Trichomonas muris infections were encountered in the oxyurid nematodes Aspicularis tetraptera and Syphacia obvelata parasitic in laboratory white mice. More rarely infections with other protozoa were encountered in the helminths. The distribution of both protozoan and helminth infections was studied, and the incidence of infected helminths determined. Dividing forms of Trichomonas muris occurred in the intestine of the worm as well as free in the lumen of the intestine of the mouse. An interpretation of certain findings in dividing forms is offered.

The invariable presence of a rickettsia in the gonads of the mosquito, Culex pipiens, is confirmed. This rickettsia is found in the cytoplasm of every germ cell in both sexes at all stages of development from larva to adult. No other species of mosquito is known to be similarly infected.

The morphology of the organism shows the following features. In addition to “typical rickettsiae”, i.e. minute rods and coccoids which multiply by binary fission, there is a variety of other forms which are interpreted as a series of stages, usually in the following sequence: small coccoids become enlarged, and instead of dividing by binary or multiple fission, undergo a condensation of their stainable substance into points or rods which ultimately appear as various combinations of sharply curved rods and coccoids retained as in a matrix within the body of the original or “parent” coccoid. As the matrix disappears, they become the irregularly curved forms and compact aggregations of “daughter” forms characteristic of this rickettsia.

The pathological germ cells previously noted in the testis by cytologists were found to occur in both sexes at all stages, and to be apparently the result of infection with this rickettsia.

The problem of classifying the rickettsiae is discussed. The similarity of the rings and curved forms of various rickettsiae to corresponding forms, as yet little studied, in other groups of organisms is pointed out.

The name Wolbachia pipientis (gen. et sp.n.) is proposed for the rickettsia of Culex pipiens.

Large, homogeneous or granular cell inclusions with an affinity for neutral red, termed NR bodies, were found in the gonad wall of nearly every specimen of Culex pipiens, but were not found in C. territans. They resemble the inclusion bodies of certain viruses and at times closely simulate masses of rickettsiae, but are apparently distinct from the species here described. Their nature is unknown but their appearance and behaviour are consistent with their being living agents or the manifestations thereof. A degenerative process, in which NR bodies replace primary ovarian follicles in whole or in part, is described.

Inoculation and cultivation experiments resulted negatively for both rickettsiae and NR bodies.

In a previous paper (1935) an account was given of the prostigmatic mites parasitic on South African lizards, belonging to the two endemic families, the Zonuridae and Gerrhosauridae. The present paper deals with the fauna of the two remaining families of lizards, the Geckonidae and Agamidae, parasitised exclusively by prostigmatic mites. Trägardh (1905) and Hirst (1926) have made the most considerable contributions to the knowledge of the parasites of Geckonid and Agamid lizards; the latter placed the group on a sound systematic basis, the former, in addition to his descriptions of new forms, contributed valuable notes on the development and morphology of the species studied. With regard to mite parasites, however, up to the present, no systematic investigation has been attempted of all the species belonging to any one family of lizards from a given zoo-geographical region. In the present paper this has been attempted; the great majority of Geckonid and all the species of Agamid lizards from the South African subregion, either as living or spirit specimens, have been examined; from most of them mite parasites have been obtained. Both families of lizards are, genealogically speaking, derived from a long-established, Saurian stock; both are, in South Africa, extremely well demarcated, homogeneous families, with their main structural characteristics varying within narrow limits. In neither of them does loss or degeneration of the limbs occur. Each family is parasitised by a single genus of prostigmatic mites, the Geckonidae by Geckobia, the Agamidae by Pterygosoma; while Geckobia is found on the Geckonidae and on no other family, Pterygosoma is also a parasite of Gerrhosaurid lizards on which three species have been found. In a previous paper by the writer (1935, p. 3) Pimeliaphilus was cited as occurring on Geckonidae. Trägardh, who erected the genus (1905, p. 31), actually found his species P. podapolipophagus living under the elytra of a beetle, Pimelia, but the other two species of the genus, P. insignis Berl. and P. tenuipes Hirst, both occur on Geckonid lizards. Neither of these two species have been found in South Africa and it is extremely unlikely that they occur in this region.

The biotic factors in the environment of the tick, Ixodes ricinus, exercise little effect on the geographical or local distribution of the species, or on its seasonal prevalence.

The local distribution is determined by edaphic factors, a wet, mossy, or peat soil and a dense mat of old vegetation or a rank growth being necessary for survival of the tick. In Britain, the critical season for survival is summer, during which the moisture factor in the microclimate acts in a limiting capacity.

The seasonal prevalence is determined by temperature. Within limits, which appear to correspond to air-temperature limits of 7 and 16° C. (weekly maxima), the unfed tick climbs the vegetation, and thus readily obtains a host. In Britain, it is inactivated in winter by the cold, and in summer it is less readily picked up by hosts because of its positive geotropic response to the stimulus of high temperatures.

The summer is, in Britain, the optimum season for development, which also proceeds to some extent in winter. Autumn and spring are parasitisation seasons. The life cycle, involving a parasitisation and a development season for each stage, requires a minimum of 1½ years. The period may extend to 4½ years.

The possible world distribution of the species is limited primarily by temperature. Thus, microclimatic extremes of — 14 and 35° C. limit the range through which the tick can survive. A period of at least 3 months with the mean air temperatures over 10° C. is necessary for development, while the mean air temperature of the coldest month must not exceed 10° C. to allow of parasitisation occurring.

Within the areas delimited in relation to the temperature requirements of the tick, distribution is governed by the moisture factor. An index of the suitability of an area within the temperature limits is afforded by the type of vegetation; forest and woodland, including grass and cultivation areas, as opposed to prairie and steppe, indicate suitable moisture conditions.

Rhabditiform first-stage larvae of Nippostrongylus muris are about 270–550μ long. The development of these larvae culminates in a moult, which occurred 36–48 hours after cultures were prepared. The first moult is preceded by an unusually early separation of the first cuticle, which is fine and unstriated, from the body tissue in the tail region and, later, by the formation of an underlying new cuticle, which is thicker and prominently striated.

Early second-stage rhabditiform larvae differ in certain morphological respects from first-stage larvae. Second-stage larvae are about 470–750μ long.

A second preparasitic ecdysis occurs and the infective larva is a true third-stage larva. This moult was the only one previously known to occur during the preparasitic development of N. muris.

The post-cervical cuticular inflation in N. muris is regarded, on the basis of a comparative study of the cuticle of this species and of Haemonchus contortus and other trichostrongyles, as resulting from the separation of the second and third layers of the cuticle. Yokogawa's view that Nippostrongylus muris has two cuticulae is rejected. N. muris has a single cuticula, apparently made up of four layers.

N. muris undergoes four complete moults and the development of this species does not differ in any essential respect from that reported for other strongyles.

1. A detailed historical account of the subfamily Haplorchinae is given. The systematic position of the various species in this group is critically reviewed. The subfamily Haplorchinae and the genus Haplorchis are accepted as valid while Monorchotreminae and Monorchotrema as synonyms.

2. Three species of Haplorchis are found in Kwangtung Province, namely, H. pumilio (Looss 1896), H. taichui (Nishigori 1924), and H. yokogawai (Katsuta 1932).

3. Throughout the work emphasis is laid on the acetabular spines or hooks which are considered to be the only recognisable constant structure practical enough for specific diagnosis.

4. It is believed that there are only four valid species of Haplorchis, namely, H. pumilio, H. yokogawai, H. taichui, and H. milvi. The validity of H. milvi, however, is in question. H. cahirinus and H. anguillarum do not fit into the diagnosis of the subfamily Haplorchinae, while H. microrchia and H. taihokui fall into synonymy with H. taichui and H. pumilio respectively.

5. A key for the identification of the known species is given.

1. Cotylaspis sinensis and Lophotaspis orientalis are described as new species of trematodes from the small intestine of the turtle, Amyda tuberculata, from Foochow, Fukien Province, China.

2. Stichocotyle cristata is described as a new species from the spiral valve of the cow-nosed ray, Rhinoptera quadriloba, from Biloxi Bay, Mississippi.

3. A new subgenus, Multicalyx, is created for the species Stichocotyle cristata, while the species S. nephropis appropriately belongs to a new subgenus Stichocotyle.

4. Evidence is provided that aspidogastrid worms are referable neither to the Monogenea nor the Digenea, but belong to an intermediate subclass group, Aspidogastrea, n.n.

5. The genus Stichocotyle has been removed from the family Aspidogastridae Poche, 1907, and placed in a new family, Stichocotylidae.

(1) Female Ixodes ricinus ticks which feed on a sheep infected with tick-borne fever are not capable of transmitting the infective agent to their progeny. Only the nymphs and females, therefore, can be infective to sheep. In the case of an infective female, the infection may have been acquired, (a) in the immediately preceding stage, or (b) in the larval stage, the infective agent remaining in the tissues of the tick during the nymphal engorgement and subsequent moult. This appears to occur irrespective of whether the host on which the nymph feeds is a susceptible or an insusceptible. animal.

(2) Ticks which have acquired infection are infective as soon after moulting to the next stage as they are able to attach to hosts. Unfed ticks were not found to be infective after a period of about 14 months.

(3) Infection is transmitted to the sheep in the course of the 2nd day after attachment of infective nymphs.

(4) The disease may be transmitted to sheep by inoculation of emulsions of infective ticks, if these have been allowed to commence engorgement. The use of saline for such emulsions appears to be inadvisable.

There is still some difference of opinion as to the classification of what Rudolphi in 1801 called the Amphistomata. Fischoeder (1901—4) regarded all “Amphistomes” as belonging to a single family, the Paramphistomidae. Stiles & Goldberger (1910) proposed the elevation of the group to the status of a superfamily, the Paramphistomoidea, comprising three families, Paramphistomidae, Gastrothylacidae and Gastrodiscidae. Braun (1911), Odhner (1911), Looss (1912) and Stunkard (1925) have maintained Fischoeder's view; Maplestone (1923) and Travassos (1934) have preferred that of Stiles and Goldberger.

In 1933 Mr C. Machattie, F.R.C.V.S., D.V.S.M., very kindly sent me a small portion of a specimen of Lymnaea tenera euphratica Musson, collected in Iraq, infected with a cercaria belonging to the heterogeneous Gymnocephalic group. Sections were cut and it was found that most of the cercariae had been fixed during various stages of encystment. An excellent opportunity was thus afforded for observing this process, particularly the initial stages.

Morphological, histopathological, and immunological studies of seven strains of rickettsial disease have placed them in two widely separated groups, for which typhus and spotted fever seem the most logical names.

Diseases of the spotted fever group are carried by ticks (Arachnoidea) and characterised by distinctive histopathological lesions—thrombonecrosis of arterioles and venules, with micro-organisms of definite and characteristic morphology, invading smooth muscle cells, as well as endothelium, mesothelium, and macrophages. Tissue culture studies show massive invasion and distention of cell nuclei, while cytoplasmic invasion of host cells is relatively sparse.

Diseases of the typhus group are carried by lice and fleas (Insecta) and characterised by the facts that the intracellular parasite which causes them invades only endothelium and mesothelium, distends the cytoplasm of its host cells without ever invading nuclei, and in guinea pigs causes only proliferative endangeitis without thrombonecrosis.

In the spotted fever tick, organisms are intranuclear as well as intracytoplasmic, invade nearly all types of tissue, and are transmitted hereditarily. In typhus-infected lice and fleas, organisms are intracytoplasmic only, infect only the lining cells of the gut, and are not transmitted hereditarily.

Neither focal brain lesions nor scrotal sac exudation is of importance in the differential diagnosis, since both occur in certain strains of spotted fever, as well as in typhus. Definite necrosis of the scrotum is of importance, since it is dependent upon the above-described arteriolar thrombonecrosis and is peculiar to spotted fever.

A review of the literature shows that rickettsial diseases and their etiological agents are being classified in an inaccurate and unjustifiably complex manner, specific names often being given to organisms without the presentation of evidence sufficient to establish them as new varieties.

A plea is made for the study of unclassified or unsatisfactorily classified diseases in all parts of the world by the methods described, and for their classification on the basis of morphologic and histopathologic observations on insect and mammalian hosts and of immunological studies on the latter, rather than on variations in clinical pictures in man and in experimental animals.

The criteria for the differential diagnosis between typhus and spotted fever are presented in tabular form and illustrated wherever possible. All reported rickettsial diseases as well as their etiological agents are classed on the basis of the criteria presented, and, with the exception of the incompletely studied mite-borne group, the available evidence strongly suggests that all are varieties of typhus and spotted fever.

The morphological, histopathological, and immunological differences between typhus and spotted fever are so great that these two diseases, as well as their causative organisms, should be widely separated from one another, and given definitely distinguishing names.

It is suggested that the well-established name, Rickettsia prowazeki, be applied to intracytoplasmic micro-organisms in Insecta causing all diseases of the typhus group, and that Dermacentroxenus rickettsi (the name originally given by Wolbach) be applied to hereditarily transmitted micro-organisms invading nuclei in Arachnoidea and in tissue cultures, causing all diseases of the spotted fever group. Specific varieties, when established by the demonstration of definite and constant biological differences, should be indicated by variety names, as Rickettsia prowazeki mooseri (for endemic flea-borne typhus in all parts of the world).

The earliest stages found are groups of small bodies, 1·8 or 2·0μ in diameter. These groups are probably taken up by phagocytic cells and pass through the epithelium of the host into the connective tissue, where a cyst is formed by the host. Within the cyst the bodies grow. In many cysts there is a differentiation into two types: the larger, when mature, attain 40μ in size, are shaped like a pear, and contain at the broad end a chromatic rod. The small bodies reach 3Oμ in length, are also somewhat piriform, and contain two similar structures attached to their nuclei. In some cysts large bodies alone are found.

Development is periodic, the youngest stages being found from January to March and the parasites not reaching maturity until September–November.

Type species P. sociabile, with the features mentioned above: found near the ventral surface of the foot of Buccinum undatum.

1. The life history of Encyrtus (Comys) infelix (Embleton), a hymenopterous parasite belonging to the chalcid family Encyrtidae, is described. It parasitises Saissetia hemisphaerica, a member of the subfamily Lecaniinae (Homoptera Coccidae). Its respiratory relationships with the host are of a quite extraordinary character.

2. The egg is provided with a hollow stalk which is left protruding from the posterior dorsal body wall of the host. The first three larval instars are metapneustic, the spiracles being placed at the tip of a pair of long caudal processes which are inserted in the hollow stalk. It has been proved that these spiracles are open and in actual communication with the atmospheric air, which enters through the pedicel.

3. The fourth and fifth larval instars are amphipneustic and the manner of respiration is entirely changed. The caudal processes degenerate and finally break away. The fourth instar larva turns round in the scale insect and becomes invested with a closely fitting transparent membraneous sheath produced by the phagocytes and fine tracheal branches of the host. This process appears to be merely a special case of the type of phagocytic activity which normally gives rise to the basement membranes and “connective tissue” membranes; such membranes are particularly conspicuous and well developed in Saissetia. The sheath becomes attached in an extraordinary manner to the main lateral tracheal trunks of the host in four (or six) places in the neighbourhood of the larval spiracles. This process is described in detail. It has been proved by an injection technique that an actual connection is established between the lumen of the host trachea and the cavity of the sheath. Bubbles of gas appear inside the sheath close to the points of attachment and near the spiracles of the larva which are thus put into functional communication with the tracheal system of the host.

4. While the production of the sheath might under certain circumstances be of value to the host the development of tracheal attachments appears to be of value to the parasite alone. The conclusion that the whole structure is an adaptation for the respiration of the parasite seems inescapable. A theory is tentatively put forward to account for the stimulation of the host tracheal epithelium by a sudden physiological change in the tension of the respiratory gases in the region of the parasite spiracles.

5. References to similar instances among the Chalcids are briefly reviewed. It is suggested that the “puparia” which have been described in the case of one or two other Chalcidoidea may be found to arise in the same way.

The commonly accepted view regarding the manner of entry of larvae of Wuchereria bancrofti (Cobb.) into the body cavity of its mosquito host is that microfilariae from human blood enter the stomach of the mosquito along with the blood meal and after casting their sheath in the stomach, they penetrate through the wall of the stomach, enter the haemocele and then find their way into the thoracic muscles. The time taken for the penetration of the stomach wall according to Looss (1914) is 6–12 hours. According to Bahr (1912) the worms appear in the thoracic muscles in 24 hours, while Lebredo (1905) recorded that in exceptional cases, worms were seen in the thorax 13 hours after feeding, but ordinarily the time interval was longer.

In a very important revision of the Streblidae, Speiser (1900) classified them into two subfamilies: (1) the Nycteriboscinae and (2) the Streblinae. To the first subfamily he referred all the genera of the world, with the exception of Strebla and Euctenodes. Upon the presence of the ctenidium and some other characters these two American genera were included in the second subfamily. This classification of the subfamilies was also adopted by Kessel (1924, 1925).

This collection, among others, was made for and kindly presented to me by Miss Miriam Rothschild during her visit to the Zoological Station, Naples, in April 1934.

Within recent years a considerable amount of work has been done on the Myxosporidia, especially, as pointed out by Kudo (1919), in North America, Asia, and to a lesser degree in Japan (Fugita, 1912, 1913, 1925). Few investigations, however, have been carried out in Australia, and consequently very little is known concerning the distribution of the group in this country. There appear to be as yet only two contributions to the subject, papers by T. Harvey Johnston and M. J. Bancroft (1918, 1919). As far as can be ascertained, my paper is the first report concerning Myxosporidia from fishes of Southern Australian waters, and contains the first record of a species of Chloromyxum from Australia.

Three pure-line strains started from single individuals, 2 of Trichomonas and 1 of Eutrichomastix, all from snakes, were isolated from mixed cultures. These strains, and another of Eutrichomastix from a chamaeleon, were under observation for periods of from 3 to 10 weeks.

The Eutrichomastix of snakes does not differ from that of the chamaeleon in any essential point, and neither developed an undulating membrane at any time during this prolonged period of observation.

The Trichomonas of the grass-snake is morphologically distinct from that of the boa. Trichomonas of the boa has an ill-developed undulating membrane with only a few shallow waves, and may simulate a Eutrichomastix owing to the accidental detachment of the bordering flagellum.

Trichomonas from snakes, as from most other hosts, readily feeds upon starch granules, whereas none of the Eutrichomastix were found to ingest unaltered starch.

Eutrichomastix from snakes and the chamaeleon never develops an undulating membrane and does not assume a Trichomonas form.

From these observations it can be definitely concluded that Trichomonas and Eutrichomastix are generically distinct.

(i) Amoebae closely resembling Entamoeba coli (of Man) have been studied in macaques (M. rhesus, M. sinicus, M. nemestrinus) and in a marmoset (Hapale jacchus).

(ii) Pure strains of these amoebae have been isolated and cultivated, and carefully compared with similar pure strains of E. coli from Man (E. coli hominis).

(iii) The complete life-cycles of these various strains—amoebae, cysts, and all stages of precystic and metacystic development—have been studied in vitro.

(iv) The “E. coli” of macaques (E. c. macacorum) was experimentally transmitted to a man, who ingested pure culture-cysts of a strain derived from M. rhesus.

(v) The “E. coli” of the marmoset (E. c. jacchi) was likewise transferred to M. sinicus, and from this host again to M. rhesus and Man.

(vi) The natural infections observed in M. sinicus, M. rhesus, and H. jacchus all died out spontaneously after persisting for various periods (4½ years, 2 years, and 1 year respectively). The experimental infection with E. coli macacorum induced in Man also disappeared spontaneously (after about 6 weeks).

(vii) Attempts to infect kittens experimentally with E. coli jacchi were uniformly unsuccessful.

(viii) All infections with “E. coli” from every source—whether natural or experimental—have proved to be completely innocuous.

(ix) Culture-cysts of various strains of “E. coli” have been found able to live at low temperatures for all times up to about 4½ months (maximum 135 days at 1°–2° C., for a strain from H. jacchus).

(x) The “E. coli” of H. jacchus—and all its experimental derivatives—ingests human red blood-corpuscles in vitro as readily as E. histolytica.

(xi) Several strains of E. coli hominis, isolated directly from naturally infected human beings, have been found to possess the same property.

(xii) Since strains of E. coli thus exist—both in men and in monkeys—which eat human red corpuscles avidly, this faculty can no longer be considered as peculiarly characteristic of E. histolytica; and the importance of this observation, for the diagnosis of amoebic dysentery, is therefore emphasized. The statement that E. histolytica ingests red corpuscles, while E. coli does not, is untrue. It is not possible to distinguish these species by this character alone.

(xiii) As it has been found impossible to distinguish various strains of “E. coli” living in macaques, a marmoset, and men, by any recognizable specific feature—all of them being so closely alike morphologically, physiologically, culturally, and in their ability to inhabit these different hosts—it is concluded that no specific difference actually exists between any of them. The “E. coli” of the Macaques and of the Common Marmoset is probably Entamoeba coli itself—the species which naturally inhabits Man.

The main theme of this series of papers will be the power of the three wellknown members of the brucei group of trypanosomes, T. brucei, T. rhodesiense and T. gambiense, to infect man. Inextricably involved with this problem are two others, both of practical importance: (1) Can the African big game serve as a reservoir for human trypanosomiasis? and (2) What are the affinities existing among these three trypanosomes? This may appear to be an inversion of the real order of importance, but a study of the literature on the brucei group, past and recent, reveals that infectivity to man dominates all other considerations.