When beginning this investigation it was my intention to make a comparison between the anatomy of the apterous viviparous form and that of the winged viviparous form, to ascertain what differences were present corresponding with the difference in habit of the Aphis in these two stages. I found, however, that the literature bearing on the group to which S. rosarum belongs, was somewhat scanty, and as a rule, rather out of date. It therefore became necessary, before undertaking the comparative work, to obtain a somewhat more complete account of the general anatomy. It is with this idea in view that the following paper has been written; but, as will be seen on perusing it, much more attention has been given to the internal anatomy than to the external parts. It is my intention, now that this first part of the task is completed, to take up my original project. I desire to take this opportunity of expressing my gratitude to Professor Bridge, Mr A. E. Shipley, F.R.S., Mr Doncaster and Mr Collinge for the valuable assistance and advice they have given me whilst this work has been in progress.

In the following notes no attempt has been made to enter into the pathology or etiology of the diseases described, as only isolated cases have been available for investigation.

I am much indebted to the Council of the Marine Biological Association of the United Kingdom for their kindness in granting me a table at their Plymouth Laboratory.

Although a good deal has been written about East Coast Fever in cattle, the literature relating thereto contains very little direct information regarding the parasite which stands in causal relation to the disease. Robert Koch (1898), who was the first to observe the parasite in cases of East Coast Fever occurring in German East Africa, regarded it as but a variety of Piroplasma bovis (= bigeminum) and described the disease as Texas Fever. Theiler (1904) was the first to distinguish clearly East Coast Fever from Redwater. He stated that “the disease has nothing to do with Texas Fever or Redwater; it is a new disease due to a parasite different to the one found in Texas Fever.” Koch (1903—1904), who gave the disease its distinctive name, reached the same conclusions as Theiler. The investigations of Theiler (1904) established the following facts: Cattle which are immune to Redwater are susceptible to East Coast Fever. East Coast Fever is not communicable by blood inoculations (30 experiments, wherein 5 to 2000 c.c. of East Coast Fever blood were inoculated). He noted the absence of haemoglobinuria in the majority of animals affected with East Coast Fever, its presence in the majority of the animals affected with Redwater. He found that in most cases of East Coast Fever, there was no appreciable decrease in the number of red blood corpuscles, this being in marked contrast to what is observed in Redwater. Theiler noted that cattle might harbour both the parasites of Redwater (P. bovis) and those of East Coast Fever (bacillary forms = T. parva). The former generally appeared in the blood “only towards the end of the fever reaction in East Coast Fever,” being previously latent in the animals which had been “salted” against Redwater. He distinguished “two groups of piroplasmosis,” the inoculable (Redwater, canine and equine piroplasmosis) and the uninoculable (East Coast Fever) by injection of infected blood. The parasites in the latter are much smaller than in the former. He named the parasites of East Coast Fever Piroplasma parvum. Theiler distinguished the parasite of East Coast Fever from P. bovis because of the frequent occurrence of bacillary forms and the minute size of the parasite, but he nevertheless retained the new parasite in the genus Piroplasma.

In a previous paper, Nuttall and Graham-Smith (Parasitology, 1908, vol. i. p. 134) described the appearances presented by P. bovis in stained films, and showed that they agreed with those of P. canis and P. pitheci with regard to the presence of the “budding” or multiplication forms. The multiplication of P. bovis in fresh blood films remained, however, to be described.

During the winter of 1908, when working in the Wellcome Laboratories at Khartoum, Dr Balfour proposed that I should examine a number of parasites both of plants and animals in order to ascertain if the former were infested with any other species of parasite; in other words, to study what has been termed hyper-parasitism.

A male Malay tiger was brought to the Clifton Zoological Gardens in July, 1908. He was then very thin and fed badly. After a time he settled down in his new quarters, and ate his food with greater relish. He appeared to be in good health and his coat was sleek and fine, but he never jumped or played in the usual way of his species. On February 4th he vomited, and had a severe attack of diarrhoea. His breathing became less abdominal and more thoracic in character, and death occurred two days later.

In an earlier paper (1908, pp. 255–257), we gave a full account of a paper by Miyajima (1907) wherein this author described experiments in which he states that he succeeded in cultivating Theileria (Piroplasma) parva. According to Miyajima, he had no difficulty in cultivating the parasite when he added the blood of cattle (containing Theileria) to ordinary bouillon in the proportion of 1 : 5 to 1 : 10, the cultures being maintained at 20–30° C. Miyajima states that trypanosomes appeared in his cultures after an interval of 3 days and underwent vigorous multiplication reaching “the maximum after the tenth to fourteenth day.” Miyajima's description of the supposed process of development is obscure, and it is difficult to understand how the diminutive intra-corpuscular Theileria can develop into a Trypanosoma. We refer the reader who desires particulars regarding these experiments to our paper already cited. In our paper we stated that “a certain amount of scepticism” appeared justified until Miyajima's results had been extended and confirmed. The warning appears to have come too late in respect to Woodcock's recent review of the Haemoflagellates in Ray Lankester's Treatise on Zoology (1909, Part I. fasc. 1, p. 260), for Woodcock appears to accept Miyajima's remarkable discovery as authentic and conclusive.

Amoeba chironomi, nov. sp., is distributed through practically the entire length of the digestive tract of the larva of Chironomus.

The body of A. chironomi varies from 15μ, to 18μ in length and from 10μ. to 12μ in breadth. The single pseudopodium may reach 15μ in length; one pseudopodium only is usually present.

Ectoplasm and endoplasm are well differentiated. A nucleus and a contractile vacuole are present. Food vacuoles are rare. The contractile vacuole resembles an iris diaphragm, consisting of a series of fine, curved, radiating canaliculi, opening into a central space. The excretory products are faintly reddish in colour. The presence of a contractile vacuole is uncommon in parasitic Amoebae.

The nucleus is poor in chromatin. A nucleolus is present.

A. chironomi is highly sensitive to the degree of concentration of the medium in which it lives. Very slight increase in density causes the organism to encyst.

Encystment occurs in the rectum of the host, and the cysts are voided with the faeces. The cysts are from 12μ. to 20μ long and from 9μ broad. The process of encystment is rapid.

The method of cross-infection of the host is probably a “casual” one, viz. by the mouth.

Early in January of the present year Professor Minchin kindly gave me for examination a portion of tissue and a mounted section taken from a case of the above disease and sent him by Dr J. Burton Cleland, then of Perth, W. Australia. The tissue was already embedded in paraffin, and both it and the section were stained by Levaditi's method and showed the presence of numerous spirochaetes.

All attempts hitherto made to infect different species of animals with P. canis have failed, and the remarkable fact remains that this parasite is apparently only communicable to dogs.

(1) Crithidia gerridis occurs in the alimentary tract of Gerris fossarwm, Microvelia and Perittopus sp. found in Madras. It is now recorded from the British water-bug, Gerris paludum, for the first time.

(2) The parasite occurs throughout the alimentary tract, in the ovaries and in the faeces of its host.

(3) There are three phases in the life-history of Crithidia gerridis, a pre-flagellate stage (Pl. IV, Figs. 1–10), a flagellate stage, and a post-flagellate stage (Figs. 64–69), the latter being adapted for life outside the body of the host, and for cross-infection.

(4) The adult flagellate has an elongate body, and possesses a large oval or round nucleus. There is also a smaller, usually rod-like mass of chromatin, the blepharoplast, near to which the flagellum arises. The flagellum may be as long again as the body, and is attached to it by a narrow, undulating membrane, in which myonemes are present (Figs. 22–46).

(5) The movements of the flagellated forms are characteristic; both the body and flagellum take part in the motion.

(6) The pre-flagellate forms are small, usually oval bodies, 3μ to 7μ long, and from 2μ to 4μ broad. Their nucleus often lies to one side and is round, the blepharoplast is rod-shaped. The flagellum grows out as a thin, delicate thread, in close contact with the body. The pre-flagellate phase of the parasite is found in the crop of the nymphs particularly, and also in the crop of the adults.

(7) The flagellate form has the general structure outlined in (4). The undulating membrane has indications of myonemes (Pl. IV, Figs. 41, 44, 45). The flagellum arises near a chromatic dot, the basal granule (Figs. 22, 30, 43). In the nucleus sometimes eight large chromatic masses may be present, or the grains may be very small. The nucleus on the whole is of the vesicular type. The blepharoplast is usually anterior to the nucleus, only very occasionally is it lateral. Chromidia are scattered in the endoplasm of the parasite (Figs. 26, 39, 46).

(8) The post-flagellate stage is a preparation for extra-corporeal life. The parasites divide in the rectum, lose their flagella, round themselves off and form a thin gelatinous cyst wall that rapidly hardens (Figs. 68, 69). These small cysts pass out with the faeces of the host.

(9) Longitudinal division is the chief method of multiplication. It may occur in all phases of the life-history and may be equal or sub-equal. The blepharoplast usually constricts first and division of it and of the flagellum follow one another very rapidly, division of the membrane follows, and then that of the body. The daughter halves gradually diverge and finally separate. In pre-flagellate division, rosettes may be formed by several rapid, repeated, longitudinal divisions (Fig. 20). The fully flagellated individuals first divide into two and repeated division may result in rosettes, but these very rapidly break up. Aggregation rosettes of mature flagellates are, however, extremely common (Fig. 47).

(10) The mode of infection is a casual one, the young nymphs taking up faeces containing crithidian cysts from the leaves of water plants.

(11) The parasite is purely a parasite of insects, occurring in two species of Gerris, G. fossarum and G. paludum. The systematic position of the parasite is near the Trypanosomes in the family Trypanosomatidae.

(12) There is much variation of form exhibited by the adult flagellate. Some of the very long parasites (Pl IV, Figs. 39, 44–46). appear to be peculiar to the Crithidia found in the gut of G. paludum, and have not been figured before so far as I know. This polymorphism needs careful attention, and is very confusing if only isolated stages of the parasite are studied. A similar remark applies to other crithidial and herpetomonad forms.

(1) The marked peculiarity in infection of Decapod Crustacea by certain parasites is the alteration of the sexual characters of the hosts (castration parasitaire of Giard). This has a twofold nature, viz.:—

(a) Sterility. In both sexes gonads dwindle and may entirely abort.

(b) Assumption of characters of other sex. In the male the external characters proper to the female are assumed in greater or less degree. The testis is transformed into a hermaphrodite gland, either while still infected by the parasite (incipient hermaphroditism of hermit crabs) or when freed from its influence (ripe ovotestis in spider crab). The reverse effects are not found in the female.

(2) This phenomenon occurs throughout the Invertebrata, but though sterility is a frequent consequence, the alteration of sex characters is never so definite as in the Crustacea. Analogous observations on parasitism in the Vertebrata have not been made.

(3) In the Insecta, parasitism appears to influence caste production. Grassi suggested that the sterility of soldiers and workers in Termites is due to protozoan parasites. In ants a giant caste is known (mermithergates) always harbouring a nematode worm.

Since we published our last notes upon new species, a considerable number of specimens have reached us from various parts of the world, thanks to many gentlemen who have interested themselves in these parasites. We propose to group our descriptions of new species according to genera as we progress with the diagnoses we are preparing for the work on Ticks which we are publishing in conjunction with Messrs Cooper and Robinson. We have already described several new species of Ixodes and Haemaphysalis and in this paper shall confine our descriptions to species belonging to these genera. Although, more recently, we have received many specimens belonging to the genus Ixodes, we have, in the majority of instances, been able to refer them to established species, but sometimes with great difficulty owing to the meagre description given by many authors, and especially to the absence of figures. We consider that too much stress cannot be laid upon the necessity of illustrative figures accompanying authors' descriptions of new species for a glance at a figure frequently suffices as a means of rapidly identifying a species, and for this purpose the simplest text figures are usually amply sufficient.

1. Regarding attempts at immunization by means of immune serum. The experiments of Nocard and Motas indicated that protective substances occur in the blood of dogs which have recovered from European piroplasmosis. These authors claimed that immune serum destroys the parasites and that it exerts a protective and curative effect upon the disease. Similar experiments conducted by Robertson in Cape Colony gave contrary results to those of Nocard and Motas. In the absence of further evidence no conclusions can therefore be drawn from these conflicting results. Two of Robertson's dogs which received injections of hyperimmune serum every day after they showed fever, died but no parasites could be found in their blood at death. This result suggests that a partial immunity may have been obtained by the treatment.

2. Regarding attempts at immunization by means of inoculations with blood containing dead parasites. Of the five dogs which we attempted to immunize in the manner we have described, three died of acute piroplasmosis; one died without parasites in its blood although parasites had been previously found; one dog died on the 36th day from chronic piroplasmosis. The absence of parasites in the blood of one dog at autopsy and the occurrence of chronic piroplasmosis in another dog may or may not indicate a partial acquisition of immunity. In any case the experiments afford no evidence that practical results are likely to follow further investigation of this character.

3. The duration of immunity following recovery is undetermined since there are no experiments to prove that animals are immune after the parasites have completely disappeared from their blood. In the socalled “immune,” “recovered,” or “salted” dogs, the animals harbour parasites in small numbers for weeks, months or years, consequently such dogs are suffering from a mild chronic form of piroplasmosis. Whilst subject to this mild form of the disease dogs usually escape acute infection when reinoculated with the parasite.

4. The parasites of piroplasmosis canis may persist in the blood of apparently recovered dogs for a considerable length of time, 6 months to 2 years, and so long as they are present in the blood the latter remains fully virulent for clean dogs. Consequently there is no evidence that the African Piroplasma canis becomes modified in its virulence during the course of chronic piroplasmosis. According to Nocard and Motas the European P. canis does become modified in its virulence in dogs suffering from chronic piroplasmosis. It should be noted that the European disease is a milder affection than the African.

5. The passage of the African P. canis through two series of dogs, that is through upwards of 90 animals in the course of two and a half years, has shown that the parasite may be communicated by inoculation from dog to dog for an indefinite period. No evidence was obtained that P. canis is in any way modified in its virulence by passage through dogs. In a number of dogs which were inoculated with post-mortem blood the disease developed more slowly than usual but nevertheless led to a fatal issue.

6. The onset of fever after inoculation with virulent blood may precede or succeed the appearance of P. canis in the peripheral circulation. Occasionally the disease may run its course to a fatal termination without the appearance of febrile symptoms.

(1) Herpetomonas jaculum is a parasite of Nepa cinerea, occurring in the alimentary tract of its host.

(2) The life-history of the parasite may be conveniently divided into three stages, the pre-flagellate, flagellate and post-flagellate stages, which gradually merge into one another.

(3) The movements of the parasite are less flexible than those of Crithidia, as Herpetomonads have no undulating membrane. The flagellum is the most active agent in effecting motion.

(4) The pre-flagellate stages of the parasite (Pl V, Figs. 1–18) are best observed in the crops of nymphs of Nepa cinerea. The parasites at first are oval (Figs. 1–6). They vary in size from about 4μ to 5μ long and from 2μ to 2·5μ broad. They show nucleus and blepharoplast, and may divide longitudinally before flagella are acquired (Figs. 2–4). The flagellum of each parasite arises from a region near to the blepharoplast but not directly from it (Figs. 5–9).

(5) The flagellate stage of the organism (Pl. V, Figs. 19–36) is that best known. H. jaculum is from 13μ to 33μ long and from 1μ to 4μ broad, the size varying according to the recency or otherwise of longitudinal division. Myonemes (Figs. 20, 21, 38) are present on the body. The flagellum is at least as long again as the body. The nucleus contains a number of grains of chromatin (Figs. 28, 34), sometimes in the form of eight large grains (Figs. 34, 36), sometimes as very fine granules (Figs. 22, 31). The blepharoplast is in the anterior, pre-nuclear, region of the parasite, and is usually rod-like (Figs. 19, 20, 21). The single flagellum (Fig. 19) arises near it but not from it. A basal granule (Figs. 22, 33, 34) is present at or near the origin of the flagellum. Chromidia are present as scattered granules in the body (Figs. 28, 32, 33).

(6) The post-flagellate stage is the form assumed by the parasite for life outside the body of the host. Preceding encystment, the organism divides twice longitudinally, giving rise to four daughter forms (Pl. V, Figs. 51–54) each of which ultimately loses its flagellum, rounds itself off and forms a cyst (Figs. 57–68). These cysts are from 2·5μ to 4·5μ long and from l·4μ to 2·6μ, broad. They occur in the rectum of Nepa cinerea and are voided with the faeces, being ingested later by other bugs.

(7) Longitudinal division is the common method of multiplication of H. jaculum. The flagellum may divide precociously, but usually division is initiated by constriction of the blepharoplast (Pl. V, Fig. 37) almost simultaneously with division of the flagellum and followed by that of the nucleus (Figs. 39, 40). A split occurs (Figs. 40–42) and the active movements of the two flagella aid in the divergence of the daughter organisms (Figs. 43, 44), which ultimately separate.

(8) I have no evidence whatever for ascribing sex to any form of Herpetomonas, but consider the occurrence of long and short and of thin and stout forms to be explicable as the results of growth and division. Also, I have shown experimentally that richly granular protoplasm is the result of a physiological condition and is not necessarily fixed as an attribute of the female sex.

(9) One mode of infection has been proved experimentally in the laboratory and also observed at the breeding grounds of the Nepa. Cysts voided in infected faeces are swallowed by other Nepa in the adult and nymphal stages. The crops of such nymphs on dissection were found to contain cysts, whereas no flagellates were present in other parts of the gut, so that primary infection occurred here in the crops of the nymphs.

Cannibalism of Nepa cinerea whereby other Nepa are devoured is also responsible for the spread of H. jaculum.

I have no definite evidence of hereditary infection, although I have found flagellate and post-flagellate forms (Pl. V, Figs. 46–49) in the ovaries of the host. The parasites were not found in the eggs. The occurrence of parasites in the ovaries may be regarded as a stage in the evolution of hereditary infection.

(10) Various environmental effects have been studied, the most important observation being that fresh food appears to stimulate the parasites and to cause their rapid division.

(11) The generic name Herpetomonas should be retained, as originally constituted, for parasites having but one flagellum and no undulating membrane (see pp. 383 et seq.).

(12) I wish to record the occurrence of a new species of Herpetomonas, H. vespae, from the alimentary tract of the hornet, Vespa crabro.

1. Trypanblau injected subcutaneously into dogs a day before or a day after they have been inoculated with blood containing Piroplasma canis effectually prevents the development of piroplasmosis by destroying the parasites at the onset of infection.

2. Trypanblau given by the mouth is ineffective, since it exerts no apparent influence either upon the parasite or upon the course of the disease.

3. Tryparosan, when injected subcutaneously or when given by the mouth, has no effect upon the parasite and is ineffective as a remedy against piroplasmosis in the dog.

One of the earliest known Spirochaetes from Lamellibranchs was that briefly described by Certes in 1882 from the crystalline style of the oyster. Certes named the parasite Trypanosoma balbianii. In 1891 the same worker recorded the occurrence of the organism in two species of Tapes, namely T. decussatus and T. pullaster. Laveran and Mesnil in 1901 showed that Tr. balbianii probably belonged to the Spirochaetes. The organism is now known as Spirochaeta balbianii.

The natives of Siam are occasionally affected by subcutaneous tumours that have been found to be due to the presence of a small nematode worm named by Levinsen (1889) Cheiracanthus siamensis. Only one description of the species occurs in literature, and as this, the original one, was based upon a solitary immature female it is necessarily lacking in many details.

1. Trypanblau promises to be an efficient remedy for bovine piroplasmosis, since it exerts a direct and obvious effect upon the parasites.

2. The effect of the drug upon Piroplasma bovis is similar to that which it produces upon the canine parasite. The dividing forms are the first to disappear, and after a few hours the pyriform parasites also disappear from the peripheral circulation; the parasites which are detected in the blood after a few hours appear degenerated and rounded or irregular; within nine to 45 hours or less all the parasites have disappeared from the blood.

3. As in canine piroplasmosis the disappearance of the parasites from the blood may be temporary. The parasites also disappear and reappear in small numbers (after two to 11 days) in animals undergoing natural recovery. In three treated animals the parasites reappeared in exceedingly small numbers after five to six days; in two they had not reappeared after 16 and 18 days respectively. The animals show no symptoms and progress towards recovery.

4. It remains to be detercnined (1) how long the blood of treated cows may contain parasites after the apparent recovery, (2) if the parasites in such recovered animals are altered in virulence, (3) if the parasites are capable of infecting ticks.

5. The experiments were conducted on nine cows, of which four served as controls and five were treated with trypanblau. Of the controls two suffered from haemoglobinuria, and one of these died of piroplasmosis; the two other controls had no haemoglobinuria and were very mild cases. All of the treated cows had haemoglobinuria and recovered. In four of the treated cows haemoglobinuria occurred before treatment began.

6. As might be expected, the drug exerts a more rapid effect when injected intravenously. The parasites disappear more slowly after subcutaneous injection of the drug. (Judging from our recent experiments on dogs, the giving of the drug per os promises to be without effect. See This Journal, p. 231.)

7. Although doses of 150–200 c.c. of a saturated watery solution of the dye were used, it is probable that smaller doses will prove efficient. The drug appears to produce no ill-effects upon cattle.

8. The drug, being a dye, has the disadvantage of colouring the tissues, more especially the subcutaneous connective tissues. How long the colouration persists remains to be determined. In any case this disadvantage can scarcely weigh in the balance as against saving the life of the animal, especially when used for breeding purposes.

9. We hope that experiments, which are about to be conducted in the field in Africa and elsewhere, will demonstrate the value of the remedy in practice.

10. Trypanblau and similar drugs should be given a trial in the treatment of Carçeag in sheep and Biliary Fever in horses.

Before describing my further results in the treatment of canine piroplasmosis by aniline dyes, I desire to mention some earlier experiments, carried out by Italian investigators whose papers have until recently escaped my attention. The results of the Italian experiments are cited in considerable detail with a view to making it unnecessary for other workers to refer to the original papers which may be difficult of access. It is well to note that the authors in question give very little information concerning their experiments; thus Memmo, Martoglio and Adani are satisfied with giving mere clinical notes, whilst Levi della Vida omits a number of essential data from his protocols.