Intestinal Flagellates from Some New Zealand Insects

Transactions and Proceedings of the Royal Society of New Zealand, Volume 84, 1956-57, Page 297

Intestinal Flagellates from Some New Zealand Insects By Marshall Laird Department of Parasitology, University of Malaya [Received by Editor August 11, 1955.] Abstract Fifty larvae of Pericoptus truncatus Fabr. (Coleoptera, Scarabaeidaé), 10 larvae of Odontria zealandica White (Coleoptera, Scarabaeidae), 20 adults of Platyzosteria novae-zealandiae Brunn. (Orthoptera, Blattidae) and 10 adults of Hemideina thoracica (White) (Orthoptera, Tettigoniidae) were collected in various localities in and about Wellington during the summer of 1950–51. On examination for intestinal Protozoa all these insects proved to be infected with Monocercomonoides melolonthae (Grassi, 1879). Four of the O. zealandica larvae harboured Retortamonas phyllophagae (Travis and Becker, 1931), while Polymastix melolonthae (Grassi, 1879) was recorded from all the larvae of O. zealandica and from all but two of those of P. truncatus Thirty-nine of the Pericoptus larvae were positive for a retortamonad for which the name of Retortamonas pericopti n. sp. is proposed. Introduction Grassi (1879) lumped together several widely differing flagellate protozoans in his genus Monocercomonas, within which he established four subgenera, Monocercomonas, Retortamonas, Schedoacercomonas and Trichomonas. He subsequently (1881) separated Monocercomonas from these other organisms, but failed to designate type species. Subsequent authors have thus differed among themselves in their application of Grassi's names, and considerable confusion and synonymy have resulted. Wenrich (1931, 1932) brought forward evidence indicating that Retortamonas Grassi, 1879—the status of which had long been obscure—has precedence over Embadomonas Mackinnon, 1911. Designating R. gryllotalpae (Grassi, 1879) Stiles, 1902, as the type, he published a good description of a flagellate which he regarded as referable to this species from Grassi's type host (the European mole cricket, Gryllotalpa gryllotalpa) in New Jersey. Although Bishop (1934) and Dobell (1935) challenged Wenrich's conclusions, the concensus of modern opinion favours the latter author. The controversy was discussed at some length by Kirby and Honigberg (1950) who, while following Wenrich, pointed out that Stiles made “no original contribution to the nomenclature of the species name (i.e., of the type species) that warrants including his name in the authorship.” Members of the genus Retortamonas are biflagellate zoomastiginids having a more or less pyriform body rounded anteriorly and usually pointed posteriorly. A caudal process is frequently present. The nucleus is anteriorly positioned, and immediately behind it is a prominent cytostome bordered anteriorly and laterally by a chromophile fibril. There are two basal granules on the cytostomal side of the nucleus, the anterior flagellum originating at the anterior granule. A recurrent flagellum, which originates at the posterior basal granule, near the anterior margin of the cytostome, is directed along the cytostome beyond which it may protrude. There has been considerable uncertainty as to the correct systematic position of Retortamonas. It has been customary to regard the flagellar number as of prime importance in the major dichotomies within the class Mastigophora. Kudo (1946) and indeed most modern protozoologists have assigned all zoomastiginid flagellates.

having either one or two flagella to the order Protomonadina Blochmann. The genus is thus usually considered to belong in that order and to the family Bodonidae Bütschli, which Kudo (1946) defined as embracing those protomonadinids lacking an undulating membrane and having two flagella originating anteriorly, one of these being directed forwards and the other trailing posteriorly. Unfortunately for this reasoning the closest affinities of Retortamonas he not with the other genera of the family Bodonidae as understood by Kudo but with Chilomastix Alexeieff Flagellates of the latter genus have three anterior flagella and a cytostomal one, and following conventional usage they must be considered as belonging to the order Polymastigina Blochmann. The members of this order never have less than three flagella and seldom more than eight, although in one family 12 or more occur. We are thus confronted with the necessity of assigning two genera of fundamentally similar structure—particularly as regards the cytostomal complex, which is of a unique type—to different orders merely on the basis of flagellar number. Alexeieff (1912, 1917) and Mackinnon (1915) drew attention to the similarities between (Embadomonas) = Retortamonas and Chilomastix, the former author (1917) suggesting that they should be assigned to a single family for which he proposed the name Embadomonadidae. Wenrich (1932), having demonstrated that Embadomonas is a synonym for Retortamonas, established the family Retortamonadidae to include the two genera. Although remarking that “the tendency to use flagellar number for the separation of larger groups of flagellates has been much overdone in the past,” Wenrich left the ordinal position of his new family unstated. The inclusion of the family Retortamonadidae, its members having two or four flagella, in either the Protomonadina or the Polymastigina would of course be no more anomalous than the accepted inclusion of the family Callimastigidae—the members of which have 12 or more flagella—in the Polymastigina. At the same time, an increasing number of protozoologists favour the viewpoint expressed by Kirby and Honigberg (1950) who recognized the artificiality of establishing arbitrary barriers between the orders of the Zoomastigina solely on the basis of the number of flagella. One solution would be the establishing of a separate order expressly for Retortamonas and Chilomastix, and Grassé (1952) has recently proposed that the Retortamonadidae and the family Cochlosomidae Tyzzer, the members of which have six flagella, should be considered together as a distinct order. However much of Grassé's drastically revised classification of the Zoomastigina ultimately comes to be accepted, it is felt that the relationships betwen Retortamonas and Chilomastix are best expressed by the retention of both genera in the order Retortamonadia Grassé, 1952. Having indicated that Eutrichomastix Kofoid and Swezy is a synonym of Monocercomonas Grassi, Travis (1932) designated M. colubroruorum (Hammerschmidt) as the genotype and established a new genus, Monocercomonoides, for certain flagellates previously included with the former ones. Later authors have mostly followed Travis in applying the generic designation of Monocercomonas to those polymastiginid flagellates having three anterior flagella and a trailing one originating at a single basal granule located in front of the anteriorly positioned nucleus, and a more or less well-defined axostyle which usually projects beyond the tapered posterior extremity of the body. The characteristics of Monocercomonoides, as exemplified by M. melolonthae (Grassi, 1879), the species selected by Travis (1932) as the genotype, are as follows: The body may be ovoidal, pyriform, spherical or subspherical; there are four flagella which originate in pairs at two anteriorly located basal granules, these being connected with one another by a rhizoplast; the proximal part of one of these flagella may adhere to the pellicle, the flagellum in question trailing posteriorly, a slender axostyle, originating at one of the basal granules, curves around the nucleus and terminates at the posterior tip of the body; in some stained examples a halo of

chromatin granules is evident at one side of the periendosomal area, between the endosome and the nuclear membrane. Butschli (1884) established the genus Polymastix with P. melolonthae (Grassi, 1879) as the genotype, Grassi (1879, 1881) having earlier designated this flagellate Trichomonas melolonthae. Polymastix is referable to the order Polymastigina, its members having four flagella. These originate in pairs at two basal granules as do those of Monocercomonoides, but the axostyle which runs posteriorly from one of the granules differs from that of the last-named genus in being quite thick near its point of origin and thereafter tapering rapidly, ending in a filament which does not reach the posterior extremity (Grassé, 1952). The body is characteristically more elongate than that of Monocercomonoides, and is usually fusiform or flattened and leaf-like. Perhaps the most striking feature of the genus Polymastix is that bacteria of the genus Fusiformis Hoelling, 1910 (Mycobacteriaceae) are always attached longitudinally to the pellicle, which thus appears ridged. Wenyon (1926) included all three genera, Polymastix, (Eutrichomastix) = Monocercomonas and (Retortamonas) = Monocercomonoides, within his family Trichomonadidae, the diagnosis of which he gave as follows: “The flagellates belonging to this family are characterized by the possession of a variable number of flagella, a definite cytostome, and a rod-like structure, the axostyle, which arises from the blepharoplasts and passes through the body to its posterior end, through which it usually protrudes In some forms, one of the flagella is directed backwards, and its axoneme may be attached to the border of an undulating membrane. In such cases there is usually a stiff basal fibre, which lies along the line of attachment of the undulating membrane to the body. Kudo (1946), on the other hand, resurrected the polymastiginid family Polymastigidae Bütschli, 1884, for uninucleate Polymastigina having axial organella but lacking an undulating membrane, cresta, rostellum, and adherent flagella He considered Polymastix, (Eutrichomastix] = Monocercomonas and (Monocercomonas) =Monocercomonoides (in part), to be referable to this family, reserving Trichomonadidae Wenyon for uninucleate Polymastrgina having both axial organella and an undulating membrane. Kirby (1947) refused to recognize a close relationship between Polymastix, Monocercomonoides and Monocercomonas, and transferred the last-named genus to the family Monocercomonadidae Kirby of his new order Trichomonadida. This order was established for zoomastiginids having from three to six flagella, one of which is typically differentiated as a trailing flagellum, an axostyle and a parabasal body of the Janicki type which originates de novo in relation to the basal granule system. Six families were included within the order, one of which, Monocercomonadidae Kirby, included “Trichomonadida with trailing flagellum free from body surface or adherent, but with no cresta and undulating membrane at the base of which there is a costa”; another, Trichomonadidae Wenyon, was to embrace “Trichomonadida with an undulating membrane and costa.” Grassé (1952) altered the spelling of Kirby's ordinal name to Trichomonadina, defining this order as including small, medium and large zoomastiginids having an axostyle, from three to six flagella—one of which is recurrent and either free or attached to the body or connected with the latter by means of an undulating membrane—and with a parabasal apparatus always well developed. He relegated Monocercomonadidae Kirby to the status of a sub-family of Trichomonadidae Wenyon, emending the definition of this family to cover those zoomastiginids having a parabasal apparatus and from four to six flagella, one of which is always reflected posteriorly, being either free and trailing or adhering to the body with or without the interposition of an undulating membrane. At the same time, Grassé retained Polymastix and Monocercomonoides in the Polymastigidae Bütschli He regarded this family as of uncertain systematic position and did not assign it to an order, defining it

as including small to medium zoomastiginids having four flagella (of which one may be directed posteriorly), not grouped in bundles and inserted apically or subapically, possessing a fibrous axostyle and a nucleus with a large, spherical endosome, and probably lacking a parabasal apparatus. There the matter rests for the present. It is not proposed to attempt an evaluation of Grassé's provocative new classification of the flagellates herein, nor yet to suggest any fresh approach to the subdivision of the zooflagellates as this would involve the consideration of many more data than are relevant to this paper. It was merely felt timely that these developments should be summarized in an Australasian journal, particularly in view of the adherence to one of the discarded synonyms, Eutrichomastix Kofoid and Swezy, in a recent New Zealand account (Anon., 1953) which listed the widespread entozoan of frogs, Monocercomonas batrachorum (Dobell) as “Eutrichomastix batrachorum (Dobell).” All three of the genera recorded herein, Polymastix, Monocercomonoides and Retortamonas, occur in the alimentary tract of their hosts. Polymastix is known from various insect larvae (particularly those of scarabaeid beetles) and from myriapods, while the other two genera have insectan, amphibian, reptilian and mammalian hosts. A species of Retortamonas, R. intestinalis (Wenyon and O'Connor), is a common and widespread endocommensal of man. Material and Methods Fifty larvae of the scarabaeid beetle, Pericoptus truncatus Fabr., and 20 adults of the “Maori bug”, Platyzosteria novae-zealandiae Brunn. (Orthoptera, Blattidae), were collected under logs above high tide mark on the beach towards Pencarrow, Wellingon, during December, 1950. Ten wetas, Hemideina thoracica (White) (Orthoptera, Tettigoniidae) were collected from pockets of native bush in the suburbs of Wellington during February and March, 1951, while 10 larvae of the “grass grub”, Odontria zealandica White (Coleoptera, Scarabaeidae) were secured from the soil beneath logs in open, grassy country near Woodside, Wairarapa, during March, 1951. All these insects were brought alive to the laboratory, where their intestinal conents (diluted with normal saline where necessary) were examined microscopically by dark ground illumination. Cover slip smears were fixed in Davis's (1947) modification of Worcester's fluid, stained with Heidenhain's haematoxylin and differentiated with picric acid. Air dried smears were also made, these being stained with Giemsa. Other protozoans noted in addition to the flagellates considered herein were an unidentified gregarine in O. zealandica and Endamoeba sp. (Sarcodina) in O. zealandica and P. truncatus. Microscopical preparations of the flagellates have been deposited at the Dominion Museum, Wellington. Systematic Account Retortamonas phyllophagae (Travis and Becker, 1931). (Fig. 2.) This species was described by Travis and Becker (1931) under the generic name of Embadomonas from the larva of a North American grass grub, Phyllophaga sp. (Coleoptera, Scarabaeidae). Kowalczyk (1938) subsequently reported it from the larva of the Japanese beetle, Popillia japonica Newm. (Coleoptera, Scarabaeidae), also in North America. Four of the 10 larvae of Odontria zealandica were hosts for retortamonads, these being present in the hind gut only. The size of 50 random examples in haematoxylin-stained preparations ranged from 4.5μ to 11.5μ by 2.6μ to 6.3μ (av., 8.2μ by 3.8μ). The body is more or less pyriform, slight torsion often being evident anteriorly (Fig. 2). In small to average sized examples the greatest breadth is attained at about a third of the total body length from the anterior extremity, while in large.

Explanation of the Plate All figures drawn with the aid of an Abbe camera lucida at a magnification of 2,530X. Fig. 1.—Retortamonas pericoptus n.sp from Pericoptus truncatus. An example near the lower limit of the size range. Iron haematoxylin. Fig. 2.—Retortamonas phyllophagae (Travis and Becker) from Odontria zealandica. An example of average size. Iron haematoxylin. Figs. 3–6.—Retortamonas pericopti n.sp. from Pericoptus truncatus. Figs. 3–5, average sized examples. Giemsa. Fig. 7.—Spore-bearing bacteria from the gut contents of Pericoptus truncatus. Giemsa. Figs 8 and 10.—Polymastix melolonthae (Grassi) from Odontria zealandica. Iron haematoxylin. Fig 9.—Polymastix melolonthae from Pericoptus truncatus. Giemsa. Fig. 11.—Fusiformis sp. from the gut contents of Odontria zealandica. Iron haematoxylin. Figs. 12–16.—Monocercomonoides melolonthae (Grassi). Fig. 12, from Platyzosteria novae-zealandiae. Osmic-Giemsa. Fig. 13, from Pericoptus truncatus. Giemsa. Fig. 14, from Odontria zealandica. Iron haematoxylin. Fig. 15, from Hemideina thoracica. Iron haematoxylin. Fig. 16, from Hemideina thoracica. Giemsa.

examples the broadest part of the body is at the central region. There is usually a short and clearly demarcated caudal spike (Fig. 2), which never exceeds 1.2μ in length, at the posterior extremity. This structure is always evident in life, but sometimes appears to be lacking in fixed and stained examples, particularly those towards the upper limit of the size range. The length of the oral pouch is about half that of the body in small examples, and about a third that of the body in large ones. A fine chromophile fibril borders the oral pouch anteriorly and laterally This fibril, although prominent in haematoxylin preparations, is relatively unresponsive to Giemsa. The nucleus has a large central endosome, which exhibits peripherally disposed chromatin granules. In the smaller examples the posterior flagellum is usually confined within the oral pouch, but in the larger ones it may protrude for a short distance. The latter flagellum is always markedly shorter than the anterior one, while the length of neither of them exceeds that of the body. The alveolar cytoplasm, which may contain small chromophilic granules and the spore bodies of ingested bacteria, stains light blue with Giemsa. Kowalczyk (1938) found the average size of 400 random examples of R. phyllophagae from the Japanese beetle to be 7.8μ by 4.0μ. This is extremely close to the size of the retortamonad under discussion, which agrees morphologically with R. phyllophagae as described by Travis and Becker and by Kowalczyk. This flagellate of O. zealandica is thus identified as Retortamonas phyllophagae (Travis and Becker), the new host and locality not being regarded as sufficient grounds for its description as new. Retortamonas pericopti n.sp. (Figs. 1, 3–7). Thirty-nine of my 50 Pericoptus truncatus larvae proved to harbour the large retortamonad described hereunder. This species has a slipper-shaped or fusiform body, which, in living examples, may measure up to 26μ in length and 9μ in breadth. Fifty random examples in haematoxylin preparations ranged from 10.9μ to 22.2μ in length and from 4.0μ to 7.9μ in breadth (av., 15.1μ by 5.9μ), while the same number of random examples from Giemsa-stained dry smears ranged from 11.1μ to 25.7μ in length and from 4.6μ to 8.9μ in breadth (av., 16.1μ, by 6.5μ) The length of the oral pouch seldom exceeds two-fifths of that of the body. The flagellate tapers posteriorly, and may exhibit spiral twisting (Figs. 3 and 6). There is usually a caudal spike which may reach 5μ in length but seldom exceeds 1μ in thickness at the base. This structure seldom appears to advantage in air-dried films, assuming a rather lighter shade of blue with Giemsa than does the body proper (Figs. 3–5), but is well marked in life and appears more or less hyaline in haematoxylin mounts. As in R. phyllophagae the anterior flagellum is the longer but is not, however, as long as the body. The posterior flagellum is rarely (Fig. 3) confined within the oral pouch, usually protruding for at least three or four microns beyond this structure. The average lengths of the anterior and posterior flagella are 14μ and 10μ respectively. The alveolar cytoplasm stains light blue with Giemsa, with darker blue maculations, especially towards the periphery. Numerous vacuoles may be present (Figs. 3, 5, 6), and food inclusions—particularly the spore bodies of bacteria (Figs. 3, 4, 6° 7)—are frequently demonstrable. The nucleus, as seen in haematoxylin preparations, has a large endosome with peripheral aggregations of chromatin (Fig. 1). It is variously distorted, and the structural details are not apparent, in Giemsa-stained examples (Figs. 3–6). The basal granules giving origin to the flagella are located against the nuclear membrane adjacent to the inner end of the oral pouch. This Retortamonas of P. truncatus differs morphologically from known species insofar as can be gathered from published data. It is of very much larger size than some of the other species from insects. Thus, while its dimensions range from c. 11μ to 26μ by c. 4μ to 9μ, R. agilis (Mackinnon) measures 4μ by 1.5μ to 11μ by

(Mackinnon, 1915), R. blallae (Bishop) ranges from 6μ to 9μ in length (Bishop, 1931) and R. phyllophagae (Travis and Becker) ranges from 4μ to 12μ in length and from 2μ to 8.5μ in breadth (Kowalczyk, 1938). Species of comparable size are R. alexeieffi (Mackinnon) (7μ to 16μ by 5μ to 9μ, according to Mackinnon, 1915, and up to 22.5μ by 8.7μ according to Ludwig, 1946); R. gryllotalpae Grassi (7μ to 19.5μ in length, the breadth varying from one-fifth to one-half the length, according to Wenrich, 1932); R. caudacus Geiman (6μ to 22μ by 3μ to 6μ; Geiman, 1932); and R. wenrichi Stabler (12.2μ to 19μ by 3.8μ to 8.4μ; Stabler, 1944). The three last-named species differ sharply from that under discussion in the length of the caudal process relative to that of the body. According to Wenrich (1932) this process is from one-quarter to two-fifths of the length of the body proper in R. gryllotalpae, which further differs from the present species in that its flagella are longer than the body, the posterior flagellum being longer than the anterior one. R. caudacus has a long, needle-like caudal process, its oral pouch is half the length of the body, and the nucleus is located right at the anterior extremity (Geiman, 1932). R. wenrichi is unique in that the caudal process may vary from a short spike to a looped and twisted filament up to 58.5μ in length. This species differs further from the one from P. truncatus in that its flagella are of approximately equal length and are generally from one-and-a-half times to twice the length of the body proper. Grassé (1952) illustrated long-tailed examples of R. gryllotalpae discovered by him together with normal and tail-less forms in the type host in Europe (his Fig. 663), and was inclined to consider that R. wenrichi may be a synonym of the former species, although he did not commit himself in this matter. He also pointed out that R. caudatus is very close to R. gryllotalpae. As far as can be gathered from the literature, the closest affinities of the New Zealand retortamonad he with R. alexeieffi (Mackinnon). This species was originally described from crane fly larvae (Dipera, Tipulidae; Tipula sp.) in England, and has since been reported from North American tipulid larvae (Geiman, 1933, Ludwig, 1946). Its size is comparable with that of the present species, according to the following figures from Ludwig (1946). As seen in haematoxylin preparations, it varies from 10μ to 22.5μ on length (as compared with 10.9μ to 22.2μ) by from 4.1μ to 8.7μ in breadth (as compared with 4.0μ to 7.9μ). The average size of R. alexeieffi in such preparations is very close indeed to that of the New Zealand species, being 13.7μ by 6.2μ (as compared with 15.1μ by 5.9μ) Trophozoites of R. alexeieffi may either have a short caudal spike (Wenrich, 1932) or else the posterior end may be rounded, this last condition being the characteristic one according to Ludwig (1946). In the retortamonad from Pericoptus truncatus, on the other hand, a caudal spike is usually present. The length of the anterior flagellum as compared with that of the body appears to be much the same in both cases, but the posterior flagellum is relatively shorter in R. alexeieffi and its extremity seldom projects out beyond the oral pouch (Ludwig, 1946) The cytoplasm of R. alexeieffi frequently contains numerous ingested bacteria, that of the example illustrated by Ludwig in his Pl. II, Fig. 13 containing two inclusions very similar to the bacterial spore bodies so commonly ingested by the species under consideration. Finally, figures of R. alexeieffi published by various authors agree in showing the nucleus located at the very anterior tip of the body. This organelle is never so far forward in the New Zealand species. Differing in detail from other species of its genus as outlined herein, the retortamonad of Pericoptus truncatus is accordingly designated Retortamonas percopti n.sp. Its closest known relative would appear to be R. alexeieffi (Mackinnon), from which it is to be distinguished by the relatively longer posterior flagellum, the relatively longer and more persistent caudal spike and the less anterior location of the nucleus. These differences might well prove to be more apparent than real following future com-

parisons of more detailed data, in which event it is not considered that the difference in hosts should be regarded as debarring the identification of the present flagellate with R. alexeieffi. Monocercomonoides melolonthae (Grassi, 1879) (Figs. 12–16). Numerous authors have recorded this species, from a variety of European and North American insects. There are also unconfirmed reports from amphibians (Swezy, 1916) and from lizards (Wood, 1935) in North America. Monocercomonoides trophozoites were abundant in the hind gut contents of all the insects examined during this study. Spherical (Fig. 15), subspherical (Fig. 13), ovoid (Fig. 12) and pyriform (Figs. 14 and 16) examples were noted, ovoid and pyriform ones predominating. Living specimens, particularly pyriform ones, sometimes have a slight axostylar projection posteriorly. The pellicle of the posterior extremity appears to be somewhat sticky, for numerous individuals were observed to have bacteria or inorganic particles adhering there. None of the internal organella could be distinguished either by bright field- or by dark field-illumination. The organism moves rapidly through the medium, three of the flagella beating anteriorly and the fourth trailing behind. This trailing flagellum was seen to serve as an attachment organelle from time to time. Giemsa-stained dry smears proved useful for the demonstration of the flagella, the axostyle and the two basal granules with their connecting rhizoplast. Although such preparations are unsuitable for the study of nuclear detail, and the flagellates concerned suffer a degree of hypertrophy and distortion, they may be employed to advantage in the rapid generic identification of these zoomastiginids. The particularly heavy staining of the flagella (see Plate) renders these conspicuous under quite low powered objectives, a useful feature for student demonstrations in teaching laboratories. Fixation with osmic vapour, followed by methyl alcohol and Giemsa resulted in the appearance of large, black-staining inclusions in the cytoplasm (Fig. 12). These—probably volutin granules—were never evident in flagellates fixed by any other method. The cytoplasm is alveolar and more or less granular, and usually contains a few vacuoles of varying size (Figs. 12–15). Ingested bacteria are occasionally evident. In haematoxylin-stained specimens the nucleus is seen to have a large central endosome, and in many instances a halo of chromatin granules is apparent to one side of this structure between it and the nuclear membrane (Figs. 14 and 15). This is a generic feature according to Kowalczyk (1938). Giemsa-stained examples, on the other hand, have the endosome hypertrophied and the nuclear membrane distorted. The anterior margin of the nucleus of such forms is usually somewhat concave, the curvature of the concavity often coinciding with that of the rhizoplast (Fig. 16). Because of this concavity the antero-lateral limits of the nucleus have the appearance of horn-like projections. This is without doubt an artefact consequent upon dry fixation, but it is nevertheless a useful aid to rapid recognition of the flagellate in Giemsa films. The trailing flagellum, which as Ludwig (1946) indicated is usually longer than the three anterior ones, originates from the same basal granule as does the axostyle. All four flagella of 20 consecutive Giemsa-stained trophozoites from Pericoptus truncatus were measured, with the following results: Trailing flagellum, 15. 0μ to 22. 5μ (av., 18. 2μ); anterior flagella, 13.1μ to 17.8μ (av., 14.8μ), 13. 3μ to 17. 4μ (av., 15. 1μ) and 12.7μ to 17. 0μ (av., 14. 6μ). The anterior flagella of course often become reflected in smears, and are so drawn in the accompanying illustrations for space economy. Three different series each of 50 consecutive trophozoites from the selfsame P. truncatus larva were measured. Living examples (made sluggish by lengthy exposure to the heat and light of the microscope lamp) were found to range from 5. 0μ to 11. 4μ in length (av., 7. 8μ) and from 3. 8μ to 10. 3μ in breadth (av., 6. 1μ).

Giemsa-stained ones in a dry film measured from 5.3μ to 10 7μ (av., 8.4μ) by from 3.6μ to 10.5μ (av., 7.1μ) Trophozoites in never-dried haematoxylin preparations, though, ranged from 4.3μ to 7.9μ in length (av., 5.8μ) and from 3.2μ to 5.7μ in breadth (av., 4.4μ). These differences according to the technique employed are in accord with those noted by Minchin (1909) for trypanosomes in air dried and never-dried blood films. No significant morphological or size differences could be ascribed to the difference in hosts. Thus 50 consecutive trophozoites in haematoxylin smears from Odontria zealandica ranged from 4.1μ to 7.9μ (av., 5.5μ) by from 3.0μ to 6.1μ (av., 4.2μ), while the same number in similar preparations from Hemideina thoracica ranged from 4.2μ to 8.1μ (av., 5.6μ) by from 2.9μ to 6.4μ (av., 4.4μ). The Monocercomonoides discussed herein is morphologically indistinguishable from M. melolonthae (Grassi) as described by numerous authors. It would appear to be of rather smaller average size than usual, the figures of some other investigators, derived from wet-fixed preparations, being: 6μ by 3μ to 9μ by 5μ (Macknnon, 1912; Tipula sp., England) 5μ to 12μ by 4μ to 10μ, av. (250 random examples), 6.6μ by 5.5μ (Kowalczyk, 1938; Popillia japonica Newm., U.S.A.) 6.2μ to 12 5μ by 4.3μ to 10μ, av (150 random examples), 10μ by 7.5μ (Ludwig, 1946; Tipula abdominalis, U.S A.). However, the size range of the present flagellate substantially overlaps the above ranges, which in any case differ quite considerably from one another; and the average dimensions of haematoxylin-stained examples are only of the order of one micron below those given by Kowalczyk (1938) Monocercomonoides globus Cleveland et al., occurring in North American wood roaches, is a distinctive species having a ribbon-shaped axostyle (Cleveland et al., 1934), while M. panesthiae, described by Kidder (1937) from a Philippine Islands wood roach, has a hyaline axostyle becoming free posteriorly and all four of its flagella appear to originate from a single basal granule. The latter species should thus be transferred to the genus Monocercomonas Grassi. Monocercomonoides cetoniae (Jollos) and M. ligrodis Travis, both described from beetle larvae, are synonyms of M. melolonthae, according to Grassé (1952) The common orthopteran Monocercomonoides, M. orthopterorum (Parisi), is of markedly smaller size than M. melolonthae, ranging from 3.7μ to 7.0μ by from 2.1μ to 3.8μ (av., 6.2μ by 3.1μ) (Ludwig, 1946) This flagellate, which on grounds of size alone bears comparison with the New Zealand one, differs from the latter in its consistently more elongate shape The organism under consideration, differing from other species of Monocercomonoides from insect hosts as indicated herein, resembles M. melolonthae (Grassi) most closely and lacks well marked characteristics which would justify its description as new It is accordingly regarded as referable to the latter species. Polymastix melolonthae (Grassi, 1879) (Figs. 8–11) P. melolonthae, which is known from the larvae of numerous genera of beetles and from tipulids in Europe and the U.S.A, was present—usually in large numbers—in the hind gut contents of all 10 Odontria zealandica larvae and of 48 of the 50 Pericoptus truncatus larvae examined. This fairly large, fusiform flagellate moves through the medium with a distinctive corkscrew motion. After prolonged exposure to the heat and light of the microscope lamp it tends to attach itself to the cover slip by the posterior tip of the body, hanging head downwards with all four flagella beating anteriorly. Perhaps its most distinctive feature is the presence of numerous, more or less longitudinally aligned ectocommensal bacteria, which are clearly visible by dark field illumination. The following description is based on haematoxylin-stained material except where otherwise stated. Although examples having the posterior extremity rounded are not

uncommon (Fig 8), the body usually comes to a point posteriorly (Figs. 9 and 10). The hinder part of the body is sometimes bifurcated, as described by Kowalczyk (1938) and figured by Grassé (1952; his Fig. 625-B). A spherical to subspherical nucleus, averaging about 2μ in diameter and having a prominent central endosome of from 1.0μ to 1.6μ in diameter, is located towards the anterior extremity. Two closely adjacent basal granules connected by means of a rhizoplast are situated just in front of the nucleus. The granules appear more widely separated in air dried smears due to distortion, and the rhizoplast is then often clearly demonstrable with Giemsa (Fig. 9). An anteriorly thickened and dark-staining axostyle originates at one of the basal granules and curves about the nucleus keeping close to the membrane (Fig. 8). The axostyle could never be traced further posteriorly than in the example illustrated in Fig. 10, and the fine terminal filament shown in Grassé's Fig. 625-G was never made out The cytoplasm is alveolar and frequently contains vacuoles and food inclusions (Figs. 8, 9), but details are difficult to distinguish because of the ectocommensal bacteria attached to the pellicle. Osmic-fixed trophozoites in Giemsa smears often exhibit dark-staining spherules similar to those noticed in such preparations of Monocercomonoides melolonthae (Fig. 9). Numbers of ectocommensal bacteria referable to the genus Fusiformis Holling (Actinomycetales: Mycobacteriaceae) are always attached to the firm pellicle as illustrated in Figs. 8–10. These have the form of elongate rods, pointed at the ends and ranging from about 2μ to 6μ in length. From two to eight siderophilous granules are distinguishable in suitably destained examples. The ectocommensals were observed to desert their host on the death of the latter, and Grassé (1952) noted that this is also the case at encystment. Morphologically identical bacteria are common in the free-living state in the gut contents of the host larvae (Fig. 11). The longitudinally attached ectocommensals of Polymastix melolonthae were referred to Fusiformis melolonthae Grassé by Grassé (1952), who gave their length as from 2μ to 7μ. It is possible that this name may prove to be a synonym of F. termitidis Hoelling, the genotype. This species, which occurs in the intestinal tract of termites, was described by Bergey (1934) as from 3μ to 5μ in length, the length increasing with the age of the organism, and as having from two to eight darkly staining granules suggestive of nuclei. A pair of flagella originates from each of the basal granules. All four flagella are often directed anteriorly, although one, which is appreciably longer than the others, may trail behind. The length of the one with the tendency to trail was found to range from 20. 3μ to 35. 1μ, and that of the other three from 13. 1μ to 22. 2μ. Fifty wet-fixed trophozoites stained with haematoxylin were measured in a preparation from Pericoptus truncatus and the same number in a slide from Odontria zealandica, no selection being exercised in either case. Those from Pericoptus proved to range from 7. 9μ to 16. 2μ by from 4.1μ to 7. 9μ (av., 13.1μ by 5. 3μ), while those from Odontria ranged from 7. 9μ to 19. 6μ by from 2. 9μ to 6. 9μ (av., 1. 9μ by 5. 3μ). These measurements exceed those of Polymastix phyllophaiae Travis and Becker, the trophozoites of this species ranging from 4. 75μ to 12. 3μ in length and from 3. 8μ to 5. 4μ in breadth (Travis and Becker, 1931), the upper limit of their size range thus coinciding with the average for the flagellate under discussion. P. wenrichi Geiman, on the other hand, an entozoan of North American crane fly larvae, attains a much larger size than does the New Zealand species, ranging from 12μ to 35μ in length and from 8μ to 22μ in breadth (Geiman. 1932). Grassé (1952) has recently described P. hystrix from termites (Neotermes aburiensis); but this species is remarkable in having a vestiture of very long bacteria attached to the pellicle by one end and is unique in its genus in possessing a parabasal apparatus—Grassé himself felt qualms at including this flagellate in Polymastix.

Kowalczyk (1938) reported that Polymastix milolontha trophozoites from larvae of the Japanese beetle range from 7μ to 19μ in length and from 3μ to 8μ in breadth (av. for 875 specimens, 11. 35μ by 4. 46μ) On measuring 25 random examples from each of a series of individual larvae Kowalczyk obtained a high mean average of 14.8μ by 4.7μ and a low mean average of 9.6μ by 3.8μ Ludwig (1946) measured 100 examples from crane fly larvae, finding the range in length and breadth to be 6. 2μ to 25μ by 3.1μ to 8.1μ (av., 12.5μ by 5μ). These figures are so close to mine, and the New Zealand flagellate compares so closely with P. melolonthae as described by other authors, that I have no hesitation in referring it to the latter species. Literature Cited Alexeieff, A., 1912. Sur quelques noms de genres des flagellés qui doivent disparaître de la nomenclature pour cause de synonymie ou pour toute autre raison. Diagnoses de quelques genres récemment etudiés. Zool. Anz., 39, 674–680. —— 1917. Mitochondries et corps parabasal chez les flagellés C. R. Soc. Biol, 80, 358–361. Anon, 1953. The Frog, Hyla aurea, as a Source of Animal Parasite. Report from a Class Project, Tuatara, 5, (1), 12–21. Bergey, D. H., 1934. Manual of Determinative. Bacteriology. Baltimore. Williams and Wilkins, xvi + 1–664. Bishop, A., 1931. A Description of Embadomonas n. spp. from Blatta orientalis, Rana termporaria, Bufo vulgaris, and Salamandra maculosa; with a Note upon the “Cyst” of Trichomonas batrachorum. Parasitol., 23, 286–300. Butshli, O., 1934. Observations upon Embadomonas intestinalis in Culture. Ibid, 26. (1), 17–25. —— 1884. Protozoa. Bronn's Klass. u Ord. Bd 1 Abt. II, Leipzig (Not seen). Cleveland, L. R, Hall, S. R, Sanders, E. P., and Collier, J., 1934. The Wood-feeding Roach Cryptocercus, its Protozoa, and the Symbiosis Between Protozoa and Roach. Mem. Amer. Acad. Arts. Sc., 17, (2). I–X + 185–342. Davis, H. S., 1947. Studies on the Protozoan Parasites of Fresh-water Fishes. U. S. Dept. Interior, Fish Bull 41, 1–29. Dobeli, C., 1935. Researches on the Intestinal Protozoa of Monkeys and Man. VII. On the Enteromonas of Macaques and Embadomonas intestinalis Parasitol, 27, (4), 564–592. Geiman, Q. M., 1932. Retortamonas caudacus (n sp), an Intestinal Flagellate from a Beetle Larva, Gyrinidae sp. Trans. Amer Micr. Soc, 51, (4), 219–224. —— 1933. The Intestinal protozoa of the Larvae of the Crane Fly. Tipula abdonali, J. Parasit, 19, (2), 173. Grasse, P-P., 1952. Phylogénie. Protozoaires. Généralités. Flagellés. Traité de Zoologie Vol. I, Fasc. I, xii + 1–1071. Paris: Masson. Grassi, B., 1879. Dei protozoi parassiti e specialmente di quelli the sono nell'uomo. Ga: Med. Ital. Lombardi, 39, (8), 445–448. —— 1881. Intorno ad alcum protisti endopaassitici ed appartenenti alle classi den Flagellati, Lobosi, Sporozoa e Ciliati. Atti della Soc. Ital. di Sci. Nat., 24. 135–224. Kidder, G. W., 1937. The Intestinal Protozoa of the Wood-feeding Roach Panesthia. Parasitol. 29, (2), 163–205. Kirby, H., 1947. Flagellate and Host Relationships of Trichomonad Flagellates. J. Parasit, 33, (3), 214–228. —— and Honigberg, B., 1950. Intestinal Flagellates from a Wallaoo. Macropus robustus Gould. Univ. Calif. Pubs. Zool., 55, 35–66. Kowalczyk, S. A., 1938. A report on the Intestinal Protozoa of the Larva of the Japanese Beetle (Popillia japonica Newm, Coleoptera). Trans. Amer. Micr. Soc., 57, (3), 229–244. Kudo, R. R., 1946. Protozoology. 3rd Ed. Illinois: Charles C. Thomas, xii + 1–778. Ludwig, F. W., 1946. Studies on the Protozoan Fauna of the Larvae of the Cranefly, Tipula abdominalis. I. Flagellates. Amoebae and Gregarines. Trans. Amer. Micr. Soc., 65. (3), 189–214. Mackinnon, D. L., 1912. Protists Parasiti on the Larva of the Crane-fly. Tipula sp. (Preliminary Note. 64) Parasitol, 5, (3), 175–189. —— 1915. Studies on Parasitic Protozoa. III (a) Notes on the Flagellate Embadomonas (b) The Multiplication Cysts of a Trichomastigine. Quart. J. micr. Sci., 61 (N S.), 105–118. Minchin, E. A., 1909. The Structure of Trypanosoma lewist in Relation to Microscopical Technique. Quart. J. micr. Sci., 53, (N S, 66). 755–808.

Stabler, R. M., 1944. A new species of Retortamonas (Protozoa) from the common mole cricket, Gryllotalpa hexadactyla. J. Parasit., 30 (3) 173–176. classes are frequently polymodal, it is considered that the former is likely to be the correct. Swezy, O., 1916. The Genera Monocercomonas and Polymastix. Univ. Calif. Pubs. Zool., 16, 127–138. Travis, B. V., 1932. A Discussion of Synonymy in the Nomenclature of Certain Insect Flagellates, with the Description of a New Flagellate from the Larvae of Ligyrodes relictus Say (Coleoptera-Scarabaeidae). Iowa State Coll J. Sci., 6, 317–323. —— and Becker, E. R., 1931. A Preliminary Report on Intestinal Protozoa of White Grubs (Phyllophaga spp.—Coleoptera). Ibid., 5, 223–235. Wenrich, D. H., 1931. Retortamonas gryllotalpae Grassi 1879, and its Relation to the Genus Embadomonas. Anat Rec., 51, (Suppl), 66–67 (Abstract). —— 1932. The Relation of the Protozoan Flagellate, Retortamonas gryllotalpae (Grassi, 1879) Stiles, 1902 to the Species of the Genus Embadomonas Mackinnon, 1911 Trans. Amer. Micr. Soc., 51, (3), 225–238. Wenyon, C. M., 1926. Protozoology. London: Baillière, Tindall and Cox. Vol. I, xvi + 1–778. Wood, W. F., 1935. Some Observations on the Intestinal Protozoa of Californian Lizards. J. Parasi., 21, (3), 165–174. Dr. Marshall Laird Dept. of Parasitology University of Malaya Sepoy Lines Singapore 3.