 reference
specimen possessed 36 somite pairs; however, its tail
was broken, and the embryo probably had an actual
total of 40 somite pairs (Fig. 2E–H). Stage 14 M. rufus
embryos therefore possess at least between 37–40 somite
pairs. Stage 14 is also easily identified by the morphology of the forelimb, which has now elongated so as to be
longer than it is wide (Figs. 2E, 4C). Additionally, hindlimb AERs are present at this stage, with the AER
becoming more obvious in late stage (Fig. 4D). The cleft
between the mandibular and hyoid arches has deepened
because of the increased size of the arches. Moreover,
the anterior margin of the mandibular arch almost eclipses the eye (Figs. 2E, 5B). The oral groove, too, is more
conspicuous, being deepened by the swelling of the proximal and distal portions of the mandibular arch. Only
the proximal base of the third pharyngeal arch is visible
posterior to the second arch (Fig. 5B). The nasal pits are
visible as shallow, broad depressions in the future viscerocranium (Fig. 5B). As opposed to stage 13, the oral
groove is not visible in ventral view, because it is
obscured by the mandibular component of the first arch
(Fig. 2F). The asymmetric shape of the hyoid arch has
given way to three apparent segments: proximal, medial,
and distal (Fig. 5B). These segments presage the development of the auditory hillocks. By late stage 14 the
cleft between arches one and two begins to widen (Fig.
5B). The ventral body cavity, in between the fore- and
hindlimbs, has distended so as to give the entire embryo
a cuboidal appearance (Fig. 2E). Moreover, the somite
pairs are difficult to discern in the anterior half of the
embryo because of body wall thickening and somite differentiation (Fig. 2G). Also noticeable is a notch along
the dorsal midline of the body opposite the first arch.
This is the external expression of the isthmus between
the mid- and hindbrain (Fig. 2E). Additionally, the cervical flexure is apparent at this stage (Fig. 2E). The anterior edge of the proximal forelimb displays a region of
thickened tissue (Fig. 4C). From this region the propatagium, or the segment of the wing membrane that extends
from the shoulder to the wrist, will likely develop.
One of the stage 14 embryos (collected from bat
MA66) provides a good example of development not
being well-coordinated with the onset of the first prolonged period of sperm-positive aspirates from the
160
NOLTE ET AL.
Figure 2.
MOLOSSID BAT EMBRYONIC DEVELOPMENT
161
Fig. 3. Stages 18 to 22. Scale bar 5 1 mm in all panels. The first
column (A, E, I, M) shows lateral views with dorsal to the left, the second column (B, F, J, N) shows ventral views, the third column (C, G,
K, O) shows dorsal views of head and trunk, and the fourth column
(D, H, L, P) shows dorsal views of the trunk and tail. A–D: Stage 18
specimen. E–H: Stage 20 specimen. I–L: Stage 21 specimen. M–P:
Stage 22 specimen. at, antitragus; chp, chiropatagium; cl, claw; el,
eyelid; mc, metacarpal; np, nasal pit; pi, pinna; pl, phalanx; plp, plagiopatagium; pop, posterior oriented phalanx; tb, thumb; urp, uropatagium; vb, developing vibrissa.
Fig. 2. Stages 13 to 17. Scale bar 5 1 mm in all panels. The first
column (A, E, I, M, Q) shows lateral views with dorsal to the left, the
second column (B, F, J, N, R) shows ventral views, the third column
(C, G, K, O, S) shows dorsal views of head and trunk, and the fourth
column (D, H, L, P, T) shows dorsal views of the trunk and tail. A-D:
Stage 13 specimen. E-H: Stage 14 specimen. I-L: Stage 15 specimen.
M–P: Stage 16 specimen. Q–T: Stage 17 specimen. at, antitragus; ah,
auditory hillock; crh, crus helix; chp, chiropatagium; cvf, cervical flexure; crf, cranial flexure; dc, digit condensation; fp, foot plate; ga, glossopharyngeal arch; h, heart; ha, hyoid arch; hp, hand plate; i, isthmus;
ma, mandibular arch; md, mandible; mt, auditory meatus; mx, maxilla;
np, nasal pit; og, oral groove; pi, pinna; pig, pigment; plp, plagiopatagium; pop, posterior oriented phalanx; prp, propatagium; st, snout; tg,
tragus; urp, uropatagium; vb, developing vibrissa.
162
NOLTE ET AL.
Fig. 4. Limb morphology. (A) Forelimb at stage 13. (B) Hindlimb at
stage 13. (C) Forelimb at stage 14. (D) Hindlimb at stage 14. (E) Forelimb at stage 15. (F) Hindlimb at stage 15. (G) Forelimb at stage 16. (H)
Hindlimb at stage 16. (I) Forelimb at stage 17. (J) Hindlimb at stage 17.
(K) Forelimb at stage 18. (L) Hindlimb at stage 18. (M) Forelimb at stage
18 Late (L). (N) Hindlimb at stage 18L. (O) Forelimb at stage 20. (P) Hindlimb at stage 20. (Q) Forelimb at stage 21. (R) Hindlimb at stage 21. (S)
Forelimb at stage 22. (T) Hindlimb at stage 22. aer, apical ectodermal
ridge; ca, calcar; chp, chiropatagium; cl, claw; dc, digit condensation;
fp, foot plate; hp, hand plate; mc, metacarpal; plp, plagiopatagium; prp,
propatagium; pop, posterior oriented phalanx; urp, uropatagium. All panels show dorsal surface of the right limb (except for panels B, I, J, L, N,
and T) with anterior toward the top and proximal at left (except for panels B, I, J, L, N, and T); views are not to scale.
mother. In this case, the mother had been killed 10 days
after the onset of this period (Table 1); however, spermpositive aspirates had also been obtained from this female
13 and 14 days prior to that. It seems most likely that conception had actually occurred in association with the earlier period of breeding activity (see Rasweiler 1988, 1990b
for the complete breeding record for this animal).
limb, the plagiopatagium primordium has developed as
a rounded bulge of tissue on the posterior side of the
proximal forelimb (Fig. 4E). The plagiopatagium is the
wing membrane section that extends from the fifth forelimb digit to the ankle. The premaxillary centers, which
are bounded by the maxillary component of the first
arch and the facial midline, are discernible as bulges
that are larger than the maxillary components (Figs. 2I,
J, 5C). The mandibular components of the first pharyngeal arch pair have fused at the midline, forming the
precursor of the lower jaw. Additionally, roundish tissue
swellings punctuate the posterior-lateral margin of the
mandibular component of the first arch; these are
aligned with tissue swellings along the anterior-lateral
margin of the second arch so that the cleft between the
two arches appears discontinuous and deeper (Figs. 2I,
5C). The external auditory canal (or meatus) will even-
Stage 15.
The most obvious external changes at
this stage involve alterations in limb geometry and pharyngeal arch modifications (Figs. 2I, J, 4E, F, 5C). The
fore- and hindlimb autopods (distal regions of each limb)
are paddle shaped, indicating that the hand plate has
developed (Fig. 4E, F). The posterior arc of the forelimb
handplate is slightly larger than the anterior arc,
reflecting anterior-posterior (A-P) asymmetries in autopod development (Fig. 4E). Between the fore- and hind-
MOLOSSID BAT EMBRYONIC DEVELOPMENT
163
Fig. 5. Craniofacial development. Panels A–C show lateral views of
developing head with anterior to the right. Panels D–I show face-on
views with anterior to the top. (A) Stage 13. (B) Stage 14. (C) Stage
15. (D) Stage 16. (E) Stage 17. (F) Stage 18. (G) Stage 20. (H) Stage
21. (I) Stage 22. ah, auditory hillock; at, antitragus; crh, crus helix; el,
eyelid; ga, glossopharyngeal arch; ha, hyoid arch; ma, maxilla; md,
mandible; np, nasal pit; og, oral groove; ov, otic vesicle; pi, pinna; tg,
tragus; vb, developing vibrissa. Views are not to scale.
tually form from the deepened and widened cleft, and
the conspicuous mounds of tissue lining this depression
are the auditory hillocks. The increased bulkiness of this
stage is most apparent when viewed from the back,
where one can see that the greatest width of the embryo
occurs between the forelimb buds (Fig. 2K, L). Lateral
body wall thickening all but entirely obscures any view
of the somites. The notch demarcating the midbrainhindbrain boundary is still present, and the cranial flexure is still evident (Fig. 2I); thus the overall contours of
the embryo remain the same as stage 14.
the pharyngeal arches, whereas the posterior margins of
the ears developed from the arches’ distal margins.
Additionally, at this stage the external ear is restricted
to the side of the head. Near a pinna’s posterior base
one can see the bulbous antitragus (Figs. 2N, 5D). Along
and near the ventral face of a pinna’s anterior base two
noticeable tissue aggregations developed from the auditory hillocks. We call the ventral-most of these two tissue aggregations the tragus (Fig. 2N). We call the second tissue aggregation, which is dorsal to the tragus
and adjoins both the side of the head and the anterior
border of the pinna, the crus helix; this structure
becomes more prominent in later stages. The maxillary
components of the first pharyngeal arch pair and the
premaxillary centers are now fused at the midline. The
mandibular process does not protrude ventrally as much
as the maxilla; and thus, the embryo possesses an
‘‘overbite.’’ The retina is obviously pigmented (Fig. 5D).
In profile view there is a noticeable, though not salient,
indentation along the profile of the head so as to suggest
to the observer a forehead above the eyes and the begin-
Stage 16. The auditory hillocks have fused to form
the pinna of each ear, as well as other external ear
structures (Figs. 2M–O, 5D). The external ears have
changed their orientation relative to other facial features. It is now more instructive to talk of the ears as
being aligned with the embryo’s A-P axis rather than
the proximal-distal (P-D) axis associated with the pharyngeal arches of earlier stages. Thus, the anterior margins of the ears developed from the proximal margins of
164
NOLTE ET AL.
nings of a snout (Fig. 2M). Five opaque ectodermal condensations are readily visible on either side of the facial
midline – these are vibrissae precursors (Figs. 2M, N,
5D). Two condensations appear above the eye, two develop medial to the eye, and one is conspicuous midway
between the nasal pits and the eye. These are presumably developing tactile vibrissae as observed on Rhinolophus ferrumequinum (Schneider, 1963). Additionally,
when viewed laterally three more condensations are visible – two just ventral to the antitragus, and one midway
along the maxilla. Although the cervical flexure is still
apparent, it is not as pronounced as in earlier stages.
The notch marking the isthmus between the mid and
hindbrain is reduced or absent. Both the foot and hand
plate exhibit opaque digit condensations (Figs. 2M, N,
4G, H). In the forelimb, future digit III is longer than all
other digits and the first and fifth digit condensations
extend in the anterior and posterior direction, respectively. Digits I and V on the forelimb autopods extend
beyond the interdigit tissue; therefore each hand plate is
spade-shaped (Fig. 4G). The distal margin of the foot
plate appears corrugated. This may be the result of
interdigit tissue recession; alternatively, the digit condensations may have grown beyond the interdigit tissue
(Fig. 4H). Because of growth along their A-P axes the
hand and foot plates contact each other along their posterior and anterior edges, respectively (Fig. 2N). The
uropatagium, which will become the segment of the
wing membrane that connects the ankle to the tail, is
evident as a slight lip of tissue connecting the bases of
the hindlimbs to the ventral base of the tail (Fig. 2N, P).
The tip of the tail is free of uropatagium. The plagiopatagium is also more distinct as a continuous layer of
skin extending from the forelimb wrist to the anterior
border of the proximal hindlimb (Fig. 2M).
Stage 17. Maturation of facial and limb structures
hallmark this stage. The rounded snout is now a prominent facial feature, and the overbite created by the
maxilla is exaggerated by a downward turn (Figs. 2Q,
R, 5E). Also, due to the rising of the head relative to
the rest of the body, the cervical flexure is absent and
the chin no longer rests on the chest (Fig. 2Q). The bulbous regions of the cranial vault are reduced so that
above the eyes the head appears rounder; the trend toward a rounder cranium continues for the rest of development (Fig. 5E). This trend is highlighted by the observation that the anterior pinnae bases are closer to
the apex of the head relative to stage 16. The antitragus is approximately equal in size to the eye, while the
tragus is now prominent and cone-shaped (Fig. 5E).
The crus helix, which is anterior and slightly dorsal to
the tragus, is larger and more elongated than the
tragus. Between stages 17 and 20 this structure fuses
with the ventral surface of the pinna (Figs. 2R, 5E).
The anterior bases of the pinnae are no longer confined
to the sides of the head, but are nearer to the midline
(Fig. 5E). The primordial eyelids are conspicuous as
thin margins of thickened tissue around each eye (Fig.
5E). The maturing bones of the forearm are evident, as
they protrude out of the membranous patagia (Fig. 2Q,
R). Particularly obvious is the flexure at the elbow, and
less evident is the flexure at the wrist (Figs. 2Q, R, 4I).
The distal margins of the autopods now meet at the
midline (Fig. 2R). Fore- and hindlimb digits are clearly
distinct from the interdigital tissue, and the interdigital
tissue of the hindlimb is clearly receding (Fig. 4I, J).
Also, the interdigit tissue is noticeably reduced between
the first and second forelimb digits, though still present. The distal tip of digit IV on each autopod conspicuously bends toward posterior, foreshadowing the final
orientation of this digit’s first phalanx in the adult
(Figs. 2R, 4I). The orientation of this digit (as well as
Digit III, described in stage 20) may aid in this species’
ability for rapid flight and quadrupedal locomotion, as
has been suggested for other bat species (Vaughan,
1966; Norberg and Rayner, 1987). The head no longer
appears disproportionately larger than the rest of the
body; this results in an overall appearance that is
developmentally more advanced, but less bulky than
stage 16 embryos (Fig. 2Q, R). Additionally, when
viewed from the back, the trunk width between the
shoulders is not markedly greater than the width of
the posterior half of the trunk (Fig. 2S, T).
Stage 18. The thumb conspicuously extends toward
the head, and depending on the specimen, may sit inside
the open mouth or juxtaposed against the neck (Figs.
3A, 4M). The tissue between the thumb and digit II has
completely receded. The forelimb digits are longer, with
the distal tips of digit III from each arm meeting or
crossing at the midline (Fig. 3B). Often, the overlying
forelimb will also cover the distal extremities of the
hindlimbs (Figs. 3B, 4K). The degree to which the forelimbs overlap increases from early to late stage 18 (Fig.
3B). The propatagium follows the edges of the stylopod
and zeugopod very closely, such that near the wrist and
the proximal base of the stylopod it is difficult to discern
(Figs. 3A, 4M). Although the first and fifth forelimb digits extend away from the center of the autopod, digits IIIV of the forelimb are juxtaposed (Figs. 3B, 4K, M). The
hindlimb digits are free of interdigit tissue (Fig. 4L, N).
In late stage 18 specimens, thumb claw primordia are
just perceptible as slight constrictions of tissue at the
distal tips of the digits (Fig. 4M). The knee-joints are
clearly articulated and as perceptible as the elbow flexure (Figs. 3B, D, 4L, N). The entire distal margin of the
pinna curves in toward the side of the head, such that
the ear ‘‘hugs’’ the head (Figs. 3C, 5F). The antitragus is
more lobate in appearance, looking like a miniature limb
bud, and is approximately equal in size to the eye. It
stands erect and is situated nearly flush against the side
of the head (Fig. 5F). The tragus appears triangular. At
late stage 18, the anterior bases of the pinnae extend toward the facial midline at least as far as the medial border of the eye (Fig. 5F). The vibrissae condensations are
more evident as white, slightly raised dots on the face
(Fig. 5F). The eyelids begin to grow over the eye (Figs.
3A, 5F). Additionally, the overbite observed in earlier
stages is not as prominent because the mandible has
grown out relative to the maxilla (Fig. 3A). Along the interior edges of the proximal region of the mandible one
can see swollen bulges of tissue flanking the tongue.
These tissue condensations may be the precursors of
the teeth. The nasal pits face forward, and the nasal
process is clearly set in relief to the rest of the snout
(Fig. 5F). In late stage 18 embryos, the indented contour that in profile view distinguished the snout and
the forehead is becoming less evident (Fig. 3A). As in
earlier stages, the snout is rounded (Fig. 3A). The uro-
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MOLOSSID BAT EMBRYONIC DEVELOPMENT
patagium that flanks the tail is straight, meeting the
base of the tail at a 90-degree angle. Moreover, the tail
appears rounder at its base and more conical in overall
appearance (Figs. 3B, 4N).
Stage 19. No reference standard specimens were
collected for this stage.
Stage 20. The pinnae have unfurled so that their
ventral surfaces are easily visible again, as in stage 16
and 17 (Figs. 3G, 5G). Moreover, the antitragus now
extends laterally away from the side of the head. Thus
the antitragus appears as a flat, table-like structure, oriented in the same plane as the mouth (Fig. 5G). The anterior pinnae bases have extended closer to the facial
midline (Figs. 3F, 5G). The tragus has reduced in size to
a bead-like nodule. Now of more prominence than the
tragus is the crus helix (Fig. 5G). Because of the unfurling of the pinnae, their proximity to the midline, and
the increasing roundness of the apical cranium, the
upper two thirds of the head look like one distinct anatomical structure – a helmet, or mushroom cap (Fig.
5G). The eyes are closed. The mouth is slightly ajar, and
the tongue is visible (Fig. 5G). Vibrissae condensations
are macroscopically visible. There is no longer any indentation between the forehead and the snout in profile
view. This welding of the snout and the forehead results
in a head that appears both more robust and teardropshaped (Figs. 3E, 5G). One or both forelimb autopods either cover the face or lie tucked underneath the chin
(Figs. 3E, F, 4O, 5G). Depending on the specimen, the
hindlimb digits may be entirely or almost entirely hidden by the overlying forelimb autopods (Fig. 3F). Welldefined claws are clearly visible on the thumbs (Fig.
4O). Hindlimb claws are also pointed and readily distinguishable (Fig. 4P). The chiropatagium is easily discernable between digits IV and V, but is not easily discernable between digits II, III and IV (Figs. 3E, 4O). The
first phalanx of digit III, like digit IV in stage 17, is now
conspicuously oriented toward the embryo’s posterior
pole (Fig. 3F, 4O). The posterior-facing tips of digits III
and IV, coupled with the approximately equal length of
the autopod and zeugopod, permit the autopod to fold
back onto the zeugopod. It has been suggested that this
compact configuration contributes to the molossids’
marked ability for terrestrial locomotion (Vaughan,
1966). Flexure in the knee, ankle, wrist, and elbow
joints is readily discernible as the patagia become thinner and increasingly membranous (Figs. 3E, F, H, 4O).
The uropatagium is folded under the ventral base of the
tail and thus no longer meets the base of the tail at a
90-degree angle (Fig. 4P).
Stage 21. The forelimbs, particularly the autopods,
are in new positions relative to the head. The wrists
have moved slightly ventral and are either positioned
alongside the open mouth or underneath the chin (Figs.
3I, J, 4Q). The autopods do not fully extend across the
midline, but instead are approximately aligned with the
embryo’s A-P axis, with the digits pointing toward the
posterior pole (Figs. 3J, 4Q). The autopod is partially or
fully folded back onto the zeugopod and the chiropatagium is folded in between the digits (Fig. 3I, 4Q), presaging the functional orientation of these limb elements
during quadrupedal terrestrial locomotion. The
hindlimbs are crossed (Figs. 3I, J, L, 4R). The thumb
and hindlimb claws appear keratinized, and pigment is
visible on their dorsal side (Fig. 4R). The ventral faces of
the pinnae bubble out, as if they had been inflated; this
is especially evident near their distal rim (Fig. 5H).
Thus the crus helix observed in the last stage is temporarily conflated with the bubbling underside of the
pinna. It is not clear whether this observation is an artifact of the fixation process or a real developmental
event. The eyes are still closed. The tongue is readily
visible in the open mouth (Fig. 5H). The regions of swollen tissue that flanked the tongue in earlier stages have
developed into a ridge of tooth primordia (Fig. 5H). The
tip of the snout is now more angular than round (Fig.
3I). The calcar, though partially obscured by the crossed
hindlimbs and creased uropatagium, is visible for the
first time extending away from the ankle into the uropatagium toward the base of the tail (Fig. 4R).
Stage 22.
Teeth have developed within the open
mouth (Fig. 5I). The upper and lower jaws are mature
and broad, appearing as wide as a line drawn from the
lateral-most corner of one eye to the other. In this
respect the snout appears as it does in the adult (Figs.
1A, B, 5I). The eyes have begun to open again (Figs. 3N,
5I). The nasal processes protrude away from the tip of
the snout, making the nose appear as a discrete facial
structure (Fig. 5I). The antitragii now stand erect, with
their distal tips projecting into the external ear cavity.
Additionally, the antitragii bases are broader and flatter
than in earlier stages and therefore appear egg-shaped,
as they will be in the adult (Figs. 1A, B, 3N, 5I). The
bubbling of the ventral face of the pinna observed in the
previous stage is now mostly absent except near the pinna’s distal rim. Therefore, the crus helix is again readily
discernible (Fig. 5I). The wrists are either juxtaposed
against the chin, or lying alongside the neck (Figs. 3M,
N, 4S). The thumb is roughly as long as the snout, and if
the forelimb is apposed to the neck the thumb may extend
dorsally around it – like a collar (Fig. 3M, O, P). The
hindlimbs are uncrossed and, like the tail, they extend
toward anterior where they contact the chin (Figs. 3M, N,
4T). In this compact configuration, which makes the entire
embryo appear spherical, the keel-like calcar is often fully
visible (Figs. 3M, N, 4T). In summary, the embryo looks
much like an adult except that it lacks fur and the pinnae
do not yet stand erect (Figs. 1A, B, 3M, N, 5I).
DISCUSSION
The external morphology described here for M. rufus
is the first embryological staging series produced for any
species of molossid bat, and is based in part on the staging system developed for Carollia perspicillata (Phyllostomidae) (Cretekos et al., 2005). Perhaps not surprisingly, the embryonic staging system for M. rufus bears
many similarities to other bat species (Tokita, 2006;
Giannini et al., 2006) that were derived from the C. perspicillata staging system. In relation to other bat species
for which staging systems exists, M. rufus embryos display a number of species-specific characters. Several of
these unique anatomical characters, identifiable during
development, have known or suspected roles in the natural history of the adult bats.
The shape and orientation of the wings of M. rufus
embryos are noticeably different from those of some
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NOLTE ET AL.
other, reasonably well-studied bat species by stage 18;
augmentation of these differences continues into later
stages. Whereas digits II-IV remain in close proximity in
stage 18 M. rufus forelimbs, the entire autopod, with
adjoining chiropatagium, is extended in C. perspicillata
and Pipistrellus abramus (Cretekos et al., 2005; Tokita,
2006). The pteropodid, Rousettus amplexicaudatus, also
passes through a stage (Stage 22) wherein the digits
and chiropatagium are outstretched (Giannini et al.,
2006). The extended autopod in the latter species reveals
their relatively rounded and broad forelimbs. This is in
contrast to the pointed, narrow shape of the M. rufus
autopod – a distinctive feature of M. rufus adults. During M. rufus development, the chiropatagium between
digits II, III and IV never seems to accumulate between
stages 16–22 as it does in the aforementioned species.
Interestingly, stage 20 of P. abramus is noted for the
folding of accumulated dactilopatagium between the digits (Tokita, 2006). Although M. rufus wings appear corrugated in later stages, this is not the case between digits II and III, suggesting that reduction of dactilopatagium development between these two digits is important
for adult bat wing shape. Additionally, the dactilopatagium between digits IV and V never develops to the
same distal extent as that observed in C. perspicillata or
P. abramus. This provides M. rufus forelimbs with a
characteristic notch between digits IV and V and a
noticeably narrower wing along the A-P axis. The long,
narrow wings of M. rufus observed during embryological
development presumably affect the flight and feeding
behaviors of the adults. As was suggested for other
molossid bats, the narrow, pointed wings of M. rufus
may aid in its pursuit of certain types of flying insects.
This may also affect foraging site preferences toward
unobstructed locales (Vaughan, 1966; Freeman, 1981a).
During stage 17 digit IV is posteriorly oriented. The
same is true for digit III at stage 20. This orientation is
unique to M. rufus when compared to the forelimb development for other staged species (Lawrence, 1991; Cretekos et al., 2005; Tokita, 2006; Giannini et al., 2006). The
orientation of the distal phalanges of digits III and IV
are believed to be an adaptation that, in concert with
other anatomical peculiarities, provide M. rufus adults,
and other bats within the Molossidae, with the marked
ability of quadrupedal terrestrial locomotion (Vaughan,
1966; Schutt and Simmons, 2006). Notably, Cheiromeles
have evolved plagiopatagium-derived pouches into which
the distal phalanges of the forelimb can be inserted during quadrupedal locomotion (Schutt and Simmons,
2006). Although no such pouches exist for M. rufus, it is
apparent from the doubled-over forelimbs of stages 21
and 22 that the orientation of digits III and IV may
effectively wrap around the elbow. In the adult, such a
configuration would prevent the tip of the wing from
being fully exposed during quadrupedal movement. In
this orientation the chiropatagium is snuggly tucked into
the autopod, and the plagiopatagium is partially collapsed
onto the zeugopod, presaging the orientation of these
limb elements during adult quadrupedal locomotion.
The pinnae (ears) of M. rufus, like the wings, become
increasingly indicative of this species in later stages of
development. At stage 16, for all species staged similarly
to C. perspicillata, the pinnae begin to develop along the
side of the head. As early as stage 17 in M. rufus
embryos, however, the anterior borders of the pinnae
have migrated nearer to the facial midline. This extension of the anterior pinnae borders to the midline continues throughout development, so that the ears are as
much a feature of the forehead as they are of the side of
the head. The conspicuous positioning of the pinnae is
complemented by the development of what we have
identified as the crus helix – the keel-like structure that
punctuates the anterior margin of a pinna’s ventral face.
In the adult this structure appears at least partly responsible for restricting the erect pinna to the side of
the head, just above the eye (see Fig. 1A, B). The development of the crus helix was not noted in the staging series reported for the vespertilionids or pteropodids,
although it is likely present in some form, as the crus
helix is a recognized structure just above the tragii in
numerous mammalian pinnae. The prominent and apparently rigid crus helix in M. rufus likely evolved to support the unique position of the pinnae along the front
and side of the head. The location and timing of the
developing crus helix suggests that its unique features –
rigidity and prominence in relation to the tragus and
antitragus – originate from a derived proliferative region
of the auditory hillocks, which already proliferate to give
rise to the external ear structures. The unique positioning
of M. rufus’ pinnae along the forehead is not without
postulated ecological importance. Vaughan (1966) suggested that the morphology of some molossid bat ears
might aid in the bats’ ability for rapid, sustained flight,
because the broad ears are drawn in toward the head and
are thus parallel to the airstream during flight. It is worth
noting that P. abramus is also an insectivorous bat and
yet, both during development and in the adults of this species, the pinnae do not migrate to the midline (Tokita,
2006). Thus, although M. rufus’ unique pinnae may aid it
during rapid flight, P. abramus apparently required no
modification of its pinnae to catch prey. Therefore, shape
differences in the pinnae are probably also driven by the
foraging and navigational needs of each species, which
rely heavily upon echolocation (Dumont, 2006).
Previous authors noted that within the family Molossidae, jaw thickness, temporal muscle size, and the size
and number of teeth correlate with and may predict well
the feeding behavior of particular species (Freeman,
1979). M. rufus crania, for example, with their thick
jaws and few, but large teeth correlated well with this
bat’s reported habit of preying upon hard-shelled insects,
such as beetles (Freeman, 1979 and references therein,
1981a,b; Fenton et al., 1998). The broad, deep jaws of M.
rufus are readily apparent by stage 20 (and less so at earlier stages). The craniofacial structure of C. perspicillata,
a frugivore, at comparable stages does not exhibit either
the broadness of the snout or the welding of the snout to
the forehead that gives M. rufus embryo crania their
robust, tear-drop shapes (cf., Cretekos et al., 2005).
Instead, at stage 20, C. perspicillata snouts are barrelshaped and do not extend laterally much beyond the
medial border of the eyes when viewed ventrally. Although
P. abramus is also an insectivore, its snout is largely
obscured during stages 18–24, and little mention was
made of its fetal snout morphology (Tokita, 2006).
The broad snout of M. rufus also helps to create a relatively large oral cavity that may be used to hold accumulations of insects. Goodwin and Greenhall (1961) reported
that these bats have large ‘‘cheek pouches,’’ which become
stuffed to capacity during foraging. The animals then
MOLOSSID BAT EMBRYONIC DEVELOPMENT
supposedly return to their roosts to chew and swallow
their food. Although Fenton et al. (1998) found some
insect remains in the mouths of M. rufus returning to
their roosts after foraging, no evidence of insects in cheek
pouches was observed. Also, Murray and Strickler (1975)
failed to find functional cheek pouches in two dissected
M. rufus. In captivity, however, these bats have often
been observed to greatly engorge their mouths with mealworms when being hand-fed and to masticate large
masses of worms for extended periods before completing
their ingestion (Rasweiler, unpubl. observ.). The ability to
temporarily accumulate at least modest amounts of food
in their mouths could be of considerable adaptive significance to M. rufus. It may enable the bats to quickly catch
large numbers of flying insects during daily periods of
peak abundance and to sufficiently chew the insects prior
to swallowing. By increasing hunting efficiency, it may
also reduce the amount of time that foraging bats must
be outside of their roosts and at risk of predation. Studies
by Fenton et al. (1998) indicate that M. rufus can forage
very efficiently. Presumably any accumulation of food in
the bats’ mouths must be balanced against their need to
use oral echolocation calls for hunting and navigational
purposes. M. rufus and a number of other molossids are
generally considered to be oral, rather than nasal, signal
emitters (Pedersen, 1993, pers. comm.; Goudy-Trainer
and Freeman, 2002).
This study is important because it provides the first
detailed embryonic staging system for a representative
of the Molossidae, one of the largest and most successful
chiropteran families. This family contains 100 species
(Simmons, 2005; Wilson and Reeder, 2005). Some of
these are extraordinarily abundant in the wild and
therefore of great ecological importance, particularly as
predators of insects. For example, summer colonies of
Mexican free-tailed bats (Tadarida brasiliensis) occupying some caves in the southwestern United States may
be the largest aggregations of mammals in the world.
These may have been even much larger in the recent
past; however, there is uncertainty about the accuracy of
historical population estimates (Betke et al., 2008). Molossid bats also differ significantly in habits, form and function from many other commonly studied bats. Prior to this
study, however, embryonic staging systems had been
devised only for representatives of three other families
(the Phyllostomidae, Pteropodidae and Vespertilionidae) of
this very diverse mammalian order. This study is also
noteworthy because the embryos were obtained from
females bred under controlled conditions in captivity.
Having a system available for precisely staging
embryos harvested either from captive-bred females or
from a reproductively-synchronized wild population (e.g.
that existing on the island of Trinidad) (Rasweiler, 1988;
Badwaik et al., 1998) opens the door to much more probing studies of significant reproductive, developmental and
evolutionary problems. It provides a means of more accurately characterizing synchronized or seasonal breeding
patterns in the wild. For example, observations made on
embryonic development in C. perspicillata bred under
controlled conditions in captivity played a major role in
the discovery of a period of developmental delay in the
wild population (Rasweiler and Badwaik, 1997).
Finally, the availability of an embryonic staging system can be of great value for studies of evolutionary developmental biology. Staging series can provide a morphological basis for functional characterization of DNA
167
sequences that control development (Cretekos et al.,
2001; Baguna and Garcia-Fernandez, 2003). Gene
expression patterns obtained throughout the development of similar structures at similar points of developmental maturity in two different species can be used as
a starting point for functional genomic studies (Cretekos
et al., 2001; Chen et al., 2005; Sears et al., 2006; Weatherbee et al., 2006; Cretekos et al., 2007). Recently, comparative mammalian embryology emboldened the decision to test the functional relevance of a putatively important genetic sequence during limb development by
replacing this sequence in one species, the mouse, Mus
musculus, with the orthologous sequence from the bat,
C. perspicillata (Cretekos et al., 2008).
ACKNOWLEDGMENTS
The authors thank the Department of Zoology, University of the West Indies, Trinidad, and particularly Mr. P.
Deoraj and Professor J.S. Kenny for generous assistance
with the field aspect of these studies. The authors are
also grateful to the Wildlife Section, Forestry Division,
Ministry of Agriculture, Land and Marine Resources of
the Republic of Trinidad and Tobago for providing collecting and export permits.
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