Background
Termites are one of the most successful eusocial insect groups today and certainly the most notorious as a result of their damage to human dwellings. Their success can be attributed in large part to microbial (especially protozoa and bacterial) symbionts harbored in their alimentary tract. Especially important are gut protists, which are essential for the survival of termites feeding on lignocelluloses. While termites do produce endogenous cellulases from salivary glands and gut cells, cellulolytic protists are crucial for the complete digestion of cellulose in wood-feeding termites
Results
Description of host termite
Damage to the termite included the loss of the left hind wing, the tip of the abdomen (including the cerci), the right side of the metathorax and the right side of the first three abdominal segments. All wings were flexed at their bases. However, it was possible to determine the venation in the distal three quarters of the right forewing and the complete right hind wing.
Based on the morphological characters of the termite (Sc absent from the hind wing, R simple in both wings, no reticulation between the veins posterior to R, R separate from the costal margin, presence of a number of regularly spaced oblique forward branches between the Rs and C), the fossil is placed in the family Kalotermitidae
Its small size, low number of antennal segments, strongly sclerotized radial sector, unsclerotized M and Cu veins, complete M vein positioned halfway between Rs and Cu, wing membrane densely covered with minute, pigmented nodules, tibial spurs lacking on the shaft of the mid-tibia and the presence of ocelli and arolia, align the fossil with the genus
Family Kalotermitidae Banks 1919
Genus
Dorsal view of
Dorsal view of
Left antenna of
Left antenna of
Claw with arolium (arrow) of
Claw with arolium (arrow) of
Lateral view of head of
Lateral view of head of
Minute black nodules covering wing membrane of
Minute black nodules covering wing membrane of
Hind wing of
Hind wing of
Alate (sex unknown): Fontanelle absent; head capsule, thorax, legs and abdomen dark brown; length of body (from anterior edge of clypeus to end of abdomen) 3.3 mm; head length, 567 μm; head width, 607 μm; eye diameter, 113 μm; ocellus small, 30 μm in diameter; located 62 μm from compound eye, length terminal maxillary palp, 135 μm; length terminal labial palp, 108 μm; right mandible with prominent terminal tooth and smaller subterminal tooth; left antennae 850 μm in length, with 12 segments, terminal segment smallest; maximum length of pronotum, 525 μm; minimum length of pronotum, 420 μm; width of pronotum, 672 μm; length scale on fore wing, 531 μm; length scale on hind wing, 292 μm; length right hind wing, 3.9 mm; width right hind wing, 1.2 mm; venation on right hind wing as follows: Sc not present; R simple, short; M with double fork near terminus; wing membrane covered with minute black nodules; 6 branches extend from Rs to C; all branches of cubitus simple except basal one; apical spines on tibia partially obstructed, at least 2 on meso and metatibia; tarsi four-jointed with first three segments short; claws 95 μm in length; arolium short; cerci missing.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Comments: The present species differs from
The wing venation, head shape, presence and size of arolia, presence of ocelli, absence of spines on the shaft of the mid-tibia, number of tarsal and antennal segments and presence of pigmented wing nodes separates
Description of protists
A variety of protists were associated with
Except for one species described in the extant genus
Terminology and classification generally follows that of Brugerolle and Lee
Phylum Parabasalia
Class Trichomonada Kirby
Order Trichomonadida Kirby
Family Devescovinidae Doflein
Photo and drawing of
Photo and drawing of
Class Hypermastigida Grassi & Foá
Order Spirotrichonymphida Light
Family Holomastigotidae Grassi
Photo and drawing of
Photo and drawing of
Order Trichonymphida Poche
Family Trichonymphidae Kent
Photo and drawing of
Photo and drawing of
Family Teranymphidae Koidzumi
Photo and drawing of
Photo and drawing of
Phylum Oxymonada
Class Oxymonadea Grassi
Order Oxymonadida Grassi
Family Oxymonadidae Kirby
Photo and drawing of
Photo and drawing of
Photo and drawing of
Photo and drawing of
Photo and drawing of
Photo and drawing of
Photo and drawing of
Family Pyrsonymphidae Grassi
Photo and drawing of
Photo and drawing of
Photo and drawing of
Photo and drawing of
"Sarcodina"
Amoeba of uncertain affinity
Photo and drawing of
Photo and drawing of
Family Devescovinidae Doflein, 1911
Description. Body asymmetrical; two anterior flagella (more could be present) arising from anterior area; trailing flagellum apparently in front of body; nucleus located in middle of body; parabasal body straight, not coiled around nucleus; axostyle extending through body.
Type species.
Description. Length, 31 μm; width, 23 μm; length of longest flagellum, 24 μm; parabasal body rod-shaped; axostyle not protruding from body.
Etymology: From the Greek "ikelos" meaning similar, in regards to the body shape resembling extant species of
Holotype: Specimen (accession # B-I-2) deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in Kachin state (26°20'N, 96°36'E), Myanmar (Burma).
Comments: The fossil resembles the body shape of extant species of
Family Holomastigotidae Grassi, 1892
Description. Body bearing emergent processes (spines), some of which bear flagella at their tips; axostyle not observed; rostrum absent; nucleus positioned at midbody.
Type species:
Description. Two adjacent individuals, upper individual (17 μm in length) with terminal process connected to girdle of lower individual (14 μm in length).
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek "akantha" for thorn, in reference to the processes on the body surface.
Comment: These two fossils are difficult to classify. Their spiraled flagellar rows and absence of an axostyle and rostrum show similarities to members of the extant genus
Family Trichonymphidae Kent, 1880
Description. Body short and broad; rostrum short; holdfast attached to intima of termite intestine; rostral flagella (only 2 visible) extend slightly over one half body length; axostyle not seen; putative nucleus positioned in upper third of body; parabasal bodies ribbon-shaped, haphazardly positioned in area surrounding nucleus.
Type species:
Description. Body acorn-shaped, length, 49 μm; width, 25 μm; posterior portion of body apparently filled with endoplasmic inclusions.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek "henos" for old.
Comment: This genus is placed in the family Trichonymphidae based on its relatively short rostral flagella, absence of an axostyle, and ribbon-like parabasals associated with the nucleus. One of three cysts of this or a related species in the same family is shown in Fig.
Possible amoebic cyst.
Possible amoebic cyst. Bar = 24 μm.
Family Teranymphidae Koidzumi, 1917
Description. Medium sized oval cell, with short rostrum attached to intestinal cells of host; rostral flagella short; post-rostral area with parallel flagellar rings without connections and giving the cell a segmented appearance; nucleus located under rostral area; putative axostyles (possibly 2) narrow, fiber-like.
Type species:
Description: Body length, 84 μm; body width, 46 μm; rostrum with rows of short flagella; post-rostral area with at least 30 parallel rows of flagella separated by ectoplasmic bands; parabasal body short.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek "rhabdotos" for lined.
Comment: Flagella inserted on parallel rows separated by ectoplasmic bands is a condition found in members of the extant genus
Family Oxymonadidae Kirby, 1928
Description. Body spindle shaped; length (including rostellum), 50 μm, width, 15 μm, length rostellum,17 μm; anterior nucleus approx 7 μm in longest dimension, positioned at base of rostellum; body with supporting fibers attached at base of rostellum; single axostyle extends length of body; faint holdfast at tip of rostellum.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek" protos" for first regarding its fossil status.
Comment: The presence of an anterior rostellum with associated fibers and the uninucleate condition are diagnostic characters of the genus
Description. Uni-nucleated flagellate with body comprising a single karyomastigont; body wider than long; axostyle short, not extending more than half body length; nucleus large, located near middle of body; rostellum with several fiber bundles.
Type species:
Description. Length, 41 μm; width, 51 μm; rostellum short, 8 μm in length; rostellum without associated fibrous structures; nucleus spherical, 11 μm in diameter.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek "geros" for old age.
Comments: The presence of an anterior rostellum with fibers arising from the base of the attachment point and the single nucleus is why this fossil was placed in the Oxymonadidae. While oxymonidids typically have an anteriorly placed nucleus, during certain phases, the nucleus may be located in the posterior portion of the body
Large spherical cyst with two nuclei
Large spherical cyst with two nuclei. Bar = 28 μm.
Small spherical cyst with a pair of nucleated cells
Small spherical cyst with a pair of nucleated cells. Bar = 14 μm.
Description. Large multinucleated flagellate with corona containing several karyomastigotes with small, anteriorly positioned nuclei; base of rostral area with filaments that extend posteriorly as axostyles; flagella attached at base of rostellum extend at least to midbody; rostral area hemi-spherical in shape.
Type species:
Description: Large cell, with knob-shaped anterior rostral area; total length, 159 μm; greatest width, 116 μm; length rostral area, 47 μm; width rostral area, 52 μm; at least 3 small nuclei positioned at base of rostral area; flagella attached to corona area beneath base of rostellum.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek "poly" indicating many nuclei.
Comment: The corona of several karyomastigotes with small nuclei, the large size and the modified rostral area align the fossil with the extant genus
Description. Axostyle prominent, thickened anteriorly, extending length of body; 4 flagella emerging from rostral area; nucleus located in lower half of body; large dark body at base of rostral area (macronucleus?); network of apparent fiber bundles throughout body; minute dark area at posterior end may represent tip of axostyle.
Type species:
Description. Length,106 μm; width approximately 1/2 length; length rostral area, 27 μm; width at base of rostral area, 36 μm; nucleus large, greatest diameter, 13 μm.
Holotype: Specimen (accession # B-I-2) deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology. From the Greek "katatonos" meaning broader than high.
Comments: This species is roughly the same size and shape as extant species of
Family Pyrsonymphidae Grassi, 1892
Description. Small rod-shaped, spindle cell with at least 4 spirally twisted flagellar cords adhering to body; small holdfast; axostyle slender; nucleus in anterior portion of body.
Type species:
Description. Body slender, 41 μm in length, 9 μm in width; axostyle extending length of cell.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek "speira" for twisted in reference to the body structure.
Comments: The fossil resembles extant species of the genus
Description. Large club-shaped, slightly spirally twisted flagellate with two nuclei positioned slightly below mid-body; with several flagella (some adhering to cell body) arising from anterior end of cell; anterior tip with thickened tubular area; axostyle not detected.
Type species:
Description. Length, 152 μm; width, 48 μm; length longest flagellum, 98 μm; length cephalic tube, 24 μm; tubular area (holdfast?) inserted in host's intestinal intima.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek " kordylinas" for club-shaped in reference to the shape of the fossil.
Comment: This fossil resembles the extant
"Sarcodina"
Amoebae of uncertain affinities (Patterson et al., 2000).
Description. Spherical nucleus in process of dividing; nucleolus not apparent; body amoeboid-like, nearly spherical, with broad pseudopodia and short protuberances.
Type species:
Description. Greatest diameter, 33 μm; nucleus undergoing division; diameter of upper nucleus, 8 μm; endoplasm containing particles of various sizes.
Holotype: Specimen B-I-2 deposited in the Poinar amber collection maintained at Oregon State University.
Locality: Amber mine in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma).
Etymology: From the Greek "proteros" for earlier.
Comments: The size and shape of the body and nucleus resemble those of extant species of
Spherical cyst with two or more nuclei
Spherical cyst with two or more nuclei. Bar = 14 μm.
Additional protists
Additional unknown protist stages associated with
Pyriform cyst
Pyriform cyst. Bar = 12 μm.
Spherical cyst
Spherical cyst. Bar = 14 μm.
Possible ciliate
Possible ciliate. Bar = 14 μm.
Possible ciliate
Possible ciliate. Bar = 12 μm.
Possible ciliate
Possible ciliate. Bar = 14 μm.
Possible ciliate
Possible ciliate. Bar = 10 μm.
A, B. Photo and drawing of a dividing cell with a double rostral area, each with an axostyle
A, B. Photo and drawing of a dividing cell with a double rostral area, each with an axostyle. Specimen partially embedded in gut of host. Bar = 12 μm.
Example of double cell with one mastigont being extruded
Example of double cell with one mastigont being extruded. Bar = 6 μm.
Unidentified flagellate, probably of the family Trichonymphidae
Unidentified flagellate, probably of the family Trichonymphidae. Bar = 28 μm.
Cell undergoing binary fission
Cell undergoing binary fission. Bar = 12 μm.
A pair of elliptical cells
A pair of elliptical cells. Bar = 13 μm.
Unknown stages, some of which could be ciliates, are shown in Figs.
A pair of dauer juvenile rhabditid nematodes were also associated with
Discussion
The present study represents the first descriptions of protists from a fossil termite and the earliest fossil record of mutualism involving microorganisms and animals
Successful establishment of protozoa in xylophagous insects necessated particular attributes. The protozoa had to withstand the chemical and physical conditions inside the insect's alimentary tract, utilize the gut contents as a food source, cause no damage to the host, and to be carried through successive insect stages and generations. These protists apparently underwent a long period of co-evolution with their hosts, since some lineages found today in the intestines of xylophagous cockroaches and lower termites are thought to have been established in Carboniferous Blattida
Today, the composition of intestinal protozoa tends to be correlated with the phylogenetic position of their termite hosts and it is possible to classify families, genera and even species of termites based on their flagellates
One striking difference in the behavior of the protist symbionts in extant termites and
Several possible scenarios could explain the presence of cysts in
A), the colony was subsocial and ingesting cysts in fecal pellets (coprophagy) was the main method for the intrastadial and intergenerational transfer of protists; B), the colony was subsocial or eusocial and both coprophagy (ingesting cysts in fecal pellets) and proctodeal trophallaxis served to transfer the flagellates to nest mates and hatchlings; C), the colony was eusocial and protozoa were transferred by proctodeal trophallaxis. The cysts were an ancestral carry-over and represent an evolutionary dead-end; D), some of the cysts could have been ingested while feeding and had no trophic association with
Materials and methods
Specimens
The amber with the fossil termite containing the protists is roughly semi-circular in outline, measuring 13 mm along the longest edge, 10 mm in width and 1 mm in thickness. Observations, drawings and photographs were made with a Nikon SMZ-10 R stereoscopic microscope and Nikon Optiphot compound microscope. Since all photographs were taken through the thickness of the amber matrix, it was not possible to get as close as desired to individual protists without polishing away adjacent ones as well as portions of the termite host. Therefore all photos were taken at 20×. With such a small image, it was only possible to obtain a single clear photo of the specimen since further fine adjustment produced blurry images. Adobe Photoshop was used to enlarge the photos and to obtain several modified images by using different settings of contrast, light intensity and resolution. The drawings were made from a combination of the modified images and that is why they contain more detail than the corresponding photographs, which represent the best single image obtained under the various settings. Thus Photoshop manipulation was used to replace optical sections, that were not possible to make with such small objects and at such a low magnification.
Locality
The amber was obtained from a mine first excavated in 2001 in the Hukawng Valley, southwest of Maingkhwan in the state of Kachin (26°20'N, 96°36'E) in Myanmar (Burma). On the basis of paleontological evidence, this new Noije Bum 2001 Summit Amber Site was assigned to the Early Cretaceous, Upper Albian
Type material
In accordance with section 8.6 of the ICZN's International Code of Zoological Nomenclature, copies of this article are deposited at the following five publicly accessible libraries: Natural History Museum, London, UK; American Museum of Natural History, New York, USA; Museum National d'Histoire Naturelle, Paris, France; Russian Academy of Sciences, Moscow, Russia; Academia Sinica, Taipei, Taiwan.
Source
Nuclear magnetic resonance (NMR) spectra and the presence of araucaroid wood fibers in amber samples from the Noije Bum 2001 Summit site indicate an araucarian (possibly
Competing interests
The author declares that he has no competing interests.