INTRODUCTION

Some years ago, a dull and weak blow at the sill of my window, interrupted my reading of the local newspaper. A small lizard of about 8 cm in length lay motionless next to the defense against mosquitoes. It was a “Brown Anolis”, the most common and abundant lizard of Cancun gardens.

There are few birds that visit our gardens, butterflies are rare, and most of the arthropods are little seen. Therefore it is nice to see the small anoles sunning on a branch, running over the white walls or between the colorful foliage, or extending its red gorge as a warning.

Of course its speed and restlessness makes difficult to capture them. So I watched carefully the animal to prevent its movements and to try to catch it. My precautions were useless: the animal was dead. Intrigued I decided to convert me to forensic studies and perform its autopsy.

The result of it, and the information I collected to understand and explain its death, form the body of this article. 

Although it is recommended that everyone find their own on-line literature (as we used to do in the normal libraries) always saves others a little work if reference links are shared. So many references are attached in the text.

THE  LIZARD

Anolis sagrei (Dumeril & Bibron, 1837) is a small grayish brown lizard that greatly abounds and moves with impunity in the gardens of Cancun and their surroundings. Apparently it is native to the island of Cuba, from where it invaded, probably by human mediation, the Cayman Islands, and then the states of Quintana Roo and Yucatán in Mexico (Álvarez-Romero, J., et al. 2005) reaching the neighbouring Belize.

It also moved northward, invading the peninsula of Florida in the United States from where it is spreading to Texas. There it competes (for food, but also as a predator of small individuals) with the local anole Anolis carolinensis (Green anole) that is being displaced (Campbell, 2002). It has been reported even in Hawaii where it probably came at a time when the careless handling of export fruit allowed easy transfaunation. (Goldberg & Burgey, 2000).
Their taxonomic position is as follows:

·        Kingdom: Animalia

·        Phylum: Chordata

·        Class: Reptilia

·        Order: Squamata

·        Suborder: Lacertilia

·        InfraOrder: Iguania

·        Family: Polychrotidae

·        Genus: Anolis, Daudin 1802

·        Species: Anolis sagrei, Dumeril et Bibron 1837

·        Subspecies: sagrei (Cuba)  and ordinatus (Bahamas).

It is worth reviewing the definition of each category in a WebQuest. An article that summarizes quite well these hierarchies is in Wikipedia (http://en.wikipedia.org/wiki/Taxonomic_rank)

Anolis sagrei shows a clear sexual dimorphism. As it is common in the animal kingdom the male is larger and has a distinctive colouring, and special attraction media, while the female looks more modest and inconspicuous.

Fig. 1 - Male on a tree, SX100is (x 10 zoom) Playa del Carmen, Quintana Roo

Fig 2 - The male torso, with insert of the unbent gorge (crop 1:1 of an SX100is 8 Mpx picture), Playa del Carmen, Q.R.

Fig. 3 – A whole Female (crop from an SX100is 8 Mpx picture), XeL-HA, Quintana Roo

Fig 4 – A closer picture of the same female

Since there are many active herpetologists, and this species is relatively easy to raise in captivity, searching by name with your browser you can find on the network many pictures and even data for maintenance in a terrarium.

FORENSIC RESEARCH – THE NECROPSY

Once dissected, I found that the lizard’s stomach contained an entangled group of nematodes (over 20 of all ages and sexes, which is admirable, given the small size of the animal and the corresponding small stomach) many of them still clinging to the stomach mucosa. Probably the poor animal could not feed and perhaps died a victim of starvation and bleeding. 

It is that, along with the lizard have travelled their parasites. One of them, the nematode Physaloptera squamatae Harwood 1932, is the subject of this note and its taxonomic position is summarized as follows:

           Kingdom:    Animalia

            Phyllum:    Nemathelmintha

   Class:    Nematoda

 Order:     Spirurida

 Family:     Physalopteridae

Genus:      Physaloptera, Rudolphi 1819

          Subgenus:     Physaloptera, Rudolphi 1819

              Species:     Physaloptera (Physaloptera) squamatae Hardwood 1932

 The family contains 7 or 8 genera. The genus Physaloptera includes parasites of batrachians, reptiles, birds and even mammals. Domestic cats and dogs are guests of some species. Its veterinary importance explains why this is the best known genus of the whole family. For some of the species, articles can be easily found online. The genus has several subgenera.  Physaloptera squamatae belongs to the Physaloptera subgenus which has assigned 126 species according to a review by the Texas A & M University. However, even looking very carefully through the network, not one acceptable image of it can be found, with the honourable exception of an image published by Denis Kunkel which is included later.

 A VISUAL DESCRIPTION OF THE ANATOMY

The fact that the structure of nematodes is very sketchy, leads almost all of the authors to focus on presenting (even in drawings that define new species) only the differences between the species described and others of the genus, so, their concern is not to describe each entity in itself, but the differential details (number of uterus; vagina position; size, shape, and type of development of the egg, spicules and papillae in the male tail; structure and cephalic organs, lips, teeth and spines of the mouth; and relative size (and relative position) of the different organs. (See for example the description of the then new species Ph. Buteonis. An abstact can be found here http://www.jstor.org/pss/3223499)

Here of course we are interested in the alternative strategy: to show visually all possible details of this species’ anatomy.

GENERAL BIOLOGY

Physaloptera squamatae is good sized, the adult female I illustrate here (fig. 5) has an approximate total length of  10 mm and a maximum width of 0.36 mm. Corresponding measurements for the male (fig. 42) are length = 7 mm and width0.3 mm. The younger female (fig. 25) measures 6 mm in length, and is 0.2 mm in width.

Apart from the body wall, which is a circular muscle coat, wrapped in a flexible cuticle, internal organization consists of a central digestive tract with a relatively simple pharynx followed by the intestine, finished, near the tail, at the anus in the female,  and at a “cloaca” (the organ that brings together the digestive and reproductive system) in the male.

The nervous system consists of a peri-pharyngeal ring (which acts as a brain coordinator) and sends a dense package of nerves to the “head”, and two posterior longitudinal nerve chains, included dorsally and ventrally in the muscle wall. ( see fig. 6  )

There are no special systems dedicated to circulation or breathing. The small diameter of the worms and the gas permeable cuticle acts as a respiratory organ. Since the pseudo cœlom has no divisions, the simple wave motion that characterizes these animals generates the necessary flow of fluids.

And the spirurida excretory system is very simple consisting of longitudinal excretory tubes that gather together in a inverted U shape at the level of excretory pore.

A much more comprehensive description, well illustrated, for anyone really interested in the topic can be found in  http://parasitology.informatik.uni-wuerzburg.de/login/n/h/0929.html#Fig-11

The reader unwilling to follow such a detailed description should check the pages dedicated to nematodes of the excellent guide to practical zoology, published online by Richard Fox of Lander University

http://webs.lander.edu/rsfox/invertebrates/ascaris.html

http://webs.lander.edu/rsfox/invertebrates/cephalobus.html

Structures detectable with the imaging media at my disposal will be presented below for both the male and female of Physaloptera squamatae. 

Preparing nematodes for observation - Nematodes are generally fixed by immersion in 70% alcohol heated to boiling. So I took a test tube a quarter full with alcohol, and (of course pointing the mouth of the tube in the opposite direction from my body) I warmed it with great caution.
Alcohol has the bad habit of boiling explosively, projecting fluid at a distance. It is best to move repeatedly and slowly the tube through the flame several times until it reaches the right temperature.

When just beginning to boil, I picked up the nematodes with a brush and soaked them in the liquid. Normally the nematodes are immediately stretched, setting straight, or slightly curved. The Physaloptera refused to respect this custom and the longest died while maintaining a contorted shape.

To observe nematodes it is best to clarify and mount them in glycerin. This is a method that, with little effort, but careful work, provides excellent results. Indeed it is the professional technique.

From alcohol 70, nematodes are passed to a solution of glycerin, 5% - 10%, in 96% alcohol. It is best to place them in a wide, shallow dish and place this inside a container (there are many small plastic containers with a width of approximately 10 cm, at least in one direction that could be useful) loosely covered, so as to allow slow evaporation of alcohol. Use enough solution so that the alcohol evaporates leaving the specimens still covered with enough glycerin. Then move them to the final container with pure glycerin. Do not forget to include a label with the corresponding data, written in indelible " China” or “India” ink, or with soft graphite pencil.

To see them, put a drop of glycerin on a slide, including a specimen. Add two or three strands of plastic, or bits of plastic film or paper, or thin aluminum foil, or small pieces of a broken coverslip, or any material of adequate thickness to allow a good compression of the animal without crushing it too. Cover with a coverslip, with care not to include air bubbles, and, placing an absorbent paper on the preparation, pass the back of your hand on it in order to expel excess glycerin.

The preparation is now ready for the 4x to 40x objectives.  But many details may require immersion oil. Therefore it is best to seal the preparation.

Place a drop of nail polish on each corner of the coverslip and let dry for half an hour. With a soft cloth, damp (not wet) in 96% alcohol clean the remaining glycerin around the coverslip. Repeat operation after a few seconds to ensure a good cleaning of the slide. Then seal the four edges of the coverslip with nail polish.  After a few hours, apply a second layer. This will provide a definitive preparation. Label it immediately.

It is always better to keep these preparations flat. Do not use common slotted boxes. In preparations kept vertically the specimens can move slowly and finish against one edge of the preparation.

The images I'll present here were taken from the better fixed specimens. Most correspond to 3 individuals: a juvenile female, an adult female and an adult male. 

THE  FEMALE  ANATOMY.

Fig 5 - A labeled picture of a PHYSALOPTERA female. Click on it to see a non labeled one.

The previous image is a mosaic of 10 pictures taken with the 10x objective and the DC3 camera and finally composed in PhotoPaint. Labels indicate the most important organs. Both pictures were reduced from the original mosaic of 3 Mpx.

 Fig 5c- Anyone who wants to see the original image of  3 MPX. can download it by clicking here.

 The front end has the mouth located between two thick lips each finished with an apical spine. Behind the buccal area the cuticle forms a fold or peri-cephalic collar within which the head can retract. The cuticle at this point widens, as shown in the following pictures taken with the Logitech 9000

Fig 6 - Crop of a picture with the 10x objective. 2 neural chains, neural ring and cephalic neural complex,
seen here as a difuse cone surrounding the pharynx

Fig. 7- dorsal view of the head, showing the two major terminals thorns. x40, Logitech

Fig. - 8 - lateral view of the “head” showing the cephalic cuticular expansion and the peri-cephalic collar that helps the animal, as if it were a suction cup, to adhere to the intestinal wall. Also look at the nervous peri-pharyngeal ring, connected to the deirids - Logitech

Figs. 9 -10 - Deirids, left and right, DC3, x 100

Deirids are characteristic sensory organs of the nematodes in the morphologically defined Secernentea. The nematodes were once divided taxonomically, based on criteria of morphological similarity in two groups (Secernentea and Adenophorea), the adenophorea lack deirids.

However the current use of the biochemical markers, as fractions of the RNA, has destroyed the apparent homogeneity of these divisions, and currently the classification is very different. Anyone wishing to initiate a review of this subject can consult Wikipedia articles on both taxonomic categories. This work is an example of the problems that face modern taxonomists:

A.Euyalem and M Blaxter - 2003 - Comparison of biological, molecular and morphological methods of identification in a set of Panagrolaimus cultured species isolates. - Journal of Nematology 35 (1): 119-128.

Five populations, all morphologically assigned to a single species (ie. that they could not be differentiated based on morphological characters) had to be separated into two different species since the study of its RNA showed that they were genetically different. There is an abstract thereof published in http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2620612/

Fig. 11 - Another picture of the thorns combining two shots with CombineZP

Fig. 12. Anterior end (“head”) of a parasitic Physaloptera from an American "racoon",  photographed with a scanning electron microscope, approx 70?x,  by Dennis Kunkel, whom we thank for permission to reproduce it. (For those which do not yet know Mr. Kunkel, his site (see the address in MICSCAPE Links front page) is full of images of the same quality, of many different biological entities. You must visit it.) Both ovoids below the spines are the anphids, sensitive structures considered of high taxonomic value.

It seems clear that the spines and the cephalic collar, plus the muscular pharynx sucking action, must injure the gastric mucosa. A study of the injuries inflicted by another species of Physaloptera, you can see in:

http://www.paru.cas.cz/folia/pdfs/showpdf.php?pdf=20977

Fig. 13-The cuticle that forms the cephalic expansion is crisscrossed by many thin transverse striations, as noted in this optical section, DC3

Fig. 14 - The picture shows the surface of the cuticle with the 100x OI objective, showing the surface grooves. Cropping 1:1 image from 1.3 Mpx. picture. Logitech

 Fig 15 - In addition to the valve between pharynx (phx) and intestine (int) may be seen one excretory tube (e) and one nervous chain (n) - Logitech

The outer layer of the intestine consists of a epithelium with very uniform cells.

Fig16 -  surface of the intestine - Logitech

Fig.17 - the tail of an adult female (Canon SX100is, 8 Mpx) objective x 4 (clipping 1: 1)

The intestine opens abroad at a short distance from the caudal end, through an extensive and folded anal pouch. The pouch is shown in more detail in the following pictures.

Fig. 18 - A detail of the previous picture. 40X, Logitech

Fig. 19 - The same with oblique Rheinberg lighting, DC3

Fig. 20 - 100x, 5 images, combined with CombineZ5 - DC3

Fig. 21 - The anal pouch of another female, DC3, x 10

 I devoted the previous 4 pictures to this anatomical detail, as the function of the anal pouch does not seem obvious to me, and is not common in nematodes. Generally such primarily cuticular structures are attached to the copulatory apparatus, but this is not the case. If we review the Fig.1 we see that the vagina and vulva are located in almost a central dorsal position, and are simple, and without ornaments.

 Another conflicting picture is this, taken with the 4x objective, of the tail of the younger female.

Fig. 22 – frontal view of the tail of the younger female. Click over the picture to see a labeled one. Logitech

It shows only a very small pouch (p) and some rays can be seen that could be perhaps interpretable as muscles to handle it. As the picture is not conclusive I put it here only to arouse the interest of some future researcher.

 Excretory system - In some individuals can be seen laterally two long strands that flank the intestine and converge  near the front nerve ring. They are the branches of the excretory system. In other nematodes (review the references) the excretory system is connected to cells called "renettes" which do not exist, or are much reduced, in the Spiruridae, so that the excretory system in them consists only of two lateral branches, gathered transversely at the height of the excretory pore. The system has then a U-shape anteriorly-based (inverted U).
In my mounted specimens the excretory system is difficult to identify. (but see Fig. 15) I could acquire only one image of the excretory pore.

Fig 23- excretory pore - DC3, x40.

Two neural chains run along the body, one in dorsal position, the other ventral.

Fig. 24 - oblique illumination, x40 – one of the neural chains - DC3

The female reproductive system in Physaloptera is quite complicated. Much of this description is based on a young female, because it was not so full of eggs, and allowed a better study of its structure. 

I superimposed over a picture of it, taken with the x4 objective, (Fig 25) several boxes that correspond more or less (they are bigger) to the levels where the descriptive pictures were taken, to make more clear the basic layout.

Sexual organs are arranged in a sequence as follows: ovaries, oviduct, (seminal receptacle), uterus, vagina and vulva, that opens dorsally abroad usually a little ahead of half of the body. There are multiple ovaries. The first portion of them, where the oogonia (primordial sexual cells) are visible is the germinal zone, it is continued by a growth zone, were the oogonia develops into oocytes (ovocytes). And they go through the oviduct to the first part of the uterus, which function as a seminal receptacle. As ovocytes pass through, they are fertilized and enter the uterus as eggs.

Fig 26 – . y.ov - young ovaries, ovd - oviduct, s.rec. -seminal receptacle, ut – uterus

Fig. 27 – Ovaries, germinal zone of young female, with visible oogonia

28 – Void oviduct, and first coils of uterus

Fig. 29- Oviduct, Seminal receptacle and Uterus.  Young female

Fig. 30 – Germinal zone, adult females. DC3, x40

Fig. 31 – End of germinal zone start of the growth zone,  x100

Fig 32 – Segmented eggs x100, DC3

Eggs with embryos are released in the host intestine and go out with the droppings. The mature eggs have inside a developed larva (larva 1 = L1). They measure aproximately 50 µ in length, are 23 µ wide and have a shell of 3 µ. I tried, but was impossible, to measure the length of the L1, it is not only contorted but folded in several planes as you can see in the transverse optical sections of fig. 34.

Fig. 33. - Eggs with already developed larvae. A couple of eggs are seen in optical cross section. X40, DC3

34. Two areas of the uterus, with fully developed L1 larvae. DC3, 100xOI

Fig. 35 - Vagina (heavely muscularized) and vulva - young female. x40, Logitech

Fig. 36 - Vulva and uterus, with mature eggs. Adult female. x40, DC3

Later development requires the consumption of a stool by an insect, in whose hemocele the larvae appears a few weeks after infection. L1 molts twice, and becomes L3, which clings to the intestine of the intermediary host. Lizards eat these insects, and free the L3 in their stomach where larvae suffer another two molts and develop to adults, beginning a new cycle. I suspect that the larvae in my garden must be hosted by ants, or by the juveniles of the American cockroach which are the unique insects with enough abundance, and permanent presence, as to guarantee the closure of the cycle.

Unlike the male which has a tail with a defined hook-shape, the tail of the female is relatively straight and gently tapered.

Fig. 37 – caudal end of adult female,Rheinberg oblique, x40, Logitech

In the free nematodes, especially the aquatic ones, freshwater or marine, there are caudal glands which download an adhesive secretion through a terminal pore. This has an obvious ecological value as this allows the nematode to cling in place even in running waters. According to my Hyman textbook, and Internet references, parasitic nematodes shouldn't have caudal glands, and therefore wouldn't have a terminal pore. It is however difficult to interpret certain images (such as that added below) without thinking that they show evidence to the contrary.

Fig. - 38 - Tip of the tail of another specimen. It could be little doubt that this shows secretory cells. The excretory pore is suggested by the final clear point.

Fig. 39 - In this picture the pore, and even the excretory duct, seems to show more clearly

THE  MALE

The male is slightly smaller, and shows the most interesting characteristics of the family.

 The head of this male had an interesting structure that allows viewing the open slit between the two lips that should lead to the mouth. And it shows well marked the edge of the cephalic collar. This made easier the interpretation of these pictures, compared to the digital picture shown below.

Figs. 40 - Right lip (wl), Left lip) ll), slit to the mouth (bs), spines (ath), muscular pharynx (mph), cuticular expansion (ce) . DC3 (somewhat reduced from the 640 x 480 originals)

The sex organs are arranged linearly: testicle, seminal vesicle, ejaculatory duct, cloaca, spicules.

Figs. 41 – Male, full length. Click on the picture to see a non-labeled version

Fig. - 42. The central section of the body with the reproductive organs, Rheinberg, DC3

These are pictures of the seminal vesicle and its continuation with the ejaculatory duct.

Fig .43 – seminal vesicle

Fig. 44 - the ejaculatory duct

The ejaculatory duct empties into the digestive system at the rectum,  and this leads to the outside. The cavity, composed by the end of both organs, is called a cloaca. The cloaca is provided with a pair of spicules that apparently have a role in opening the gonopore of the female. The cloaca is enclosed within an outer frame (or bursa) consisting of two membranous wings (which Hyman calls “alae”), transparent and slightly ornamented. Four "pedunculated papillae" on both sides of the cloaca help provide rigidity and functionality to the copulatory bursa.

Fig. 45 - Tail from a specimen stained with eosin. One can see the wings (alae) of the copulatory pouch, the cloaca and the stalked papillae. The focus is made on the upper surface (proximal) of the worm – DC3

Fig. 46 - Cloaca and stalked papillae at a high magnification, with a partial view of the ornamentation of the alae. DC3

Fig. 47 - caudal end and copulatory complex. de - ejaculatory duct (comes from the seminal vesicle, ends in the cloaca), i - intestine, pg - sessile glands, pp - stalked glands. There are also seen the alae and the spicules. DC3

Fig. 48 - Spicules and glands with greater detail. spc, spicules, sp, one sessile papilla pp, pedunculated papillae, c, cloaca, a, alae DC3

Fig. 49 - A couple of pre-spicular sessile genital papillae  (sp) DC3

Fig. 50 - And, finally, an  image showing one caudal sessile papillae (at least 3 other papillae can be spotted on my specimen, but I have no references of how many or in which position are located those that are normal for Physaloptera squamatae – Hyman draws 6 post anal papillae for an unnamed species) DC3. 

I think that this article, that lay waiting for so many years on my hard disk, is almost certainly my last one on parasitic nematoda. But, for the parasitologists-to-be that could be amongst the readers, I recall that I published a short series on the nematoda and other parasites of the cockroach Periplaneta americana.

http://www.microscopy-uk.org.uk/mag/artjun05/wdparasite.html

http://www.microscopy-uk.org.uk/mag/artjul05/wdparasite2.html

http://www.microscopy-uk.org.uk/mag/artaug05/wdparasite3.html

http://www.microscopy-uk.org.uk/mag/artsep05/wdparasite4.html

http://www.microscopy-uk.org.uk/mag/artoct05/wdparasite5.html

and one on the parasites of the “tropical gar” a “living fossil” from Mexico

http://www.microscopy-uk.org.uk/mag/artjun09/wd-pelegarto.html