If not otherwise stated in the caption the microscopical pictures were taken through the 100x HI objective.

 It is raining in Cancún. The rainy season has started and it is now time to hunt for ….. tree holes. Where a branch had fallen or has been cut; many times a more or less deep hole develops. It is a cup waiting for the rains.

 For eight or perhaps more months the hole has received dust and leaves, or other detritus that was contributed by the wind, including the cysts and spores of living things. Now the rain fills in the hole with neutral or acid water, in a city were all natural water is hard. (Over 30 DH.)

 It is an opportunity to see if there is something living in those special microhabitats.

 The first tree hole is no more than twenty meters from my house were a young doubly branched tree was pruned to allow only one stem to grow.  With time at 30 cm from the ground an oval hole with 25 x 15 cm diameter has developed with a depth of 6 cm.

Fig. 2

 Four days ago it was dry with a consolidated earthy bottom and some small leaves in.

 Today it is filled with rainwater. And the water looks a little muddy and with a superficial film of bacteria. It is an invitation to see the initial fauna of a recently flooded tree hole (fig.  2  above).

  With my “sampler” (a little ladler) I took more or less 50 ml of water, and a very little sample of dirt from the bottom, which I install in a 100 ml cylindrical flask for a couple of hours before starting the examination.

Bacteria - stained with Gentian Violet.	Astasia - showing the density in a typical sample. Obj. x4

The immediate exploration of the superficial and intermediate waters shows plenty of bacteria (mostly bacillus rods and some spirillum) and an indefatigable population of flagellates that swim at a velocity that made it impossible to shoot even one in-focus picture. To show the density of the population I only had to resort to adding a drop of GALA, to definitely stop the out of control movement. This also permits a better understanding of its morphology and permits us to assign the majority of the population to an euglenoid flagellate of the genus Astasia.

 
Astasia Ehrenberg 1838, is a small flagellate, of extremely fast movement, which reached great densities in this culture. We can see this in fig. 3 taken with the 10x objective.

 In order to see its morphology in more detail I must use at least a magnification of x1000 which requires the immersion objective, a very thin preparation and a coverslide solidly fixed with a sealant. I used nail polish varnish.

 In normal swimming Astasia appears fusiform, with the anterior end more or less blunt, and an acute caudal end (fig 3). In the anterior end there is one short invagination of the pellicle (canal, or reservoir) erroneously denominated in English as the "gullet". When some euglenoid species ingest foods, (like Peranema, by ex.) they do it generally with a temporal buccal opening, or one that is permanent and provided with reinforcing rods (trichites) always placed outside and below the canal.

Fig. 3-Astasia klebsii: alive (at right) and a big specimen fixed with GALA at left

  Instead the reservoir provides space and protection for the insertion of the flagella.

 Astasia species are practically colorless, because they do not have chloroplasts like Euglena nor do they possess euglena's characteristic red stigma generally found in the base of the reservoir.

 The position of the reservoir allows the differentiation of two subgenera of the genus Astasia. These are Euastasia and Euglenoidea. The population in my tree hole, by the apical position of the reservoir (fig 4) and the flagellum, belong to the subgenus Euastasia Christen 1963. In the subgenus Euglenoidea Christen 1963, the reservoir and therefore the emergence of the flagellum is subapical, as it occurs in Euglena. 

Fig. 4 - Astasia (Euastasia) klebsii - extended and fixed individual, and two pre-palmelloid states

Alive, the most remarkable anatomical elements, aside from the flagellum are the large contractile vesicles located next to the flagella canal, and the grains of paramylon that tend to group itself at the rear. Paramylon is a complex carbohydrate that is easily recognized because it does not give the typical bluish black coloration of the starch, when tested with iodine.

 The individuals that live in the substrate, or even some swimming ones, show a typical movement called "euglenoid movement" that consists of a local expansion of the cell, shaped like a knot, which moves up and down throughout the cell. Although it is shown spontaneously in cells that have rejected its flagellum, it's easy to initiate the euglenoid movement by adding to the drop with the euglenoids a very small drop of fixative. Irritation causes the movement.

 In the individuals with euglenoid movement the grains of paramylon tend to concentrate themselves in the posterior end, which in these cases is spherical.

 The nucleus is only visible in favorable individuals, and it is not very often well defined.

The size of individuals varies of course with their contraction state, from 30 to 40 micrometers in individuals in normal swimming (fig 3) up to 60-65 micrometers in totally extended individuals (fig 4). The flagellum is at least one and a half times as long as the swimming cell, and the grains of paramylon are short cylindrical rods barely 3.5 to 4.0 micrometers in length with a diameter of 1.5 to 2.0 micrometers. The nucleus has an average diameter of 6 micrometers.

The previous description and the captured images are consistent with the species Astasia klebsii Lemmermann 1910.

 The effect of asphyxia.- if one leaves alone a sealed preparation for some minutes the individuals react to the lack of oxygen in a quite homogenous form. They shed the flagellum, and they adopt a shape that goes from an ellipse to a sphere. This is a defense state called palmelloid, that generally is completed by the secretion of a mucilaginous sheet, which allows the protist to subsist until finding itself again in favorable conditions. They accumulate paramylon in the posterior end and the spherical nucleus is clearly visible in preparations fixed with GALA, with a large endosome and a granular content (fig.4).

 
Khawkinea Jahn & McKibben 1937

 On the day of harvesting, the population was formed by myriads of small Astasia klebsii. But after three days in the laboratory, the dominance switched to a somewhat bigger species, wider and with a more tapered shape, Khawkinea sp.

 Khawkinea has a subterminal (lateral) reservoir, in a position similar to the one of Euglena, and it also has one red colored ocular spot (stigma). Its only difference with the euglenas is that it does not have chloroplasts. In laboratory experimental conditions Euglena can be induced to shed its chloroplasts, and those individuals are hardly distinguished from a Khawkinea.

Fig. 5 - one Khawkinea alive, and one recently fixed. Notice the red ocular spot and the subterminal position of the flagellum emergency point. In the left picture the canal can be seen.

 To study this species more easily I added to a 1 ml sample 10% of Rhode’s fixative which induces the sedimentation of the suspended specimens, be they euglenoids or ciliates, and gives them a mostly brown color.

 The length of a typical specimen is around 45 micrometers, with a flagellum at least of the same length, and even longer. In the fixed individuals the nucleus measures 7 mic., and a grain of paramylon taken at random measured 5 x 3 mic.

 An individual stigma (fig 5, right) is seen in front view, and this allowed the measurement of its diameter which was 2.2 mic. As the stigma is always found at the bottom of the flagellum reservoir, the measurement from stigma to the flagellum point of emergence gives an estimation of its length, which turns to be near 9 mic. 

Fig. 6 - A Khawkinea, fixed with Rhode's. 3 amalgamated images to show the entire flagellum length.

 As seen in the pictures the iodine present in the Rhode’s hasn't colored blue any inclusion.

 When fixing the specimens, or when these die by asphyxia they contract and adopt a tennis racket shape. Generally dead individuals have shed the flagellum, but most of Rhode’s fixed ones show it very well (fig. 6). The high density of the grains of paramylon prevent the nucleus or the contractile vacuole to be seen.

 Euglena sp.

  In only one preparation appeared one lonely individual of a phototrophic flagellate. It was a species of Euglena of 100 microns length, with small chloroplasts and cigarette shape. Perhaps it was the announcement of a future change in the population.

 Ciliates from the surface. 

 At much lower density (only one or two individuals to each two or three preparations) appeared what I think are two species of Dileptus, a large and narrow one (Dileptus sp.1), and another smaller but wider one (Dileptus sp. 2). When little drops of fixative were added to stop them, the individuals rounded up and edematized. (fig. 7). Only individuals fixed with Rhode's maintained a suitable morphology which allows them to be shown in a picture.

fig 7 - bad reaction to anaesthesia intent

 Dileptus  moves near the surface below the bacterial veil. It swims impelled partly by his cilia, but extending its long neck and waving it in a helical manner (akin to the movement of a flagellum) while rolling in the water as they advance. This movement allows us to easily distinguishing this genus from Amphileptus, of very similar aspect, but of shorter and rigid neck. In the base of the Dileptus neck a great mouth is opened, oriented forwards, circular and reinforced by trichites that turn it into a distensible funnel.

The smaller but wider species measures 200 to 250 micrometers of length with a maximum width of 50 to 58 mic. In the photographed individual the neck represents 32 to 40% of the total length.  In the longest and narrowest species the corresponding measures were: Total length 330 to 360 mic, maximum width 35 to 39 mic. Neck/total length 40-48 %. The proportion of wide to total length was 10-12% in the narrow one and 20-24% in the wider.   Measurements were taken on pictures of live individuals, swimming normally, that cannot be reproduced here because the movement of the protists doesn’t allow sharp enough images to be captured. Both species have a thick and long macronucleus which seems to be built of two sections slightly displaced one with respect to the other.  The bottom and its inhabitants. The accumulated sediment in the bottom showed it is composed fundamentally by ejections of previous inhabitants. By its form and size they seem to be faeces of some mosquito species. (fig. 9) Amongst them they teem Khawkinea, and also exists an abundant population of one small Vorticella with annulated pellicle, and with a hardly 40 mic. chalice length (fig. 10). By its general appearance, estriation and position of the nucleus it seems to be V. cupifera Kahl, 1935 (You can see an illustration in pag. 724, fig. 9 of Wimpertiere oder Ciliata, on-line facsimile).  In the water immediately in contact with the sediment it moves with enormous speed and executes true zigzag jumps, (bouncing like a ball it is said sometimes to resemble) one abundant population of Halteria grandinella, a spectacular small ciliate, with a diameter not greater than the length of the flagellates.

Fig. 8 - a big individual of the Dileptus sp. 2

fig. 9 - another Dileptus sp 2, labelled

But like Dileptus, this species is impossible to photograph alive without the aid of an electronic flash. Nevertheless Halteria is fixable in an acceptable shape by means of Rhode’s fixative. Although the fixative distorts a little the ciliation in fig. 11, easily recognizable are the Adoral Zone of Membranelles, its Macronucleus and the Equatorial Band of Stereocils that allows its acrobatic antics.

fig. 10 - detritus in the bottom of the tree-hole. obj. x 4. Dark stop 12 mm	fig. 11 - remains of arthropoda in the bottom detritus. x 4 obj.

Previous inhabitants.  Aside from the evidence of the existence of dipteran larvae commented above, they appeared between the bottom sediments, fossil-like remains of other previous colonizations, a small Centropyxis and the remains of 2 bdelloids, totally lacking of all content except for trophi. From the three fingers of the foot and the lack of spurs it seems to belong to the genus Rotaria.

fig. 12 - Halteria grandinella. Fixed with Rhode's.	fig. 13 - Vorticella cupifera alive.

IN SUMMARY

 Four days after its first filling, our tree hole of 15 x 21 cm with 6 cm of depth, supports an enormous and diverse bacterial population, three species of flagellates and four ciliates. Both flagellates do not have chlorophyll, neither mouth, nor other elements for the capture of particulate food, and they are known to be osmotrophics.

  Along with the bacteria, they exploit the abundant dissolved organic and inorganic matter the rainwater extracted from the existing bottom sediments of the tree hole.

fig 14 - remains of a bdelloid rotifers in the bottom detritus x 40 obj.	fig. 15 - the shell of a Centropyxis in the bottom detritus x 40 obj.

Euglena is obviously phototrophic, although it can also participate with the absorption of dissolved substances, and the ciliates are all heterotrofic. Vorticella and Halteria they are bacteriophages and microdetritivores.

Dileptus is considered a predator. Being no other visible prey we must consider that they consume the so abundant flagellates and Halteria. But I did not see them in any capture activity, nor do its vacuoles reveal digested Khawkinea remains. Its enzymes must be really active.