cockroaches parasites 3

The materials used for this work were 6 specimens of Periplaneta from Durango and 6 others from Cancún.

Anesthesia of the just captured animals (Durango) or those collected from material immobilized by the insecticide in Cancún was induced by putting the specimens in the refrigerator at 4º C, in a closed capsule of course.

In general it is best to initiate the work with anesthetized insects, not dead ones, to avoid any damage to the parasites of our interest. But see next paragraphs.

In spite of the immobility of anesthetized animals, which does not respond to the external stimuli, the antennas, legs, and buccal palps continue moving, because in the long nervous ventral cord there are many independent ganglia that govern them. (A similar phenomenon occurs in the batrachians, in which, even after anesthesia and decapitation, the heart continues beating for hours, due to the presence of intracardiac ganglia, independent of the brain.)

If this makes you uncomfortable, euthanasia of the animals anesthetized by cold can be completed by submerging them for a minute in a more or less concentrated solution of domestic detergent (dishwasher). The detergent is immediately absorbed by the tracheal system and it kills quickly without affecting the hosts of the digestive tract.

Head is first severed out, and next are the legs, with the help of fine pointed forceps and scissors (the best ones, are the professional instruments used for iridectomy, but, see the Appendix). Next the body must be pinned to a small dissection tray with thin but rigid pins (the best are the kind used by entomologists to pin their specimens, but you can use the thinnest and longest steel hand sewing needles. Do not use ordinary stitching pins)

With the scissors you must cut the ligaments on the righthand side of the abdominal sternites, beginning at the rear end, and the ventral plate so released is hinged towards the left side, clearing its adhesions to the internal organs with sharp needles or with a microscalpel, and is discarded or pinned down. You may want to see the centrally placed ventral nervous cord with its many ganglia and nerves.

All this can be done in the air, but the following manipulations give better results if they are made under water. A physiological saline solution designed to work with insects can be used to avoid damages to tissues by osmotic pressure differentials. (See the APPENDIX.)

Nevertheless due to the annoying reflections of light on the surface of the water, even sacrificing some resolution, the following photo was made in the air.

With thin and sharp teasing needles and (or) very fine pointed forceps, the fat that surrounds the abdominal organs is removed. The alimentary canal is easily isolated and it can be set free from its ties. If you want to separate it more or less completely, the gizzard must be moved with the tweezers until the esophagus is seen and then it must be cut distally, the rest of the digestive tract can now be liberated with the needles, and cut at the other end, at the level of the anus (or cloaca). It is then possible to end with a preparation like this:

But, given that gizzard and crop normally do not have parasites of interest to us, you may wish to separate only the intestine and the rectum.

Identify the intestinal cecae. With the help of the forceps and the scissors, make two cuts in the intestine: one below the cecae, and another one at the level of the cloaca. The separated intestine is transferred to a capsule with clean physiological solution. With the two rigid teasing needles, fine and sharpened, the intestine must be open alongside, releasing its content. Do not use a micro-scalpel; you risk cutting in pieces the parasites. With due care remove the intestinal tissues and discard them.

The material in the capsule is preferably examined over a matt dark background (a black box ( see Appendix), or a matt black velvet) with the help of an upper light, this will make it easy to see the nematodes which are wriggling most of the time, or the ciliates swimming in the bottom of the capsule.

Separate the most individuals you can, with a fine pointed pipette or a fine pointed brush, with the help of a magnifying glass of 6 to 10 powers (or the low power - 8.5 x on my microscope - see "Köhler microscope" article). Those who have a stereomicroscope already have the really effective tool.

Collect the nematodes into a small capsule in a few of milliliters of physiological solution.

An old, but always useful method to fix nematodes is to warm water or alcohol 50%, to 50ºC in a small test tube over a low flame. (Take care and protect your skin and eyes from the hot liquid projections. The mouth of the tube must always be directed away from your body.)

Gathering the nematodes with a fine brush, submerge them quickly in the fixative. The organisms are thus fixed in extension.

Now pour off most of the liquid without losing nematodes and replace it with AGA (alcohol 70% with 10% of glycerin and 1% of acetic acid). Set apart the specimens in a well stopped vial to examine them later.

Return to the sediment in the capsule and prepare now 3, 4 or more thin wet mounts. Seal them using molten paraffin wax, from a candle, or better VALAP or VAPA (see the APENDIX). These preparations will allow you to investigate inmediately the sample with the 40x and even 100x objectives, to search for the parasitic protozoa. Make your drawings, take your notes, shoot your pictures. The individuals will be alive for a while.

If there were sufficient individuals in your material, and you have left some live nematodes, the moment to place them under the objective has now arrived. Observation of live worms can give interesting data, but in such transparent animals that accept a perfect clarification in glycerin, it is not essential for arriving even at a specific determination.

To examine in detail the nematodes, you can pass after an hour the fixed material to glycerin 20%, and after another hour to 30% glycerin. If you want to make permanent slides use the method of Seinhorst for glycerin mounting that was detailed in my article on mounting media. It is the professional method to mount and preserve nematodes.

In only 12 dissected Periplaneta americana I found two protists and two species of nematodes that I describe in detail below.

The genus is Endamoeba Leidy, 1879, whose type species is exactly Endamoeba blattae; and by no means Entamoeba, Cassagrandi & Barbagallo, 1895, which is the genus that is normally applied to the most common parasitic amoebas of man ( E. hystolitica, E. coli, E. gingivalis) and to other several species parasitic on other vertebrates.

Apparently the International Commission of Zoological Nomenclature considered as a valid genus for all those amoebas, the one founded by Leidy. But the common use in the bibliography, particularly in North America, favors Entamoeba for the human and other vertebrate parasites.

In what is inappropriate from every point of view, many authors extend the use of Entamoeba to the amoebas of invertebrates. Do not follow this trend.

Endamoeba has two known species, both parasitic on insects. The other is E. simulans, parasitic on termites.

Endamoeba blattae is a big species, easy to observe, with a clear and homogenous cytoplasm and a globular nucleus with a wide peripheral clear ring and a darker center. It belongs to the kind of amoebas that move like the free living A. limax.

They do create a unique and ample "rolling" cytoplasmic front. Its cytoplasm, still being amoeboidal, that is to say, easily deformable in any sense, does not produce the common pseudopodia expected for most of the free amoebas.

Three aspects of the locomotion of Endamoeba blattae.
At bottom one individual is crawling over a Malpighian tubule. In the center it has freed itself and floats in the liquid. And at right the amoeba has settled over the slide, developed an uropod and start gliding to the right.

The similarities with the free living amoeba are clear.

It is not probable that this amoeba causes serious damage to the cockroach. Apparently their food is grains of carbohydrates not digested by its host, and probably also bacteria. It seems also not to be very common because I only found it in one individual. Any type of oblique illumination allows satisfactory images.

Nyctotherus is a genus of parasitic ciliates very common in other invertebrates, and also in different vertebrates (frogs for example, which hosts another very common species: N. cordiformis).

It has cytological characteristics that have turned it into the star of important investigations on the life in anaerobiosis. The favorite species to conduct those studies is exactly the species that inhabits the Periplaneta.

N. ovalis is a big species, pear-shaped, but somewhat laterally compressed. It is enveloped by a thick and stiff cuticle, that bears the numerous and long cilia. The thin end is the anterior one. It has a nucleus, visible alive, which in lateral view looks fusiform and granular.

The nucleus is surrounded by a membrane, that some denominate suspensorium, which adheres to the “dorsal” cuticle at a higher level than to the “ventral” one.

a full picture (two stitched images) of Nyctotherus ovalis with the x 100 HI objective

So, the nucleus and its suspensorium separate the ciliate in two well defined zones. An anterior and smaller one filled with darker particles, and a bigger posterior one full with lots of clear granules.

In lateral view one can see along the frontal side a long, deep, and clear furrow (the peristome) that lodges an undulating membrane reaching the suspensorium base. The peristome ends in a funnel open to the exterior creating a false cytostome, that gives access to a long cytopharinx also provided with an undulating membrane, which extends under the nucleus almost in contact with its inferior wall, and finally curves downwards to open in the cytostome in which the alimentary vacuoles are born. These are practically never distinguished, because they are obscured by a countless multitude of small opaque grains that fills all the later part of the protozoan.

At the rear end can always be seen a big and clear excretory vacuole extended cross-sectionally.

What makes the Nyctotherus famous is the fact that the opaque grains that fill their cytoplasm are of a very special nature.

Nyctotherus lives inside an intestine, in the middle of a mass of decomposed food, practically without oxygen. How it can even fulfill its vital functions to be even the most frequent and numerous inhabitant in the intestine of most cockroaches?

It does because it has been able to obtain energy for its vital functions from the hydrogen of the methane, produced by bacterial fermentations in the intestine of cockroaches (and also in that of human beings). As methane fluoresces it can be detected inside the Nyctotherus with UV light. See an example in this site.

Nyctotherus do not have true mitochondriae. (See part 1) Its cytoplasm is full of prokaryotic derived symbionts named hydrogenosomes, which uses as a source of energy hydrogen, not oxygen.

That is the secret of its adaptation to the LIFE IN THE INTERIOR. That secret has been investigated by tens of scientists that have been able to determine, from the morphological and chemical structure of the hydrogenosomes, that these are probably derived from the mitochondria of other ancestral cells, or from a former prokariote that was at the same time the ancestor of both mitochondria and hydrogenosomes, as an adaptation to the metabolic exigencies of the life in intestinal media. The battle over the nature and origins of the hydrogenosomes has not ended and is investigated using different fragments of DNA from hydrogenosomes and mitochondria.

Nyctotherus was present in most of the specimens dissected in Durango, but it did not appear in any of the Periplaneta dissected in Cancún. This is suggestive and provoking, but of course the small number of specimens dissected in each locality does not allow us to do affirmations, or even hypothesis, of any kind.

Any metallic container of about 15 to 18 cm. wide by 20 to 22 cm in length and 3 to 4 cm deep can become a useful tray for dissecting adults and larvae of insects, oligocheta, small vertebrates, etc.

The only one essential adaptation is to provide the tray with a floor that does not float, that receives well the dissection pins, and that preferably has a dark color, so that the removed pieces of the dissected subject stand out better. In shops that sell products for crafts (artistic candles), you can found a good enough paraffin. In

Mexico

and other religious countries there are sold big votive paraffin candles (here called “veladoras”). Buy the necessary paraffin to produce about 400 milliliter of melted paraffin. In order to obtain the black, coffee, or very dark green color you need, you can resort to pigments soluble in the paraffin. The best source are the same shops for handicrafts. But if necessary you can resort to buying the "wax pencils”, or “crayons” children used in schools, and to mix the suitable ones with melted paraffin. Mix well to have a homogeneous color. According to the hardness of paraffin, you can add solid petroleum jelly to it (does not matter if it is perfumed or not) until you have a paste that solidifies but admits easily and without cracks the pins necessary to hold the dissected bug. After a time of use the adhered impurities and the holes left by the pins will ruin the surface. As the tray is metallic it is simple to place it over the fire or in the oven to melt the paste. All the impurities will fall to the bottom and the surface when cooling will be totally recovered.

With some skill it is possible to construct or to adapt the few instruments necessary.

Scissors. The stainless steel scissors with fine curved points that manicurists use could do a good job if you select them with care, and take time to fit screws and to sharpen the cutting edges with a small fine grained stone.

Micro-scalpels. Shaving blades of one or two edges can be cut in fine triangles each with an edge, if you press them between the jaws of a bench vice (a jewellery “baby” bench vice is ideal), and bend them with the aid of a piece of hard wood. It is of course a task for a careful adult. Cut pieces of 12-15 cm from a wooden rod of 3 - 4 mm in diameter (here in Cancún they are easily obtained from stores that sell articles for schools). If you want, with a cutter or a pencil sharpener give a conical shape to one end. Press the blades edges between the jaws of the bench vice with their points down. With a hammer, and care, fit the wooden handle to the blade. If you can not find shaving blades, pieces of steel springs from more or less old clocks provide a steel with magnificent qualities. It is only necessary to have a rotatory sharpening stone to give a good shape to it.

Teasing needles. Hand sewing needles, of steel, long and slender, can be mounted in wooden handles with the same procedure used for the scalpels.

Micropipettes. You must follow the clear indications of Jean Marie Cavanihac:

Transparent plastic dishes. To substitute the small Petri dishes of prohibitive prices now; in the handicraft shops and those selling fantasy articles for children's birthdays, small boxes are sold to package candies or small gifs. They serve very well for these aims. Add some plastic plates recovered from packages of medicine pills or tablets, with cavities of different form and size. They will be most useful to separate specimens or to deal with them.

Of course who can make required investment will do well in following the detailed instructions of R. L. Howey for the adaptation and improvement of relatively cheap surgical instruments.

You must use preferably a physiological solution for insects. The professional prescription is the following one:

Dissolve 9g sodium chloride (NaCl), 0.2g potassium chloride (KCl), 0.27g calcium chloride (CaCl₂•2H₂O) and 4g glucose. Add enough distilled water to bring final volume to 1000ml. Adjust pH to 7.2 with sodium bicarbonate (NaHCO₃)

But this one is an amateur formula which works: in 1000 milliliter of filtered rainwater, or bottled plain water (do not use distilled or demineralized, we need the minerals normally present in the water) dissolve 9 grams of a good and fine kitchen salt and 4 milliliters of syrup for breakfast with fructose or glucose. Caution: do not use the domestic water supply; it has much deleterious chlorine and chloramines.

Make pH slightly alkaline with sodium bicarbonate (7.2-7.4 by ex.) To measure pH the best thing is to use a kit of those used for aquariculture, selecting a pH paper or solution with a rank akin for our necessities. David Walker reviewed the subject for the

It is difficult to obtain a perfectly black background. All the materials reflect the light in greater or smaller measure. The ingenuity of physics, and photographers – mainly the ones specialized in macrophotographs – produced the "black box".

A "black box" is a relatively big box, with the interior walls covered with matt black velvet, whose upper face has a relatively small orifice. The size of the black box depends on the use to which it is destined, for that reason the term "relatively" is used here. The most useful shape for you is a cube, but the former scientific black boxes were spherical, that shape optimized the light extinction.

Any light that goes through the orifice will be absorbed by the internal walls, so offering a perfect black background useful for numerous applications. If the intestinal content is placed in a capsule of glass or plastic placed over the orifice of the upper box face, and is illuminated with a hand torch or another appropriate light, the content will be seen very clearly illuminated, allowing to distinguish by its form and movement ciliates and nematodes. The amoebas can only be detected observing samples at the microscope with the 40 and 100x objectives.

In order to seal a number of definitive preparations VAPA or VALAP can be used. Both formulae adhere very well to glass even whetted with glycerin, and allows a complete an easy recovery of both slide and coverslip.

VAPA. - is a mix of equal parts of solid petroleum jelly and paraffin (or 60% petroleum jelly and 40% paraffin) both substances are placed in a container and warmed up until melted and well mixed. Pack it in a wide mouth container, and let it solidify until it is needed.

VALAP. - is a mixture in equal parts of petroleum jelly, lanoline and paraffin. It is prepared and packed the same way the previous sealant was. It is less fragile than pure paraffin and adheres much better to the glass.

A sealing tool, very efficient, constructed with an office paper clip, was described by Olivier Messmer in his Message of 08/11/2004 to the Forum Mikroscopia (the address is on the front page of Micscape). The illustrations are so clear that even not knowing a word of French Language you can make and use it. So I take the liberty to reproduce it here. (If the browser resizes image to screen, use feature usually on bottom right of image to expand to full size .)

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