Electron microscopic studies of mummified tissues in amber fossils

AMERICAN MUSEUM
Novitates

PUBLISHED BY THE AMERICAN MUSEUM OF NATURAL HISTORY
CENTRAL PARK WEST AT 79TH STREET, NEW YORK, N.Y. 10024

Number 3097, 31 pp., 84 figures May 19, 1994

Electron Microscopic Studies of
Mummified Tissues in Amber Fossils

DAVID GRIMALDI,! ELIZABETH BONWICH,?
MICHAEL DELANNOY,? AND STEPHEN DOBERSTEIN?*

ABSTRACT

The degree and consistency of fine and ultra-
structural preservation of primarily soft tissue re-
mains preserved in amber fossils were examined,
using scanning (SEM) and transmission electron
microscopy (TEM). 16 insects of various taxa and
structure were studied with the SEM, as well as 4
plant specimens, which were from two chemically
distinct ambers: 25—30 million year old (myo) am-
ber from the Dominican Republic, and ca. 40 myo
amber from the Baltic region. A new technique
was used for “‘exhuming” a specimen in order to
examine the in-situ preservation of whole organs
and tissues. Remarkable preservation found in both
ambers confirmed earlier reports based on a few
specimens. Preservation of all soft tissues in Do-
minican amber insects appears to be more con-
sistent than in Baltic amber insects, several of which
were virtually hollow “‘casts.””» Membranous struc-

tures preserved in the insects include air sacs, un-
collapsed tracheae, and various portions of the gut,
as well as the brain and bundles of muscle fibers
in their original origins and insertions. Very little
shrinkage was observed. Specialized pockets, the
mycangia, in wood-boring beetles (Platypodidae)
still possessed spores and conidiophores of their
symbiotic fungus. For plants, columnar cells of leaf
mesophyll were found in their original positions
and sizes, pollen grains retained the exine sculp-
turing, and mats of epidermal cells were preserved
in anthers.

At the ultrastructural level, both flight muscle
and brain tissues show substantial degradation of
cytoplasmic components due to dehydration. Al-
though sarcomeres are easily identifiable in native
muscle samples, the sarcomeric repeats disappear
upon hydration, indicating that the repeated struc-

' Associate Curator, Department of Entomology, American Museum of Natural History.

? Student, Department of Biological Sciences, Barnard College of Columbia University, New York, NY.

> Department of Cell Biology and Anatomy, Johns Hopkins School of Medicine, Baltimore, MD 21224.

*4 Johns Hopkins School of Medicine; presently: Postdoctoral Fellow, Department of Molecular and Cell Biology,
519 LSA, University of California, Berkeley, CA 94720.

Copyright © American Museum of Natural History 1994 ISSN 0003-0082 / Price $3.20

2 AMERICAN MUSEUM NOVITATES

tures are probably composed of inorganic salts de-
posited on thick filaments during dehydration.
Membranous structures are generally better pre-
served than proteinaceous ones. Flight muscle mi-
tochondria are particularly well preserved with
tracheoles and T tubules also identifiable. Axon
tracts in the central nervous system can be distin-
guished from cytoplasmic regions, and parallel

NO. 3097

strips of membranes surrounding cytoplasm are
abundant in rehydrated brain samples.

Mode of tissue preservation appears to be a rap-
id and thorough fixation and dehydration, sufh-
cient for preserving DNA in amber more consis-
tently than any other kind of fossil. Pollen was not
found viable, but the possibility remains that vi-
able bacterial and fungal spores occur in amber.

INTRODUCTION

In general reviews of taphonomy, reference
is often made to Conservat Lagerstatten, or
fossil deposits of exceptional preservation
(Allison and Briggs, 1991; Briggs, 1991).
These include, for example, the Eocene oil
shale of Messel, Germany; the fine-grained
Lower Cretaceous limestone of Ceara, Brazil
(Grimaldi, 1990; Maisey, 1991; Martill,
1988); and the intricately preserved carbo-
naceous flowers from the Turonian (mid-
Cretaceous) of New Jersey (Crepet et al.,
1992). For deposits like the shale and lime-
stone, impressions of soft tissues may remain
(“extraordinary”’ preservation, sensu Briggs
[1991]), but these are only low molecular
weight residues and carbonized films. The
flowers from the Raritan-Magothy Forma-
tion of New Jersey are actually charcoalified:
turned to pure carbon after forest fires swept
over them while buried in leaf litter. A sed-
imentary deposit with unique preservation
are the arthropods from the middle Devo-
nian shale of Gilboa, New York (ca. 378 mil-
lion years old): sockets of setae, sensilla, and
other microscopic details are preserved, ap-
parently as the original cuticle (Shear et al.,
1984; Schawaller et al., 1991).

Three rarely-discussed modes of preser-
vation retain features of organisms more life-
like than any other kinds of fossils: freezing,
dehydration, and preservation in amber. All
apparently prevent hydrolysis of complex
molecules, by either suspending water as a
solid or removing it. Very often frozen or-
ganisms, such as the carcasses of mammals
in cold deserts, are partially dehydrated, due
to the sublimation of ice crystals. Frozen and
dehydrated organisms rarely extend past the
Holocene or Pleistocene; thus, their paleon-
tological value is limited. Yet, no preserva-
tion is more celebrated than the highly ritu-
alized burials of the ancient Egyptians,

particularly those from the 21st dynasty (ca.
1050 sBc)(David and Tapp, 1984), although
exceptional mummification occurs in other
ancient cultures around the world (Cockburn
and Cockburn, 1980; Hansen et al., 1991).
Egyptian cadavers were first eviscerated, then
washed in wine, and the thoracic cavity stuffed
with incense, cassia and other spices, and
crushed myrrh (a fragrant, resinous gum from
Commiphora [Burseraceae]). The cadavers
were also stored for about 70 days in dry
natron (hydrated sodium carbonate, Na,CO,-
10H,0, a natural deposit of salt lakes). Linen
wrappings around the body were likewise im-
pregnated with resin, or with pulverized am-
ber. The success of Egyptian mummification
was revealed by histological (Sandison, 1955;
Zimmerman, 1973) and ultrastructural stud-
ies (Curry et al., 1979; Lewin, 1967; Riddle,
1980). Cells, albeit shrunken, contained nu-
clei and nuclear membranes, although mi-
tochondria were not readily observable (Lew-
in, 1967). DNA, too, has been extracted and
sequenced from Egyptian mummies (Paabo,
1985).

How the Egyptians began the use of amber
for mummification is intriguing, especially
since amber does not occur in Egypt. Amber
is the highly polymerized, fossil form of tree
resins; hundreds of deposits occur around the
world, varying in age, botanical origin (Lan-
genheim, 1969), and the kind and quality of
small organismal inclusions in it. Very an-
cient amber (ca. 125 million years old) occurs
in Lebanon, Jordan, and Israel (Schlee and
Dietrich, 1970; Bandel and Vavra, 1981; Nis-
senbaum, 1975), but the early Egyptians
probably traded for it with the Phoenicians,
who may have acquired it from more exten-
sive deposits in Sicily. Amber and resin were
certainly not as important as preservative
agents of the mummies as was the natron,

1994

but their use was perhaps inspired by the re-
markable external preservation of small or-
ganisms, like insects, that the Egyptians prob-
ably saw in the amber. They certainly were
unaware of the great antiquity of amber, and
of the preserved details of internal tissues.
Here, we report the preservation of soft, in-
ternal insect and plant tissues fossilized in
lower Tertiary ambers, with unexpectedly
consistent, lifelike fidelity: a process of nat-
ural mummification, with finer preservation
than is found in human mummies, and per-
haps the ultimate in Conservat Lagerstatten.

ACKNOWLEDGMENTS

It is a pleasure to acknowledge the help of
several people who were instrumental in
bringing this study to fruition. At the AMNH,
Peling Melville provided a great deal of time
and expertise to the scanning electron micro-
graphs; and Jacquie Beckett produced prints
from the SEM negatives. Jake Brodzinsky was
a very helpful source of study specimens. We
thank Drs. Rob DeSalle (AMNH), George
Eickwort (Cornell University), and Jean Lan-
genheim (University of California, Santa
Cruz) for their critical review of the manu-
script; and Drs. Thomas D. Pollard and Da-
vid B. Weishample of the Johns Hopkins
School of Medicine, who provided support
and stimulating discussions.

Costs for the work at the AMNH were
payed for by an NSF grant to Melanie Stiass-
ny, which sponsored Elizabeth Bonwich in
the REU program in Comparative and Evo-
lutionary Biology. The personal generosity of
Dr. Herbert Axelrod is gratefully appreciated,
which allowed for the purchase of the spec-
imens used here and numerous other rare
ones which will never, ever be marred.

ABBREVIATIONS

an antenna
as air sacs
at atrium (of spiracle)

b brain
cm _ cibarial muscle
cr crop

ef eye facets
es esophagus
fl flagellomere
ge genitalia

GRIMALDI ET AL.: AMBER FOSSILS 3

gl glossa

g gut

h head

Imd_ longitudinal median dorsal muscle
my mycangium

oc ocelli

oim oblique intersegmental muscle
olm oblique lateral muscle

pe pedicel

ph phragma

pr pyloric region
th thorax

tr trachea

ve ventriculus

PREVIOUS STUDIES

There have been several reports on the
preservation of tissues in amber fossil ar-
thropods, each of which is based on only one
or two specimens. Kornilowitsch (1903) was
the first to report the fine structure of tissue
preserved in amber fossil insects. He thin-
sectioned the legs of Diptera and Neuroptera
in Baltic amber and found remarkable stri-
ations in the muscles. Mierzejewski (1976a)
was the first to apply electron microscopy to
tissues “‘exhumed”’ from amber fossils. He
reported “optic cells, pigment cells, or crys-
talline cones” in the facets of a dolichopodid
fly preserved in Eocene Baltic amber, as well
as the minute tracheae that deliver oxygen to
these and other insect cells. (Unfortunately,
the SEM in his plate II, fig. 2, offers little
resolution; secondary iris cells in the middle
of each optic cell do not appear to be pre-
served). Elsewhere (Mierzejewski, 1976b) il-
lustrated with the SEM an entire book lung
from a Baltic amber spider: these are an in-
vaginated series of thin parallel plates with a
thin cuticular covering. The reports by Poin-
ar and Hess (1982, 1985) and Poinar (1992)
are based on a single sample of tissue which
adhered to the inner wall of the abdomen of
a fungus gnat (Diptera: Mycetophilidae), also
in Baltic amber. Using transmission electron
microscopy (TEM), they reported nuclei,
“lipid droplets” (histochemical tests for lip-
ids were not done), mitochondria and cristae,
and apparent endoplasmic reticulum in epi-
dermal cells. Muscle banding was not well
preserved. They considered such preserva-
tion to be anomalous, since it was believed
that the resin would need to be in direct con-

4 AMERICAN MUSEUM NOVITATES

tact with tissues, which might occur if the
body wall was traumatically opened. The
possibility was mentioned that “‘sugars and
terpines [sic] present in the original resin”
could have dehydrated the tissues. Poinar
(1992) also presented a TEM of tissue from
the abdomen of a braconid wasp in Canadian
Cretaceous amber (ca. 80 myo), in which
“folded membraneous [sic] structures adja-
cent to the vacuolated cytoplasm” (p. 270)
were identified.

Schliiter (1989) presented SEMs of a ter-
mite and possible ant head preserved in Cen-
omanian amber from France (ca. 100 myo).
Fine structure of the epicuticle that was ob-
served included microtrichiae, and reticula-
tions on the original cuticle. The preservation
of soft tissues was not examined. He also
found minute crystals of marcasite in some
specimens, which is often associated with the
iron-rich sediments of ancient deltas where
amber deposits usually occur. Ground water
with dissolved minerals seeps into fine cracks
and permeates an inclusion where crystalli-
zation later occurs. In fact, Baroni-Urbani
and Graeser (1987) described with SEMs a
pyritized cast of an ant in Baltic amber.
Enough detail was preserved to determine
that the cuticular microsculpturing of this ex-
tinct species differed from that of any living
species. The work of these authors implied,
though, that electron microscopy of the cu-
ticle of amber insects would depend on pyr-
itization, since this mineral would conduct
electrons well.

Studies by Henwood (1992a, b) used SEM
and TEM on three insects preserved in Oligo-
Miocene amber from the Dominican Repub-
lic. In the TEM study, the flight muscle of an
empidid (““dance’’) fly was cross-sectioned,
revealing the hexagonal ultrastructure of my-
ofibrils and the mitochondria densely packed
among them, even showing the intricate fold-
ing of the internal cristae. Longitudinal sec-
tions of the flight muscle, which could have
shown additional ultrastructural detail, were
not made. In the SEM study, Henwood stud-
ied two beetle specimens, a cantharid (“‘sol-
dier beetle”’) and a nitidulid (“‘fungus beetle’’).
Nitidulids are as heavily sclerotized as most
other beetles (the cantharid less so), and both
these specimens were completely intact, in-
dicating that the viscous resin does not need

NO. 3097

to contact the internal organs directly. Some
tissues were found in their original positions,
but shrunken to about 50% of the original
size. Most interesting was the observation of
a proventriculus in the nitidulid, which is
posterior to the esophagus and is very slightly
sclerotized (virtually membranous). Blunt,
heavily sclerotized teeth lining the interior
serve as a grinding mill and food filter, and
the entire structure was remarkably intact.
Unfortunately, Henwood’s method of cutting
entirely through the specimen with a saw
abraded much of the internal remains, and
she concluded by acknowledging the need for
an alternative method of extraction.

The present study expands upon the pre-
vious ones by using many more specimens
of insects of various taxa, sizes, internal anat-
omy, and degree of cuticle sclerotization as
well as several plant specimens. The insects
derive from two chemically distinct ambers,
which allows additional taphonomic com-
parisons. This approach more fully addresses
the question of consistency of soft tissue pres-
ervation in amber fossils. Also, we used a
procedure for examining whole tissues and
organs that was minimally destructive, leav-
ing the internal structures more intact. The
remarkable tissue preservation is apparently
due (at least partially) to dehydration, which
is an ideal condition as well for long-term
storage of pollen (Shivanna et al., 1991). Thus,
we examined possible viability of Hymenaea
pollen in Dominican amber, which is the tree
that gave rise to this amber (Langenheim,
1969; Hueber and Langenheim, 1986) and
whose anthers are a relatively common in-
clusion.

MATERIALS AND METHODS

Amber fossilized insects and plant parts
were acquired from several sources. Baltic
amber was acquired from Jorge Wiinderlich
(StauSenhardt, Germany), and Dominican
amber from Jacob Brodzinksy (Santo Do-
mingo, Dominican Republic), Manuel Perez
(Orland, FL), and several other dealers in
Santo Domingo and Santiago. Specimens
were selected according to several criteria,
most important of which was an abundant
supply of individuals. Particular effort was
made to use only common species (e.g., where

1994

a series of at least 20 specimens existed in
the AMNH collection and which could be
easily replaced). Secondly, selected speci-
mens had no fine cracks between the surface
and the inclusion, which might have allowed
invasion of the amber seal by bacteria, mois-
ture, or other elements of decomposition.
Third, specimens represented a great taxo-
nomic diversity, which generally correspond-
ed with great variation in basic body form,
habits, relative amount of muscle mass,
etc.... Fourth, species of significantly dif-
ferent sizes were chosen, since the proportion
of muscle mass to body surface area would
apparently be a significant factor in mum-
mification. Lastly, species were chosen based
on differences in the degree of sclerotization
and thickness of cuticle, beetles being the most
heavily sclerotized and termites being the
least.

The following specimens were examined:
For Dominican amber—three stingless bees
(Proplebeia dominicana [Wille], Apidae: Me-
liponini), two termites (Reticulitermes sp.,
Termitidae), three platypodid beetles, two
fungus gnats (Mycetophila sp.: Mycetophili-
dae), two scuttle flies (Megaselia sp.: Phori-
dae), one leaflet of Hymenaea (Legumino-
sae), and seven anthers of this tree. For Baltic
amber—two dolichopodid flies, and two my-
cetophilid fungus gnats.

Internal organs of the insect inclusions and
the pollen contents of stamens were exposed
using the following method. Each specimen
was first photographed with light microgra-
phy using a Zeiss SV-8. Amber was ground
to within 3-4 mm on all sides of the inclu-
sion. A groove ca. 1.5 mm thick was circum-
scribed around the mid-sagittal line of the
specimen (while observing under 10 mag-
nification), using a 2 in. diameter emory wheel
on a motorized flexible shaft (Dremel, Inc.).
The groove formed a circle less than 1 mm
from the inclusion. Fine powder from the
cutting was blown away with compressed air.
A sharp, pointed X-Acto blade was used to
carefully score the internal edge of the groove,
even closer to the inclusion, until slight lev-
erage between the two halves ofamber caused
the piece to split, generally along the axis of
the groove and through the middle of the
inclusion. Very little particulate debris from

GRIMALDI ET AL.: AMBER FOSSILS 5

the cutting contaminated the preparation and
no abrasion of the internal parts occurred.

For observation with the SEM, the intact
end of the amber piece was mounted to a
stub using liberal amounts of silver paint. A
5A coating of gold-palladium was applied.
Amber has excellent insulative properties, as
best seen when it gathers static charges upon
rubbing (hence the Greek name for amber,
elektron). Thorough grounding will reduce
electron charging, although additional pre-
cautions were made using low voltage (2-3
kV). Use of a Zeiss DSM (Digital Scanning
Microscope) 950 with electron-collection en-
hancement features allowed study at such low
kVs with little loss of resolution.

To observe the ultrastructure of tissues and
cells with the TEM, two specimens of the
extinct Dominican amber bee, Proplebeia
dominicana, were freshly opened (this is one
of the most common inclusions in this am-
ber). Bundles of the longitudinal median dor-
sal muscles were carefully lifted from the tho-
rax using hand-sharpened watchmaker’s
forceps, with the tips first washed in alcohol
and dried. Brain tissue was removed from
the head in the same way. Tissue specimens
were immediately stored in dry Eppendorf
tubes and express shipped from New York
to Baltimore (1 day delivery). They were not
frozen or refrigerated. Numerous possible
“controls” could be used to compare to the
amber specimens: air dried and freshly fixed
specimens; and specimens stored for varying
amounts of time in tree saps, various fresh
resins, Canada Balsam, etc. ... That is a fu-
ture project beyond the scope of the present
one; it would address the chemical factors
responsible for the preservation we report
here.

Samples were prepared for transmission
electron microscopy by two protocols. Some
were embedded directly in LR White resin
(Newman et al., 1983; Polysciences Inc.,
Warrington, PA) by immersing the tissue in
unpolymerized LR White for at least 8 hours.
A second set of tissue samples was rehydrated
and dehydrated before embedding. These
samples were rehydrated overnight at room
temperature in Ruffer’s solution (30% etha-
nol, 1% NaCO, in distilled water [Lewin,
1967]). Following rehydration, the samples
were dehydrated by progressive 60 minute

6 AMERICAN MUSEUM NOVITATES

incubations in 30, 50, 75, and 90% ethanol
in distilled water. The final dehydration was
carried out by three 20 minute incubations
in 100% ethanol followed by one 20 minute
incubation in 50% ethanol/50% LR White.
The samples were then infiltrated in 100%
LR White for at least 8 hours. Each set of
embedded samples was polymerized at 50°C
overnight in fresh resin in gelatin capsules.
Ultrathin (90 nm) sections were cut from the
polymerized blocks on a Reichert-Jung Ul-
tracut E microtome using a Diatome 35° an-
gle compression-free knife, then transferred
to Formvar-coated EM grids and allowed to
air dry. The sections were stained with 1%
OsO, and uranyl acetate. All samples were
viewed and photographed on a Zeiss TEM
10A transmission electron microscope at 60
kV.

To test for pollen viability we attempted
to induce pollen tube growth on the following
medium: | ml. of ‘‘mother’” solution (0.1
mg/ml boric acid, 0.3 mg/ml CaNQO,, 0.2 mg/
ml MgSO,, 0.1 mg/ml KNO, dissolved in
sdH,O), which was added to: 9 ml sdH,O, 9
g sucrose, 0.8 g gelatin. Pollen controls were
from fresh petunia flowers. Negative controls
were made by microwaving the petunia pol-
len on high for 2 minutes, which rendered
the pollen inviable. Four anthers of Hymen-
aea in Dominican amber were cut open and
the tissues scraped out using an ethanol-
cleaned pin. Controls and experimental sam-
ples were incubated on the culture medium
for 72 hours at room temperature and ex-
amined for pollen tube growth. Experimental
samples (from amber) were tested in an area
of the lab separate from the controls, to re-
duce any possible contamination.

Mounted specimens were retained on SEM
stubs and are deposited in the amber fossil
collection of the AMNH, should future ex-
amination be of interest. Each has a catalog
number, to which we refer in the text and the
figures. The following references were con-
sulted for interpreting morphological and cy-
tological structures; terminology derives from
these references: Bold (1973); Chapman
(1982); Smith (1968); Snodgrass (1925, 1935).
The SEM plates are arranged according to the
specimen and type of organism, even though
the following discussion is presented by types

NO. 3097

of tissues. TEM plates are presented last, but
ultrastructure is discussed under sections
concerning the appropriate tissues.

THE AMBERS

Amber from the Dominican Republic and
the Baltic were chosen because of the differ-
ences in age and chemistry, the latter feature
of which is well documented. Dominican am-
ber was unquestionably formed from an ex-
tinct species of the living genus Hymenaea
(Leguminosae) (Langenheim, 1969; Hueber
and Langenheim, 1986). Although cited often
by some as being Eocene in age (e.g., Poinar,
1992), the available evidence on stratigraphy
suggests an origin around the Oligo-Miocene
boundary for the oldest deposits, and Hen-
wood (1992b) cautioned against an Eocene
age. Hymenaea trees today produce copious
amounts of resin, containing arrays of ses-
quiterpene hydrocarbons and diterpenoid
resin acids (Langenheim, 1981; 1990).

Baltic amber, by contrast, derives from a
conifer, possibly a pine (Pinus) or close rel-
ative, but probably an araucarian (Gough and
Mills, 1972; Mills et al., 1984). It has a par-
ticularly high concentration of resin acids,
such as succinic and communic acids (Gough
and Mills, 1972). The chemical differences
between Dominican and Baltic amber are
readily observable via the external preser-
vation of the insects in them. Insects in Do-
minican amber are typically perfectly pre-
served, whereas Baltic amber specimens often
have a milky coating on the body. This milk-
iness is due to a froth of microscopic bubbles
(Mierzejewski, 1978), presumably the prod-
ucts of microbial decomposition and/or au-
tolysis of internal tissues.

Much older amber fossils from the Turo-
nian (mid-Cretaceous, ca. 90-94 myo) of New
Jersey and the Neocomian of Lebanon (ca.
125 myo) are in the AMNH collections, but
were not used for these tissue studies. The
paleontological value of this material is great-
er than that of the Tertiary material, and it
is difficult (or impossible) to replace. Any
studies causing partial or entire destruction
of a Cretaceous specimen should be done only
after exhaustive morphological study on a
large series of specimens of one species.

1994

INSECT PRESERVATION

Cuticular Structures. Preservation of cu-
ticular structures (external as well as invagi-
nated into the body) would not seem sur-
prising in amber fossil insects, given the
intricately preserved external detail of in-
sects. The natural positions of structures, and
the detail of their preservation, however, were
unexpected. In the stingless bee (specimen
B1), the row of imbricate plates on the glossa
(or tongue) was found, with a fringe of fine
hairs along its left edge—the hairs function
in imbibing nectar (fig. 6). Also seen in the
stingless bees (B1, B3) was a curious detail
of the mesothoracic phragma (a paddle-
shaped apodeme). It possessed a geometric
pattern of eight cells, each having a finer pat-
tern of roughly hexagonal cells on it (figs. 17,
18). These must be imprints of the muscle
bundles, fibers, and myofibrils. Similar geo-
metric patterns are observed on insect egg
chorions, which are the imprints of the fol-
licle cells (Hinton, 1981); and the epicuticle
of various insects bears the hexagonal im-
prints of epidermal cells. It has always been
assumed that the original cuticle was intact
in amber fossil insects, but the degree of pre-
served detail and molecular composition has
not been examined. In one of the scuttlefly
specimens, the reverse and obverse images
of the antennae showed that actual sensilla
and setulae can be preserved not just as im-
prints (figs. 26, 27). This specimen clearly
shows how the actual material is separated
from and smaller than the amber “cast.” Since
it is unlikely that the cuticular surface would
shrink, even during dehydration, it is more
likely that the cast surface represents expan-
sion of the amber, probably due to polymer-
ization of the original resin. How much orig-
inal chitin remains in the “amberized”’ insect
cuticle has yet to be determined. Insect cu-
ticle is composed of 25-40% chitin, which is
a B-pleated polysaccharide sheet intercalated
with proteins. Miller et al. (1993) found that
the cuticular remains of Pleistocene beetles
buried in sediments (ca. 15,000 years old)
contained half the amount of chitin that would
be expected from fresh material.

Fine structural details of the original cuticle
can also be seen by the microtrichiae pre-

GRIMALDI ET AL.:

AMBER FOSSILS 7

served on the wing membrane of a dolicho-
podid fly in Baltic amber (fig. 56). A row of
six minute campaniform sensilla occur on the
wing vein of a mycetophilid midge, also in
Baltic amber (fig. 61).

Musculature. Insects have a complex mus-
culature, possessing approximately twice the
number of muscles as do mammals (Snod-
grass, 1935). Much of this musculature is in
the thorax and serves to power the legs and
wings. Commonly seen was preservation of
the longitudinal median dorsal muscles of the
thorax; in stingless bee specimen B1 (fig. 4)
it is seen as closely joined bundles forming a
large sheet occupying most of the thorax. This
muscle mass is completely intact. Smaller
muscles are also well preserved, with their
insertions and origins intact. A bundle of 10—
11 muscles is attached to the mesothoracic
phragma and postnotal wall of stingless bee
B1 (fig. 10). In B3, the slender oblique lat-
erals, oblique intersegmentals, and portions
of longitudinal median dorsal muscles were
seen (fig. 17). Small muscle fibers attached to
the membranous sucking pump (pharynx) of
stingless bee B1 showed transverse striations
at higher magnification (fig. 8). A compact
bundle of small muscles was found dislodged
from the mostly empty thorax of a myceto-
philid midge in Baltic amber (fig. 55). Not all
muscles appeared so intact: some thoracic
muscles of platypodid beetle P1 were very
fibrous and shredded. Shrinkage of muscles
was not discerned in any specimen that pos-
sessed them, although the muscle and other
soft tissue was always a very dark red or tar
black. Causes of the discoloration are un-
known.

At the ultrastructural level, native Prople-
beia dominicana flight muscle is easily iden-
tifiable, with striking patterns of repeated sar-
comeres present in longitudinal sections of
all myofibrils (fig. 69). The Z-and M-lines are
present as spaces between the electron-dense
remains of the thick filaments in the A-band.
Higher magnification reveals almost no fi-
brillar appearance to the A-bands, suggesting
that the electron-dense material present there
is composed primarily of inorganic salts de-
posited on thick filaments during dehydra-
tion (figs. 70-71). Strips of electron-dense mi-
tochondria separate the myofibrils. The

8 AMERICAN MUSEUM NOVITATES

mitochondria have well preserved, densely
packed cristae characteristic of insect flight
muscle (fig. 72).

Membranous structures in flight muscle are
generally better preserved than proteinaceous
ones. In addition to mitochondria, tracheoles
are present, and the membranes of epithelial
cells lining these tracheoles are apparent (fig.
73). In some sections, T-tubules are pre-
served (fig. 74). Extensive membranes are of-
ten found superficial to the muscle cells them-
selves (fig. 75).

Samples that were rehydrated prior to em-
bedding are essentially composed of strips of
mitochondria, separated by spaces with al-
most no electron-dense material (fig. 76). The
material composing the sarcomeres is almost
completely extracted by the rehydration step.
We suspect that the extracted material is
composed of inorganic salts, with perhaps
some remaining proteins. It is possible that
fixation with glutaraldehyde during rehydra-
tion might preserve some proteinaceous
structures like sarcomeres while extracting
inorganic salts.

Several flight muscle samples dissected
from Proplebeia dominicana had an addi-
tional tissue structure attached to the surface
of the muscle bundle itself (fig. 77). We sus-
pect this tissue to have connected the flight
muscle to the axillary sclerites of the wing,
since cuticle is often found attached (fig. 78).
This tissue consists primarily of amorphous
fibrillar material (probably collagen fibers)
interposed with small electron-dense parti-
cles (figs. 79-80). We do not know the origin
of these particles, which may be nuclei, cells
that have been extensively distorted by the
rehydration process, or precipitated inorgan-
ic salts. In any case, the fibrillar matrix is
apparently quite well preserved, and is an
attractive candidate tissue for isolation of
fossil polypeptides.

Nervous Tissue. Nervous tissue is notori-
ously difficult to preserve well. It was very
surprising, therefore, to find several speci-
mens with the brain (protocerebrum) intact
(stingless bee B1 [figs. 5, 9]; phorid fly [fig.
25], platypodid beetle, P1 [fig. 30]), and my-
cetophilid midges [fig. 59]). In the stingless
bee and platypodid the tissue was loosely fi-
brous, indicating loss of some interstitial ma-
terial, although there was virtually no shrink-

NO. 3097

age of the entire structure. In the other
specimens this structure was dense and amor-
phous.

Brain tissue removed from a specimen of
Proplebeia dominicana was embedded and
sectioned for TEM and was rather poorly pre-
served, as expected. Some histological fea-
tures are still observable, however. Most ar-
eas of the sections are covered with a dense
outer layer of electron-dense salts (fig. 81).
Notable exceptions to this are regions of con-
voluted membranes, which we suspect to be
the remains of the major axon tracts of the
central nervous system (fig. 81).

In brain tissue that was rehydrated before
embedding, some other interesting details be-
came apparent after extraction of the inor-
ganic salts. In some areas, extensive tracts of
parallel membranes surround patches of elec-
tron-dense cytoplasmic remains (figs. 82, 83).
We believe these to be membranes of oli-
godendrocytes wrapping large axons. Anoth-
er possibility is that these membranes are ar-
tifacts occasionally seen in TEM of necrotic
tissue (Commonly called “myelin figures’’),
caused by extraction of lipids from dying cells.
Cytoplasm of neural cells is generally poorly
preserved, with almost no cytoplasmic de-
tails. The putative axon tracts observed in
brain sections are also preserved after rehy-
dration, although the membranes appear
swollen and distended compared to native
samples (fig. 84).

Membranous Structures. These include
portions or most of the digestive, excretory,
and reproductive systems, as well as the dor-
sal aorta. In several specimens, but seen best
in B1 (figs. 11, 12), were the folds of fine
membranous air sacs, which occupy the pos-
terodorsal half of the thorax in bees. Unlike
the other parts of the respiratory system (tra-
cheae and tracheoles), these membranes have
no trusswork of minute, chitinous rings (taen-
idia). Excellent examples of preserved tra-
cheae are seen in a Baltic amber mycetophilid
(figs. 67, 68). The tracheae are hardly col-
lapsed. Cross sections of tracheoles were seen
in TEM thin sections of Proplebeia bee flight
muscle from Dominican amber (fig. 73).

Portions of digestive tracts of several spec-
imens were remarkably intact. In one my-
cetophilid the ventriculus possessed a geo-
metric pattern of folds on the outer surface,

1994

each fold forming a cavity with a papilla in
it (figs. 21, 22). The papilla is probably a
crypt, perhaps of regenerative cells. A portion
of the thin esophagus was seen in one termite
(specimen T1, not figured); and a more ex-
tensive portion of the foregut, including the
crop, was found in stingless bee B2 (figs. 16,
17). In one platypodid beetle, an extensive
portion of the gut in the abdomen was ob-
served, which included the entire midgut and
hindgut (fig. 38). The region where the fine
malpighian tubules occur had an odd pres-
ervation, which was an amorphous arrange-
ment of stacked mounds; holes; and flat, hex-
agonal crystals (fig. 41). Other parts of the
midgut showed unusual formations of deep,
fine pleats (fig. 39). The detail of membra-
nous tissue preservation is best seen from an
unidentified structure from a termite thorax
(fig. 24). The tissue is very thin with one mar-
gin frayed, showing loose fibers.

Other organisms. Intimately associated
with arthropods in amber are a host of or-
ganisms, such as parasites and symbiotic mi-
crobes, as well as evidence of other ecological
relationships. It is not uncommon, for ex-
ample, to find stingless bees in amber with
clumps of pollen adhering to the hairs on the
hind legs and abdomen. One bee specimen
(B1) was examined that had a clump of pollen
on the ventral side of the abdomen. The exine
was intact enough to observe that two dis-
tinctly different pollen types were collected
(fig. 13), indicating that the bee had been vis-
iting two kinds of flowers. The exine of the
most common pollen type is very finely pit-
ted and. quite similar to that of living Hy-
menaea (Langenheim and Lee, 1974) as well
as to the amber Hymenaea (fig. 48); the other
type has coarse sculpturing (fig. 14).

Some wood-boring beetles feed not on the
wood, but on the fungus cultures that they
inoculate in the galleries. Ambrosia beetles
(families Scolytidae and Platypodidae) are
quite common in the Dominican amber; no
doubt they were living in the amber tree itself,
since amber pieces are occasionally found
with beetle galleries in them. The beetles
transmit the fungus via specialized struc-
tures, the mycangia (Batra, 1963). Mycangia
are pockets of invaginated cuticle that occur
in various insects, mostly beetles, that harbor
inoculum of symbiotic fungi. The beetles feed

GRIMALDI ET AL.: AMBER FOSSILS 9

on the fungi and also serve as the main dis-
persal agent. Mycangia vary tremendously in
the Coleoptera, with structures occurring on
the mandibles, head, elytra, abdomen, and
thorax (Crowson, 1981). Perhaps no fungal-
beetle symbiotic relationship is more spe-
cialized and intimate than that of ambrosia
beetles and their ambrosia fungus (subclass
Hemiascomycetidae). The various ambrosia
fungi are known only from the beetle’s my-
cangia and galleries and are specific to the
species of beetle, not the host tree (Batra,
1963).

Specificity of the ambrosia fungus to the
beetle may be due to the apparent glandular
nourishment that the fungus receives in the
mycangium. In fact, the shape and location
of saclike mycangia in the Scolytidae and Pla-
typodidae are often species-specific (Batra,
1963; Francke-Grosmann, 1956). Batra clas-
sified five categories of mycangia in these bee-
tles based on their location on the body.

In an unidentified platypodid beetle (P2),
two large ventral mycangia were found on
the insect’s left side (mycangia occur in pairs;
see fig. 33). The anterior one was largest, ap-
proximately 200 um long, drop-shaped, and
was lying between the meso- and metathorax
(fig. 34). The posterior mycangium was round
and lying between the metathorax and ab-
dominal sternite 1 (figs. 35). The size and
general location correspond with other re-
ports based on living species (cf. figs. 5, 16,
and 17 in Batra [1963]). Both mycangia were
replete with spores and conidiophores (fig.
36). Viability of the spores has not been test-
ed.

PLANT PRESERVATION

Leaves typically have a heavily cutinized
(waxy) epidermis, beneath which there is a
layer of mesophyll. The mesophyll is the pho-
tosynthetic layer of cells, and comprises of a
spongy layer of squamous cells and a palisade
layer of columnar cells. In a small leaf of
Hymenaea in Dominican amber, an entire
surface was exposed, which was charcoal-
black, finely cracked, and crumbling (fig. 44).
Close examination, however, revealed that
the palisade layer of mesophyll was intact in
places: columnar cells were easily seen and
showed no distortion (fig. 45).

10 AMERICAN MUSEUM NOVITATES

The anthers of Hymenaea are, not surpis-
ingly, rather common in Dominican amber.
According to Jean Langenheim (personal
commun.), during the pollination period the
area beneath a Hymenaea tree is covered with
dehisced stamens (the anthers do not fall from
the filaments). Seven specimens were ex-
humed: four for testing pollen viability, three
for study under the SEM. In five of the an-
thers the material inside had a black, gummy
appearance, but was actually brittle and very
dry (e.g., figs. 46, 47). Under the SEM this
material was amorphous, and presumably is
a dried film lying over pollen and tissue. In
anther specimen Al most of the pollen was
not clearly visible because it was obscured by
this apparent film. Some pollen grains that
were dispersed into the amber were found to
have the exine less obscured (this anther was
chosen for study, in fact, because it was de-
hiscent and apparently mature). The exine of
the pollen of the extinct Dominican amber
tree, Hymenaea protera, has a surface of
dense, fine pits very similar to that of Hy-
menaea courbaril and H. verrucosum (fig. 48;
cf. fig. 4 in Langenheim and Lee [1974]). This
is also very similar to the pollen found on
stingless bee specimen B-1 (fig. 14). In the
other two anthers (1 dehiscent, the other not;
see figs. 49-51) there was completely different
preservation. Internal tissues were not a tarry
black, but chalky and brown. Extensive mats
of dense, fine, hairlike structures filled most
of the anther, but no pollen grains were ap-
parent. These structures are probably colum-
nar epithelial cells. In the test for viability,
pollen tubes grew on all the positive control
samples. As expected, no tubes were found
in the negative controls or in the experimen-
tal samples removed from the Hymenaea an-
thers in Dominican amber. Despite the de-
hydration properties of amber, enzymes and
other labile molecules in the pollen probably
undergo autolysis.

CONCLUSIONS

The observations reported here and else-
where reveal tissues, cells, and cellular ultra-
structure in amber insects and plants, with a
startling lifelike fidelity. In two of the four
small (< 4 mm body length) Baltic amber
flies that were examined here, the thoracic

NO. 3097

and abdominal cavities were largely vacant
of tissue, but internal tissues and organs of
the other Baltic amber flies were as intact as
is typically found in the Dominican amber
insects. Thus, the preservative qualities of
ambers are not equivalent, although the tis-
sue which did remain in the Baltic amber flies
was as well preserved under the SEM as that
in Dominican amber flies (TEM of Baltic am-
ber fly muscle, for example, was not done in
our study). In the insects with preserved tis-
sues, the organs showed little or no shrinkage,
contrary to the observations of Henwood
(1992b) who reported up to 50% shrinkage
of muscle from a Dominican amber fly. The
general lack of shrinkage or autolysis, and
preservation of such delicate structures as air
sac membranes and brain tissue, indicate a
very rapid mummification, which tempts ex-
planations on the possible chemical process
involved.

The best scenario we can provide to ex-
plain such apparently rapid and thorough cel-
lular fixation is that the most volatile, low
molecular weight fractions (mono- and ses-
quiterpenes) in the original resin readily dif-
fused through intact body walls and perfused
the tissues. For insects, the intersegmental
membranes would be an important area of
diffusion (even though they are thinly cutic-
ular and possess a waxy layer, the interseg-
mental membranes are the thinnest parts on
an arthropod). These volatile fractions must
have replaced the cellular water. This can be
seen in many amber fossilized insects: often
there is a transparent, light brown ‘‘halo”
around them, which must be aqueous com-
ponents of the body fluid sequestered by the
surrounding resin. Monosaccharides, alco-
hols, aldehydes, and esters also occur in res-
ins (Langenheim, 1990), and a few investi-
gators have implicated a role for at least some
of these in “‘amberization.”’ Currently, stud-
ies have begun to examine the infiltration of
the volatile sesquiterpene hydrocarbons and
some oxygenated forms into insect tissue. The
role of monosaccharides and other com-
pounds is probably much less significant than
that of terpenes, because they occur in much
lower concentrations, and (with the exception
of alcohols) would perfuse tissues more slow-
ly than volatile terpenes. Henwood (1992a),
for example, mocked a sap flux with maple

1994

syrup, in which flies were preserved and their
muscles later thin sectioned. Muscles in the
syrup-preserved flies were not nearly as well
preserved as the muscles of amber insects
reported here and by Henwood, and the sugar
concentration of the syrup is much higher
than any found in natural plant exudates, let
alone in resins.

The ultrastructural results prompt ques-
tions as to the actual molecular preservation
that has occurred. What proteins, if any, are
present? In what state might they be? Im-
munological evidence suggests preservation
of protein tertiary structure in brachipod shells
that are up to 4 million years old (but no
older) (Collins et al., 1991), and glycoproteins
from an 80 million year old mollusk shell
were reported (Weiner et al., 1976). The den-
sity of mollusk and brachiopod shells may
uniquely provide for the preservation of such
macromolecules, particularly since the mode
of fossilization in marine sediments would
seem less than ideal for protein preservation.
Contrary to this are the plant fossils from the
fine, dense, anoxic clays of the mid-Miocene
of Clarkia, Idaho. Chloroplast preservation
is reported from leaves found there (Niklas
et al., 1985), as well as two cases of DNA,
from a magnolia (Golenberg et al. 1990; Go-
lenberg, 1991) anda bald cypress (Taxodium;
see Soltis et al., 1992). However, Logan et al.
(1993) found that preservation of biomole-
cules is highly selective in Clarkia fossils, with
no evidence of polysaccharides, polyesters,
or proteins, but only lignins and an aliphatic
biopolymer present. Logan et al. questioned
the DNA results from the Clarkia plant fossils
and, indeed, DNA preservation in them is
hardly consistent: among “hundreds” of ex-
tractions, these two published examples and
several unpublished ones are the only suc-
cesses (P. and D. Soltis, personal commun.,
1992; E. Golenberg, personal commun. 1993).
Quality of protein preservation is dependent
on how one analyzes the molecule. Amino
acid sequencing might reveal short chains of
a-amino acid units, a result of peptide bond
hydrolysis, and it would be useful only if hy-
drolysis occurs preferentially in some bonds.

GRIMALDI ET AL.:

AMBER FOSSILS 11

Or, side groups such as amino and carboxyl
groups could be lost. If analytical methods
are dependent on tertiary structure for de-
tecting protein preservation, one would also
be measuring the degree to which H bonding
and crosslinking are still intact (which varies
with the kind of protein). Ambler and Daniel
(1991) reviewed successful extractions of
protein from ancient materials.

There is little doubt that amber will pre-
serve DNA more consistently than any other
kind of fossil. Four examples of DNA from
insect and plant tissues in amber have been
published, three of them from Dominican
amber: 1. A large, primitive termite, Mas-
totermes electrodominicus (DeSalle et al.,
1992); 2. A stingless bee, Proplebeia domin-
icana (Cano et al., 1992); 3. A leaf of the
amber tree, Hymenaea protera (Cano et al.,
1993b: an unrefereed report in which se-
quences were not presented); and 4. A ne-
monychid weevil in 125 million year old Leb-
anese amber (Cano et al., 1993a). Three other,
unpublished successes are known thus far, all
concerning Dominican amber: a drosophilid
fruit fly (Y. Shirota, Hirosaki University); an
anisopodid woodgnat, Valeseguya disjuncta
(DeSalle and Grimaldi, unpubl.: AMNH); and
several chrysomelid beetles (B. Farrell, per-
sonal commun.: Univ. Colorado). Such con-
sistent preservation of DNA must be attrib-
utable to the unique chemistry of resins. The
fact that the primary structure of DNA re-
mains reasonably intact in amber fossils (ap-
proximately 250 base pair segments on av-
erage), indicates an ability of the amber to
preserve the phosphate-ester bond between
the deoxyribose sugar units. This bond is the
one most susceptible to hydrolysis (Eglinton
and Logan, 1991; Lindahl, 1993), and points
mostly to the dehydration properties of the
resin.

What proteins remain, and in what state,
is the subject for future studies. Regardless
of this, all evidence still indicates that amber
very consistently and exquisitely mummified
the small organisms that became entombed
in the ancient resin.

12 AMERICAN MUSEUM NOVITATES NO. 3097

Figs. 1-6. Stingless bee, Proplebeia dominicana (B1) in Dominican amber. 1: Intact specimen, show-
ing cut circumscribed around mid-saggital line. 2: Freshly opened specimen (light micrograph). 3, 4:
Opposite halves of entire bee. 5: Detail of head. 6: Detail of glossa. Scales: 3,4: 500 um; 5: 100 um; 6:
20 «wm.

1994 GRIMALDI ET AL.: AMBER FOSSILS 13

Figs. 7-12. Details of stingless bee (B1). 7: Sucking pump with attached muscles. 8: Detail of muscle
(note transverse striae). 9: Protocerebrum (brain). 10: Bundle of small muscles in thorax. 11: Thorax.
12: Detail of thorax, showing membranous air sacs. Scales: 7: 50 nm; 8: 10 wm; 9: 50 wm; 10: 20 um;
11: 200 wm; 12: 100 um.

14 AMERICAN MUSEUM NOVITATES NO. 3097

Figs. 13-18. Stingless bees, P. dominicana, in Dominican amber. 13: Clump of pollen from abdomen
of B1. 14: Detail of one pollen grain. 15, 16: Specimen B2. 15: Head and thorax, lateral view. 16: Detail
of esophagus and crop seen in 15. 17, 18: Specimen B3. 17: Transverse section through thorax. 18: Cell
imprints in cuticle of phragma seen in 17. Scales: 16, 17: 200 um; 18: 5 um.

1994 GRIMALDI ET AL.: AMBER FOSSILS 15

Figs. 19-22. Female fungus gnat (Mycetophila sp.: Mycetophilidae) in Dominican amber (M1). 19:
Habitus of intact specimen. 20: Lateral view of entire, opened specimen. 21: Portion of the membranous
ventriculus. 22: Detail of ventriculus. Scales: 20: 500 wm; 21: 20 um; 22: 5 wm.

16 AMERICAN MUSEUM NOVITATES NO. 3097

Figs. 23-27. Insects in Dominican amber. 23, 24: Isoptera (termite), Reticulitermes sp. (T1). 23:
Thorax, showing unidentifiable muscle bundles. 24: Detail of the edge of a sheet of membrane. 25-27:
Fly, Megaselia sp. (Phoridae). 25: Head. 26, 27: Antenna, opposite halves. Note the actual structures
and corresponding impressions. Scales: 23: 200 um; 24: 10 um; 25: 50 wm; 26, 27: 20 um.

1994 GRIMALDI ET AL.: AMBER FOSSILS 17

Figs. 28-31. Platypodid beetle in Dominican amber (P1). 28: Entire, intact specimen. 29: Entire

specimen, opened. 30: Protocerebrum (brain). 31: Detail of fibrous tissue in or on brain. Identity of the
tissue is uncertain—it does not resemble nervous tissue. Scales: 29: 500 um; 30: 100 wm; 31: 20 um.

18 AMERICAN MUSEUM NOVITATES NO. 3097

Figs. 32-36. Platypodid beetle in Dominican amber (P2). 32: Entire, intact specimen. 33: Entire
specimen, opened. 34: Detail of head and part of thorax, showing anterior mycangium. 35: Detail of
posterior mycangium. 36: Detail of fungal spores and conidia in mycangium. Scales: 33: 200 um; 34:
100 wm; 35: 50 wm; 36: 10 um.

1994 GRIMALDI ET AL.: AMBER FOSSILS 19

ie

Figs. 37-42: Platypodid beetle in Dominican amber (P3). 37: Entire beetle (lateral), opened. 38: Apex
of abdomen separated from amber cast. 39: Wall of ventriculus, showing group of fine, deep pleats. 40:
Gut. 41: Area near pylorus, showing amorphous structures and some crystals, but not malpighian tubules.
42: Detail of amorphous structures near pylorus. Scales: 37: 500 wm; 38: 100 wm; 39: 10 wm; 40: 200
um; 41: 50 wm; 42: 10 wm.

20 AMERICAN MUSEUM NOVITATES NO. 3097

1994 GRIMALDI ET AL.: AMBER FOSSILS 21

Figs. 46-51. Anthers of Hymenaea protera in Dominican amber. 46, 47: Opposite halves of specimen
Al. This specimen was clearly dehiscent and dispersing its pollen. 48: Detail of pollen released near
edge of anther. The pollen was not viable (see text). 49: Portion of extensive matting from specimen
A2, which was an immature anther. 50, 51: opposite halves of anther A2. Scales: 46, 47: 500; 48: um;
49: 10 um; 50, 51: 200 um.

22 AMERICAN MUSEUM NOVITATES NO. 3097

a

Figs. 52-56. Dolichopodid fly in Baltic amber (D1). 52, 53: Opposite halves of whole specimen,
opened. The bright area at the ventral part of the thorax is the milky impurity, which is due to numerous
fine bubbles. The thorax was largely empty. 54: Head, partly exposed showing original cuticle, and
globule within (presumably the dried remains of liquified decomposition). 55: Bundle of fine muscles
dislodged from the thorax. 56: Detail of original microtrichiae on wing. Scales: 52, 53: 200 um; 54: 100
um; 55: 50 wm; 56: 10 um.

1994 GRIMALDI ET AL.: AMBER FOSSILS 23

Figs. 57-62. Mycetophilid fungus gnat (Mycetophila sp.) in Baltic amber (M-B1). 57, 58: Opposite
halves of entire, opened specimen. The thorax is largely hollow. 59: Detail of head. 60: Detail of bases
of mid and hind legs. Original cuticle is present, but no remnants of soft tissue remain. 61: Detail of
wing membrane pressed into surface of amber, showing line of sensilla on vein (arrows). 62: Male
genitalia, showing internal sclerites and apodemes, but not muscle bundles. Scales: 57, 58: 500 um; 59:
50 wm; 60: 100 um; 61: 20 um; 62: 100 wm.

24 AMERICAN MUSEUM NOVITATES NO. 3097

mia

imen, opposite halves. 65:
Detail of thorax. 66: Detail of head. 67: Detail of thorax, showing tracheae and atrium. 68: Detail of

another trachea. Scales: 63, 64: 500 um; 65: 200 um; 66: 50 wm; 67: 100 wm; 68: 50 um.

1994 GRIMALDI ET AL.: AMBER FOSSILS 25

ay

Figs. 69-72. TEM thin sections of Proplebeia flight muscle from Dominican amber. 69: Myofibrils
with sarcomeric repeats (2200 x). 70, 71: Detail of fig. 69. (70: 16,000 x; 71: 35,000 x). There is a lack
of fibrillar material in the dark banded areas, suggesting replacement by inorganic salts. 72: Detail of

mitochondria tightly packed among myofibrils. Finger print patterns are the internal cristae of the
mitochondria.

26 AMERICAN MUSEUM NOVITATES NO. 3097

ae

Figs. 73-76. TEM thin sections of Proplebeia flight muscle and associated tissue in Dominican amber.
73: Tracheoles and associated epithelial cells lying between myofibrils (2500 x ). 74: T-tubules (11,000 x).
75: Extensively folded membranes superficial to muscle cells (10,000 x). 76: Rehydrated muscle tissue
(52,000 x). Note that the sarcomeres are almost completely extracted, suggesting these are preserved
mostly as inorganic salts.

1994 GRIMALDI ET AL.: AMBER FOSSILS 27

Figs. 77-80. TEM thin sections of a connective tissue attached to flight muscle of Proplebeia in
Dominican amber. 77: Whole section (10,000 x). 78: Portion with cuticle attached (32,000 x). 79, 80:
Details of tissue, with amorphous fibrillar material (collagen fibers?) and electron-dense structures, which
are possibly distorted cells, nuclei, or salt crystals (79: 12,000 x; 80: 30,000 x).

28 AMERICAN MUSEUM NOVITATES NO. 3097

Figs. 81-84. TEM thin sections of brain tissue from Proplebeia in Dominican amber. 81: Whole
section, showing nervous tissue embedded in a thick layer of electron-dense salts (3500 x ). 82-84: Brain
tissue after rehydration, which extracts the inorganic salts. 82, 83: Extensively laminated membranes,
perhaps of oligodendrocytes surrounding large axons (82: 22,000 x; 83: 80,000 x). 84: Swollen mem-
branes surrounding putative axon tracts (3500 x).

1994

GRIMALDI ET AL.: AMBER FOSSILS 29

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