The biofilms that upholster and cover the walls of some caves can be inhabited by a wide variety of microorganisms, although green algae and cyanobacteria generally abound in these communities.
In the surroundings of the "Ares Station", an operations center of the company Astroland Agency, located in the “Cueva del Escalón”, Valle de Asón (Cantabria, Spain), part of astrobiological research work is carried out that has as its objective the study of the biofilms found in their environment.
The facilities of the "Ares Station" fundamentally serve as support for sampling activities and preliminary studies of microscopic research in situ.
They test different protocols related to microbiological research, the appearance of life on our planet, or the search for signs of life on Mars.
The facilities that make up the "Ares Station" are respectful of the environment in which they are located and try to protect the biodiversity of biofilms through different monitoring and study actions.
In the surroundings of the "Ares Station", these biofilms have proved to be a treasure trove of microbial biodiversity. They are mostly made up of a complex and varied tapestry of cyanobacteria, many of which are little known, and which are currently being investigated in several projects, but also host other microorganisms of great interest, including tested amoebas such as Difflugia alhadiqa, the subject of today's article, discovered and described for the first time by colleagues Carmen Soler-Zamora, Miguel González-Miguéns, and Enrique Lara, very recently, in the Cueva del Hundidero, Montejaque (Málaga, Spain).
Discovering this species in Cueva del Escalón is like finding a jewel among the treasure of biofilms that inhabit its damp walls.
Fig.1 - Difflugia alhadiqa from a fresh sample of cyanobacterial biofilm. Photographs taken at 400x magnification with the epifluorescence technique, FLED module, and the Motic 40X/0.65/S (WD 0.6mm) dry objective. Equipment used: Motic Panthera CC trinocular. In the upper left part and under the theca of the amoeba - in greenish color and in which the xenosomes that cover it can be seen - the cells of some colonies of Gloeobacter violaceus stained with an intense yellow color can be observed.
This is the first time that Difflugia alhadiqa has been photographed for science, using the epifluorescence technique, which makes it possible to observe some details that are very difficult to appreciate with other microscopic observation techniques, in this case using a Motic Panthera CC trinocular equipment, equipped with the Motic FLED fluorescence module.
The one photographed and described here is very likely the second record of this species in the Iberian Peninsula and the first time it has been reported from the north of the peninsula.
Fig.2 - The same previous sample of Difflugia alhadiqa photographed in brightfield technique with the Motic 40X/0.65/S (WD 0.6mm) dry objective. Equipment used: Motic Panthera CC trinocular. In the upper left part and under the theca of the amoeba, the colonies of Gloebacter violaceus are not well appreciated, which with the Motic FLED module are stained with intense yellow color. The xenosomes are not very well differentiated either, although they can be seen in the previous image, due to the use of a different lighting technique.
Amoebae of the genus Difflugia represent the oldest and most extensive group of shelled amoebae known. Although now subject to revisions, fundamentally based on genomic sequencing studies which try to establish how many taxa it includes, it is estimated that it encompasses a number close to 300 species, all of them characterized by the presence of a theca built on a thin membrane from of the small mineral fragments that these amoebas find and collect in their environment, and with which they make, like the expert architects and potters that they are, the house that shelters them.
Fig.3 - Difflugia alhadiqa from a fresh sample. Photograph taken at 400x magnification with the epifluorescence technique, FLED module, and the Motic 40X/0.65/S (WD 0.6mm) dry objective. Equipment used: Motic Panthera CC trinocular. It is shown the contours in its middle section and some isolated cyanobacteria of Gloeobacter violaceus in yellow.
The shape of this stone house that covers it, made of uncarved blocks, has served to establish a very elementary taxonomy of the group, and not very precise, since on many occasions it is insufficient to be able to accurately determine each species, given that the grains of quartz that generally make up this shell, are opaque and do not allow to recognize other internal characters that may be important when it comes to knowing it.
Difflugia theca is formed by an agglutinated and cemented layer of mineral particles, quartz fragments, or diatom frustules that are generically called xenosomes.
Fig.4 - The same previous sample of Difflugia alhadiqa, but in a different shot, showing another more superficial layer of its theca, photographed in brightfield with the Motic 40X / 0.65 / S (WD 0.6mm) dry objective. Equipment used: Motic Panthera CC trinocular. In the upper left part and under the theca of the amoeba, the colonies of Gloebacter violaceus are not well appreciated, which with the module with the Motic FLED module are stained with intense yellow color. The xenosomes are not very well differentiated either, although they can be seen in the previous image, due to the use of a different lighting technique.
Today the amoeba Difflugia alhadiqa has retracted its thick arms of soft branches, its pseudopods, inside the theca. But when they come out of it, they spread out like slow rivers, imitating the ramifications of some of the cyanobacteria among which they live, bringing their bodies closer to the light. He has nothing to fear, his stone house is a wandering cave, within another cave.
Fig.5 - The appearance of the cyanobacterial masses inhabited by this amoeba in the surroundings of the Ares station. The greenish and yellowish masses correspond to Stigonema informe and the pink ones to Gloebacter violaceus.
Photography by Jesus Rocandio.
Photography by Jesus Rocandio.
Difflugia frequently select and organize these xenosomes according to their size and shape to build a shell with a unique morphology for each particular species. This shell always has a terminal opening through which Difflugia alhadiqa sticks out its broad hands and feet. Its pseudopods are always oval, round, or lobed, but never slit-shaped or with an internal diaphragm as in other closely related genera.
The nucleus is generally oval, but it may be vesicular in larger species. Some species are multinucleate, and often the largest freshwater species can establish symbiosis with green algae that will end up living inside them.
Difflugia alhadiqa is a very little known taxon and with this publication, the knowledge of it spreads. Its outlines are rough and its silhouette more or less pear-shaped, with a shell composed of angular quartz fragments of very different sizes, joined together by an organic cement.
With dimensions ranging from 25–35 µm wide and 45–60 µm long, 13–25 µm aperture diameter, this circular section in D. alhadiqa is very different from the trilobed one in D. baculosa. D. alhadiqa resembles D. glans, but the dimensions of the former are notably smaller; furthermore, the necklace of small particles surrounding the opening is not as conspicuous as that of D. glans. Difflugia hiraethogii is much larger and its theca has a very well differentiated neck that is not well appreciated in D. alhadiqa.
Despite its rough appearance, Difflugia alhadiqa is a real gem, like an unpolished diamond, which will shine with further study.
All the photographs have been taken at a magnification of 400, with bright field and epifluorescence techniques, with a Motic Panthera CC trinocular equipment and come from the samples collected inside the Escalón cave, in the Ares Station environment, in a very dimly lit area where the Astroland Agency is developing an approach to learning about Mars in its astrobiological project.
Fig.6 - Observing Difflugia alhadiqa samples with the FLED module and the Motic 40X/0.65/S (WD 0.6mm) dry objective. Students of 1º of Baccalaureate of the subject Scientific Culture in the “IES Escultor Daniel" (Logroño, Spain)
Today this amoeba is shown for the readers of this publication, and for the 1st year Baccalaureate students of the “Escultor Daniel” High School in Logroño (Spain), who have participated in these observations handling the Motic equipment, learning to handle this equipment both with field lighting conventional light, as with the FLED epifluorescence module, essential to identify and see the mineral particles that form the theca of this amoeba.
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