A Redescription of the Genus Parathelohania

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Agricultural Research Service U.S. DEPARTMENT OF AGRICULTURE CONTENTS

Page Abstract 1 Introduction ...... 2 Methods ...... 3 Light microscopy ...... 3 Scanning electron microscopy ...... 4 Transmission electron microscopy ...... 4 Results .... , ...... , ...... , . 5 Parathelohania Codreanu 1966 ...... 8 Parathelohania legeri (Hesse 1904) ...... 8 Parathelohania anophelis (Kudo 1924) ...... 10 Pa1'(l.thelohania obesa (Kudo 1924) ...... 14 Parathd:hania illinoisensis (Kudo 1921) ...... 16 Pa'rathelohania anornala (Sen 1941) ...... 18 Parathelohaniaindica (Kudo 1929) ...... 18 Pamthelohania obscura (Kudo 1929) ...... 19 Parathelohania pericu.losa (Kellen and Wills 1962) ...... 19 Parathelohania ajl"icanus sp. n ...... 20 Par'2thelohania octolagenella sp. n ...... 22 Literat\,re cited ...... 24 Achllowledgments ...... 26

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Stock Number (j 100 - 03364 A Redescription of the Genus Parathelohania Codreanu 1966 (Microsporida: Protozoa) With a Reexamination of Previously Described Species of Thelohania Henneguy 1892 and Descriptions of Two New Species of Parathelohania From Anopheline Mosquitoes

By EDWIN I. HAZARD and DARRELL \Y. ANTHONY, 1'esea1'ch entomologists, Insects Affecting iVlan Research Labomt01'Y, Agricultuml Research Serv ice, U.S. Department of Agriculture, Gainesville, Fla.

ABSTRACT

The genus Parathelohania is redescribed as representing species that, in mosquito larvae, have eight characteristically ridged spores enclosed in a persisting sporont membrane and that, in adult female mosquitoes, have smooth-walled spores not enclosed in a sporont membrane. The type species is Parathelohania legM'i (Hesse) from Anopheles rnaculipennis Meigen. Anopheline mosquitoes are the most common hosts of these microsporidian parasites. Two new species of Paratheloha,nia from Anopheles garnbiae Giles and Ano pheles pretoriensis (Theobald) al'e described. The species name "illinoisensis Kudo" is restored to represent the Parathelohania species in Anopheles punctipennis (Say). All representative species of Parathelohania and their synonymies are listed. The revised or ganization of Parathelohania was made possible by new biological information and new diagnostic characters provided by h'ansmis sion electron microscopy and confirmed by scanning electron micro scopy. Photomicrographs of spores from host larvae (made with light microscopy and scanning and transmission electron micro scopy) are presented to show the characters that distinguish the two new species and three species described earlier.

1 2 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE INTRODUCTION Thelohania legeri Hesse (1904a) 1 was the first microsporidium known from anopheline mosquitoes and was reportedly taken from larvae of Anopheles rnaculipennis Meigen collected in France. A second species, from the larvae of Anopheles lJUnctipennis (Say) collected in Illinois, U.S.A., was described by Kudo (1921) and named Thelohania illinoisensis. After examining what he believed were two additional host species, Anopheles cTucians Wiedemann and Anopheles quadrimaculatus Say, Kudo (1924a) made T. illin oisensis a synonym of T. legeri. In the same year Kudo (1924b) described three additional microsporidia from N ore1. American anophelines, which he named Nosema anophelis, Thelohania obesa, and Thelohania pyriformis. The host of N. anophelis was A. quadri maculatus, and both late instal' larvae and adults were found to have stages of the microsporidium. The Anopheles species that were hosts of the two Thelohania species were undetermined. Missiroli (1929) described a second species from A. maculipennis, but unlike T. lege?'i, which occurs in the fat body of larvae, this species was found in the ovaries and eggs of adult females. Missiroli named it Thelohania grassii after Grassi (1901), who first observed the spores in adult females collected in Italy. Kudo (1929) described two new Thelohania species from ano pheline larvae collected in India: Thelohania indica from Anopheles hyrcanus (Pallas) and Thelohania obscum from Anopheles varuna Iyengar. In the same paper he added the mosquitoes Anopheles barbi1'ostris Van del' Wulp, Anopheles annularis Van der Wulp (=A. fuliginosus of Kudo), Anopheles ramsayi Covell, and Ano pheles subpictus Grassi, as well as both A. varuna and A. hyrcanus, to the list of hosts of T.legeri. Sen (1941) reported another species, Thelohania anomala, from an Indian anopheline, A. ramsayi. Sen also added Anopheles vagus Donitz to the list of hosts of T. Zegeri, and Tour et al. (1971) included Anopheles labmnchiae atroparvus Van Thiel. The last Thelohania to be described from anopheline mosquitoes was Thelohania periculosa Kellen and Wills (1962), from the adipose tissue of the larvae of Anopheles franciscanus McCracken collected in California, U.S.A. Later, Kellen et al. (1967) described Nosema chapmani, from adult female A. francis canus, but Hazard and Weiser (1968) have since demonstrated that the sporonts and cylindrical spores are the stages ofthe transovarial cycle of T. periculosa. Codreanu (1966), believing that Thelohania octospom Henne guy (1892) was the type species of Thelohania and noting that it

J Dates in parentheses refer to "Literature Cited," p. 24. REDESCRIPTION OF PARATHELOHANIA 3 has spores with filamentous appendages, chose Thelohania legeri Hesse as the type species for a new genus he named Parathelohania. Codreanu was not aware that Gurley (1893) many years before had designated Thelohania giardi Henneguy (1892), which does not have spore appendages, as the type species of Thelohania. Cod reanu's mistake, however, did not produce complications for the taxonomy of Microsporida because those Thelohania found in Ano pheles mosquitoes are sufficiently different from the Thelohania species described from decapod crustaceans by Henneguy. The spores of T. legeri and other species in anopheline mosquitoes havn a characteristic constricted posterior end, and this difference, as well as other characteristics, justifies their placement in a genus separated from Thelohania. The original problem Codreanu at tempted to resolve, however, remains untouched by his changes in the generic placement of T. legeri. T. octospora should now be placed in a new genus if Codreanu or other investigators continue to believe that spore appendages are important generic characters. Pleistophora and Nosema diseases have been reported in colon ized anopheline mosquitoes by Canning (1957), Fox and Weiser (1959), Vavra and Undeen (1970), Canning and Hulls (1970), and Hazard (1970) ; these are well-established Nosema and Pleistophora species and do not need further discussion in this paper. Some spe cies formerly placed in the genus Thelohania, however, must be re viewed here because of the new information presented by Hazard and Weiser (1968) concerning the unusual sporonts that produce spores in adult female mosquitoes unlike those commonly known in male mosquito larvae and because of new diagnostic characters found on and in the spores from male larvae in scanning and trans mission electron photomicrographs. The sporonts of all Parathelo hania species in male larvae are similar and do not pl·esent diag nostic characters for the separation of species. The spores from these larvae, however, and the shapes and tissue sites of the spores in adult females have many structural characters that can be used as criteria for species differentiation. A complete description of the genus Parathelohania is presented, and two new species are described. Synonymies are also listed for existing species. METHODS Light Microscopy Infected mosquito larvae were cut into small pieces for preparing Giemsa-stained and Heidenhain's hematoxylin-stained smears. Giemsa smears were prepared by smearing the infected tissue on a 4 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE microscope slide, fixing with 95 % methanol for 1 min, staining with a 10 % Giemsa solution, made with buffered water (pH 7.41), for 10 min, and washing with tapwater. Heidenhain's hematoxylin stained smears were pl'epared by smearing the specimen on a glass cover slip, immediately fixing in aqueous Bouin's fluid for 6 h, wash ing in 70 % ethanol, staining with Heidenhain's hematoxylin, and mounting on a glass microscope slide with mounting medium. The spores from each infected specimen were examined under the phase contrast microscope as fresh smears to record their size, shape, and structure. The fresh spores as well as fixed spores were measured with an A.E.!. Cook image-splitting micrometer at a magnification of 1,000 X. Scanning Electron Microscopy Infected larvae were smeared in distilled water on glass-covered metal specimen stubs or pelleted by centrifugation and smeared on stubs in a small amount of distilled water. The water 'vas allowed to evaporate, and the specimen stubs were placed on a small rotating table in a Denton DV-502 high-vacuum evaporator where the spores were coated with 200 to 300 A of gold at a vacuum of 2X10-5 torr. Other spores were either pelleted by centrifugation, fixed with 1 % osmium tetroxide (OS04) in 0.1 M phosphate buffer, dehydrated with increasing concentrations of ethanol, and smeared on speci men stubs or simply smeared on stubs, fixed in the vapors of OS04, and coated with gold as described above. The specimen stubs were placed in a Cambridge Steroscan electron microscope, and the sporer, were photographed at accelerating voltages of 5,10 or 20 kV.

Transmission Electron Microscopy Infected larvae and adults were cut into small pieces (1 mm3) and fixed for 3 to 4 h at room temperature (or overnight in the refriger ator) with 4 % glutaraldehyde in 0.1 M phosphate buffer and trans ferred to 0.1 M phosphate buffer overnight. The following day the pieces of larvae or adults were postfixed with 1 % OS04 in 0.1 M phosphate buffer for 2 h, dehydrated by passage through increasing concentrations of ethanol to propylene oxide, and embedded in epon araldite mixture (Mollenhauer 1964). Ultrathin sections (600-900 A) were cut on a Sorvall MT-2 microtome with glass or diamond knives and stained with saturated uranyl acetate, followed by lead citrate (Venable and Coggeshall 1965) . The sections were examined and photographed with an Hitachi 125-E electron microscope at an accelerated voltage of 50 kV. REDESCRIPTION OF PARA'I'HELOHANIA 5 RESULTS When examined in Giemsa-stained smears, spores of most species of Parathelohania from anopheline mosquito larvae are distorted because of artifacts from shrinkage. Structural differences are bet ter observed when the spores are fixed as fresh wet smears in aqueous Bouin's solution and stained with Heidenhain's hematoxy lin because the natural.ghape of the spores is better preserved. From observations of these stained smears, we find that the spores of the microsporidium (formerly Thelohania legeri Hesse, Kudo 1924a) from A. quadrimaculatus larvae are elongate and have a moderately constricted and truncate posterior end (fig. lA). The spores of Parathelohani.a obesa (Kudo) from A. crucians larvae are broadly oval with a short, truncate, and only slIghtly constricted posterior end (fig. IB). The spores of a new species from the larvae of Ano pheles gambiae Giles are small and oval with only a slightly con stricted posterior end (fig. Ie). The other new species from Ano pheles p1'etoriensis (Theobald) larvae has spores that are elongate and are greatly constricted at the posterior end, forming a promi nent attenuated point (fig. ID). The surface structure of the four species mentioned above is viv idly illustrated by scanning electron microscopy. The spore of the species in A. quadrimaculatus has four thick keellike structures originating near the middle of the spore and continuing to the con stricted posterior end, where they connect with four more equally thick ridges, giving the appearance of a cratelike structure at the constrioted posterior end of the spore (fig. 3). The spore of P. obesa from the larvae of A. crucians }las a similar structure. However, its shorter constricted posterior end usually has five longitudinal ridges instead of four (fig. 5). The spores of the microsporidium from A. gambiae larvae lack the distinct ridges of the previous two species. Instead, they have numerous shallow and incomplete ridges, giving the spores a wrinkled appearance (fig. 8). The Parathelohania spe cies from A. pretoriensislarvae is unique. Its spores have thick and prominent ridges running nearly the full length of the spore and joining at the posterior end to form a blunt attenuated tip (fig. 10). The spores are also more elongate and larger than the spores of the other species. Transmission electron photomicrographs confirm the character istic shape and structure of the Parathelohania species and provide additional information concerning their internal structure. The spore wall consists of a thick, electron-dense outer coating; a some what less thick, electron-lucid inner coating; and two spore mem branes. The spore wall, however, becomes very thin at the point 6 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE

J '

B D PN-3901 FIGURE l.-Bouin's-fixed and Heidenhain's-stained spores of Parathelohania. X 2,000. A, P. anophelis (Kudo). B, P. obesa (Kudo). C, P. a!1'icamts sp. n. D, P. octolagenella sp. n.

where the polar cap is anchored to the anterior end (fig. 2). When observed in transmission electron photomicrographs, cross sections through spores show the polar filament in all species to be a coiled structure with a broad basal portion that abruptly constricts to a REDESCRIPTION OF PARATHELOHANIA 7

PN-3902 FIGURE 2.-Transmission electron photomicrograph of P. anophelis (Kudo) showing polar cap at anterior end. >~ 79,200. PC, polar cap. narrower and longer apical end. The length of the polar filament and the ratio of the length of the basal portion to the totalleugth of the polar filament are different in each species (figs. 4, 6, 7, 9, and 11). The spores of all species are uninucleate, and the polaroplast consists of many lightly packed, laterally alined lamellae (fig. 4) . 'We believe that the differences in the structure of spores from A. c1"1.wians, A. gambiae, A.1)1'etoTiensis, A. punctipennis, and A. quad rimaculatus larvae are sufficient to establish these as distinct spe cies. It also appears that these species are relatively host specific and that P. lege')'i is not universal. Because of the new information we have obtained in our studies with the ultrastructure of the spores of the microsporidia in A.1)lLnctipennis and A. quadril1taCulatus, we reinstate Kudo's 1921 name, illinoisensis, to represent the species in A. punctipennis. \Ve also propose two new species for this genus from two-anopheline species collected in Africa. 8 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE

Parathelohania Codreanu 1966 The genus consists of microsporidia with two developmental se quences, one producing eight oval spores enclosed bya sporont mem brane in mosquito larvae and a second producing a variable number of elongate and slightly bent or curved spores not enclosed by a spor ont membrane in adult females. Spores in adult females are 1)1'0 duced by mUltinucleate sporonts having 6 to 25 nu.clei. All species are transmitted to the progeny of infected females via the ovaries for one generation or more. The oval spores in larvae have a con stricted and ridged, or keeled, posterior end; their structure takes different forms in each species, and this is clearly resolved in scan ning and transmission electron photomicrographs. The diagnostic structure of these spores in some species is distinguishable with the light microscope, but not in others. The oval spores are retained in the sporont membrane at least until they become fully developed. In transmission electron photomicrographs, the polar filament has a broad basal portion that abruptly constricts to a thinner apical end. The broad basal portion never exceeds one-half the length of the polar filament and usually constitutes the first one to four coils of this structure inside the spore. Upon mechanical extrusion of the polar filament, the broad basal portion rarely escapes from the spore. The spores associated with the transovarial stages in adult females have thin, smooth walls with no surface structure. The polar filament of these is of uniform thickness and is often very short, as seen in electron photomicrographs. In immature spores the vacuole occupies one-half or more of the spore, but as the spores mature, the vacuole becomes progressively smaller. These spores, in adult fe males, have various lengths, shapes, and tissue sites in different species. They average 2.0 to 3.5 J.lm in width regardless of their length, which sometimes exceeds 9.0 rtm. Species of Pamtheloh((nia are primarily parasites of mosquitoes, particularly those of the genus Anopheles. Type species: P. legeri (Hesse 1904a) Codreanu 1966, by original designation.

Parathelohania legeri (Hesse 1904) T helohallia /egel'i Hesse, 1904a, C. R. Soc. BioI. 57: 570; Hesse, 1904b, C. R. Soc. BioI. 57: 571; Kudo (in part), 1924a, Arch. Protistenk. 49: 157; Kudo (in part), 1924b, Ill. BioI. Monogr. 9 (2-3) : 143; Kudo (in part), 1925, Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Aht. 1. Orig. 96: 431; :\1issiroli (in part), 1929, Rev, .Malariol. 8: 395; Weiser (in part), 1947, Pl'. Moravske Pfir. Spol. 18: 38; Thomson (in part), 1960, J. Insect Pathol. 2: 359; Weiser (in part), 1961, Monogr. Angew. Entomo1. 17: 113. Toxoglugea, missi1'oli Weiser, 1961, Monogr. Angew. Entomo1. 17: 117; Weiser, 1963, Bull. W.H.O. 28: 125. New synonymy. ~ , REDESCRIPTION OF PARATHELOHANIA 9

Host.-Anopheles 11wculipennis Meigen. Locality.-France, Italy, and Czechoslovakia. Tissue.-Adipose tissue and oenocytes in males; oenocytes and ovaries in females. Spore size.-12X 5 fJ.m (.fresh?, macrospores in larvae) Hesse; 6 8X3-4 ~lm (fresh?, microspores in larvae) Hesse; 3-4X 1.5-2.0 ,1m (fresh?, in adult females) Missiro11; 4.8-6.0X3.4 /Jom (fresh?, in larvae) 'Veiser. This species was originally found in the fat bodies of the larvae of Anopheles maculipennis Meig. Kudo (1924a) reported observing the spores in stained smears of Anopheles bijuJ'catus [apparently a synonym of Anopheles cla'l.:igeT (Meig.), Stone et al. 1961] from Hesse's collection. Kudo referred to an imminent publication of Hesse 'where this species would be reported as an additional host of T. legeri; however, the paper never appeared. Hesse (1904a) says the spores are ovoidal with nearly equally rounded ends and that the spore membrane is thick and distinct. He found both macrospores and microspores, which measured 12 X 5 and 6-8X 3-4 11m, respec tively. The polar filament ,,'as reported to measure 50 /1m in length. In a second paper Hesse (190c1b) said of the early developmental stages: "Meronts are rounded bodies, 3-41.L in diameter, with a strongly colorable cytoplasm and a fairly small nucleus, in the form of a mass of chromatic grains surrounded by a clear zone. The mer ant gro"'s in size and can reach up to 6". in diameter. As in Stempell's interpretation of Theiollania mulle1'i L. Pfeiffer, the meronts divide by a transversal constriction of the cytoplasm preceded by the direct division of the nucleus. Sometimes strings of three meronts are observed which then separate from each other. The nuclear division does not al\,-ays immediately follow that of the cytoplasm." As for the sporonts, he said: "At first the sporonts are oval bodies without diifG-rential walls, measuring 9-10" on the large axis and 4-6/l on the small axis. They possess a much clearer cyto plasm than do the meronts, and a large nucleus with a colorable membrane. Besides having small chromatic grains arranged into a network, the nucleus also contains a complex karyosome made of four chromatic masses." 1'.:Iissi1'oli (H)29) in the first part of his paper described a micro sporidium, TheZo/zania {j/'assii, from the eggs of A. ))z({clilipennis. Near the end of this paper he described "'hat is apparently T.Zegeri, because in the last sentence of the paper he claimed it was not suffi ciently different to be a new species. Because of the two separate descriptions, the paper is misleading and confusing, and at first 10 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE reading, we get the impression that both descriptions concern the species T. gmssii, as did Weiser (1961). This is not the case, how ever, as Missiroli described two parasites: one in A. maculi1Jennis eggs (T. gmssii) and another in the blood cells of adult female A. ?r/,aculipennis, which al'e the stages of T. lege1'i Hesse. \Ve believe that these are the spores of the transovarial cycle of T. lege1'i, be cause the shape and size of the spores are characteristic of what we see in other Pamthelolwnia species and also because he found these spores developing in blood cells, the site of the infection of other Pamthelohc~nia in adult female mosquitoes. Missiroli gives the length of the spores as 3 to 4 lJ.m without giving their width, but, if we use the length to compute the width of the spore shown in his illustration, we find that it would measure about 1.5 to 2.0 Itm. The length and width ratio, then, of these spores is similar to what we have observed in other species of P(£1'((thelohania. We have observed a microsporidium similar to Missiroli's T. g1'assii in the ovaries and eggs of An01Jheles funestus Giles and A. gam,biae collected in Nigeria, Africa. In our material the spores were extremely small, perhaps not more than 1.5 fJ.m in length. Mis siroli did not give the measurements of the spores of T. gmssii. but from his illustration of the spores in eggs, these also could be no more than 1.5 I,m. The spores of this species do not have thB charac teristics of spores of Pamthelohania in adult females :md, therefore, do not represent a species of this genus. It is doubtful that the anopheline species A. annuZcL1'is, A. barbi1' ost1'is, A, hYCmnu8, A. labncnchiae at-ropm''l.'U8, A. 1'amsClyi. A. sub pictus, A. vagus, and A. v((ntna are hosts of P. legeri. The micro sporidia in these mosquitoes need to be reevaluated by electron microscopy. Parathelohania anophelis (Kudo 1924) Nosema an07Jhelis Kudo, 1924b, Ill. BioI. ;\1:onogr. 9 (2-3) : 110; Weiser, 1947, Pl'. 1.\1:ol'a:vske PfiI.. Spol. 18: 37. New synonymy. Thelohania illinoisensis Kudo (in part), 1922, J. ParasitoJ. 8: 74. New synon ymy. Thelohania lege1'i Kudo (in part), 1924a, Arch. Protistenk. 49: 148; Kudo (in part), 1925, ZentralbJ. Bakteriol. Parasitenk. Infectionskr. Hyg. Abt. 1. Grig. 96: 431; Chapman et al. (in part), 1966, J. Invertebr. Pathol. 8: 453; Hazard and Weiser, 1968, J. Protozool. 15: 818. New synonymy. Nosema stegomyiae Fox and Weisel' (in part), 1959, J. ParasitoJ. 45: 21; Thomson (in part), 1960, J. Insect PathoJ. 2: 352; Weiser (in part), 1961, Monogr. Angew. EntomoJ. 17: 108. New synonymy.

Host.-Anopheles quad1'i111,aculatus Say. Locality.-Georgia, New York, Louisiana, and Florida, U.S.A. REDESCRIPTION OF PARATHELOHANIA 11 Tissue.-Adipose tissue and oenocytes in males; oenocytes and ovaries in females. Spore size.-4.7-5.8X2.3-3.2 ,.,.m (fresh from adult females) Kudo; 5.18+0.04X3.16+0.02 !-,.m (fresh from male larvae) Chap man et al.; 3.6±O.04X2.1±O.03 p.m (fixed from male larvae) Haz ard and 'Yeiser; 4.0-5.5X2.5-3.6 !tm (fresh from male larvae) Haz ard and 'Yeiser; 4.5X2.2/l.m (fresh from adult females) Hazard and 'Yeiser.

The spore in male larvae is an elongate oval with a moderately constricted and truncate posterior end (figs. lA, 3, and 4). The pos terior end has fonr prominent ridges running from the middle ohhe spore to the tip, where these ridges are connected by four ridges of equal prominence to form a structure that appears cratelike in scan ning electron photomic:rographs (fig. 3). In transmission electron photomicrographs of thin sections the polar filament has eight coils, the first four being formed by the broad basal end (fig. 4). Kudo (1925) described this species from two larvae and an en gorged adult and said it. was found in the epithelial cells of the gas tric pouches of lanae and in the epithelial cells of the anterior por tion of the midgut of the adult. ViTe have had considerable experience

PN-3903 FIGURE 3.-Scanning electron photomicrograph of P. anophelis (Kudo) spores. X 19,000. CPE, constricted posterior end. I-' t>:)

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~ ..... 0 c:: t3 c:: ::u PN-3U04 tIl FIGURE 4.-Trunsmissipn electron photomicrograph of P. anophclis (Kudo) spore. X 30,000, CPE, constricted posterior end; N, nucleus; P, polaroplast; PF, polm' filament. REDESCRIPTION OF PARATHELOHANIA 13 with this microsporidium in A. q'l.ladrimac'l.llat'l.ls in the southeastern part of the United States and recognize his mistake in identifying the site of infection. We have followed the infection in females (both larvae and adults) and have seen the parasites in the location of the gastric ceca of mature larvae and in the anterior portion of the mid gut of newly emerged adults. We, too, first believed that the infec tions were in the digestive tract, but later discovered, after examin ing stained sections of larvae and adults, that the infection was in oenocytes closely adhering to the epitheliai cells of the digestive tract. Sometime after the female takes a blood meal, the infected oenocytes dislodge from the ventral diverticulum (organ replacing the gastric cecum of the larva) and tlle anterior midgut cel1s and migrate to the ovaries, where they become involved in the transov arial passage to the progeny of the infected female. These observa tions were made from specimens obtained from our infected colony of A. quadTinwculat'l.ls maintained for 75 generations. The infection is carried to the progeny of infected females continuously each gen eration. This species has meronts and sporonts in male larvae similar to those of Kudo's T. illinoisensis, which he adequately described (1921, 192401) . The meronts reproduce in blood cells in early instal' male larvae, and after the blood cell becomes filled with these stages, the blood cell wall breaks, freeing the stages, which migrate to the fat body. In the fat cells they divide by nuclear division to form octonucleate sporonts and eventually spores enclosed in a subpersis tent sporont membrane. The meronts remain dormant in the hemo coel of female larvae, however, until the last larval molt and then enter oenocytes. In females the number of divisions of merogony are fewer, and only a few oenocytes initially become invaded, pl'oducing small numbers of meronts. These meronts reenter other noninfected blood cells and become sporonts. The sporonts divide by nuclear divi sion, forming mUltinucleate stages with 8 to 25 nuclei. The sporo blasts and spores are not enclosed in a sporont membrane. After the adult female emerges from the pupal skin and takes its first blood meal, the infected oenocytes migrate to the ovaries, which they enter by undetermined means. Once in the ovaries, we theorize that the spores are extruded by the pressure exerted upon them from the enlarging ovarioles, thus releasing the sporoplasm that enters the developing eggs. Kudo (1924b) described the spores of this species as being oblong \vith one end slightly narrower and sides asymr. etrical (one side slightly concave and the other convex). He also reported that the spores have thin walls and that some have large vacuoles. This de 14 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE

scrilJtion is very characteristic of the spores in the adult female A. quadrimaculatus ,ye have studied in our infected colony. Also, we have not found, after 7 years of searching, similar :-.:osema spores in A. quctdrimaculatZls or in other AnophelEs species collected in Flor ida, Georgia, and Louisiana. We therefore place S. ano]Jhelis Kudo in the genus Paratlzelolzania to represent the species found in A. qlladrimaculatus.

Pamthe~ohania obesa. (Kudo 1924)

Thelohania obesa Kudo, 1924b, Ill. BioI. :\Ionogr. 9 (2-3) : 161; Kudo, 1925, ZentraJbl. Bakteriol. Parasitenk. Infektion;;kr. Hyg. Abt. 1. Orig. 96: 432; l\Iissiroli, 1929, Rev. :\IalarioL S: !J9G; \\·ei~el'. 1947, Pl'. :\Iora"ske Pi·fr. Spol. 18: 38; Thomson, 1960, J. Insect Pathol. 2: :JG;J; Weiser (in part), 19G1, :Uonop:l'. Angew. Entomol. 17: 111; "Wills and Beaudoin, 1965, J. Invertebr. Pathol.. 7: 12; Hazard and Weiser, 1968, J. ProtozooJ.l5: 820. Thelohania legeri Chapman et aI., 196.6, J. Invertebr. Pathol. 8: 453. Condi tional synonymy.

Host.-Anopheles CJ'llcians \riedemann and A. quadrimaClliatus Say. Locality.-Georgia, Pennsylvania, Florida, and Louisiana, CS.A. Tissue.-Adipose tissue and oenocytes in males; oenocytes and ovaries in females. Spore size.-1.0-4.5X3.0-3.5 I'm (fixed from male lan-ae) Kudo; 4.23=O.04X3.24=O.03 1,m (fresh from male larvae) Wills and Beaudoin; 4.64:::0.05X3.22=O.02 :,m (fresh f!'om male lar\'ae) Chapman; 3.6:::0.01X2.6:::0JJ1 !'m (fixed from male larvae) Haz ard and Weiser; 3.5-5.7>' 2.8-1.2 !,m (fresh from male lan-ae) Haz ard and 'Yeiser; 7.9X3.5"m (fresh from adult females) Hazard and Weiser.

This species has meronts and sporonts in larvae simjjar to those of the t\,·o preceding species; unlike the others, the octonuc!eate sporonts contain many dense granules that Main intensely red in Giemsa smears. These granules remain during sporulation, ,,-here they are easily seen surrounding the sporoblasts enclosed in the sporont membrane. ~Ieronts im-ade blood cells in early instal' larvae, where they reproduce and form many sporonts that migrate from the ruptured blood cells to the fat cells. In the fat cells they become octonucleate stages and produce eight spores enclosed by a sporont membrane. Tbe spores are broadly oval (fig. lB) anel have a short, constricted posterior end (fig. 5) _ The polar filament makes se\-en coils inside the spore, three of which are formed by the broad basal end (fig. 6). REDESCRIPTION OF PARATHELOHANIA 15

PN-3905 FIGURE 5.-Scanning electron photomicrograph of P. obesa (Kudo) spores. X 15,600. CPE, constricted posterior end.

Sporonts in oenocytes of infected female larvae and adults have six or eight nuclei. These stages produce six or eight spores. A verag ing 7.9X3.5 p'm, with some as long as 10 ,urn, these are the largest Pamthelohania spores found in female mosquitoes. The spores are elongate, broader at one end, and bent or curved, and they have a large vacuole in the broader end. The vacuoles in young or immature spores are larger than half the spore length, but become smaller as the spores mature. These spores are seen in greatly hypertrophied oenocytes in the hemocoel near the ovaries. No spores are found in the ovaries, although other stages similar to sporonts are seen in Giemsa-stained smears of these organs. Sporonts and spores are seen in field-collected adult female mos quitoes, but not in the female progeny of infected individual females. We believe, therefore, that female larvae must become infected when they feed on either the spores from adult females or from other larvae, but we have not been able to produce infections in uninfected mosquitoes by feeding them spores from larvae. 16 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE

PN-3906 FIGURE 6.-Transmission electron photomicrograph of P. obesa (Kudo) spore. X 30,000. CPE, constricted posterior end; PF, polar filament.

Pamthelohania illinoisensis (Kudo 1921)

Thelohaniaillinoisensis Kudo 1921, J. Morpho!. 35: 167. Thelohania lege1'i Kudo (in part), 1924a, Arch. Protistenk. 49: 148; Kudo (in part), 1924b, Ill. BioI. Monogr. 9 (2-3) : 143; Kudo (in part), 1925, Zentralbl. Bakteriol. Parasitenk. Infektionskr. Ryg. Abt. 1. Orig. 96: 431; Missiroli (in part), 1929, Rev. Malariol. 8: 395; Weiser (in part), 1947, Pl'. Moravske Prir. Spol. 18: 38; Thomson (in part), 1960, J. Insect Patho!. 2: 359; Weiser (in part), 1961, Monogr. Angew. Entomo!. 17: 113; Kudo, 1962, J. Insect Pathol. 4: 353; Chapman et aI., (in part), 1966, J. Invertebr. Pathol. 8.: 453; Anderson, 1968, J. Invertebr. Pathol. 11: 452. New synonymy. Host.-Anopheles punctilJennis (Say). Locality.-IIIinois, Louisiana, and Connecticut, U.S.A.; Quebec, Canada. Tissue.-Adipose tissue and oenocytes in males; oenocytes and ovaries infemales. REDESCRIPTION OF PARATHELOHANIA 17

Spore size.-4.75-6.00X3-4 pm (fresh from larvae) Kudo; 4.94+ 0.05X3.18+0.02 .urn (fresh from larvae) Chapman et al.; 4.6 +0.05X2.9+0.04 .urn (fresh from larvae) Anderson; 4.3 +0.07X2.9 +0.07 .urn (fixed from larvae) Anderson.

We restore Kudo's name illinoisensis for this species, believing it must be distinct from P. legeri in the European mosquito A. 11WCU lip ennis since it is obviously distinct from other species in closer host relatives in the United States. The spore in larvae is an elongate oval and similar in shape to those of P. anophelis and P. lege1'i, but is different in ultrastructural characteristics (fig. 7) . The spore has a longer constricted posterior end and does not have the distinct cratelike structure seen in scan ning electron photomicrographs of P. anophelis. Also, the polar fila

;~l ~ .t . ' PF

.. .

I· •

>, ">t;

PN-3907 FIGURE 7.-Transmission electron photomicrograph of P. illinoisensis (Kudo) spore. X 28,500. CPE, constricted posterior end; PC, polar cap; PF, polar filament; P, polaroplast. 18 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE

ment is very short, making five coils in the spore, only two of which are formed by the broad basal end.lVreronts and sporonts are similar to those of P. lege'ri, and they were sufficiently described by Kudo (1921). Little is knmvn about the spores or sporonts in the adult females, but because of the low incidence of infection in larvae colleded in the field, we believe it also is transmitted to larvae via the egg.

Parathelohania anomala (Sen 1941)

Thelohania anomala Sen, 1941, J. Malar. Inst. India 4: 258; Thomson, 1960, J. Insect Pathol. 2: 356.

Host.-Anopheles '/'(.rnsayi Covell. Locality.-India. Tissue.-Oenocytes in the area of the adipose tissue of larvae. Spore size.-5.1-6.1X2.0-2.1 /-tm (fresh from female larvae).

Sen (1941) placed this species in the genus Thelohania even though the sporonts have a variable number of nuclei (8, 10, and 12 in number) producing 8, 10, or 12 spores enclosed in a "thin mem brane." In our opinion these are the spores associated with the transovarial stages in females, because the sporonts give rise to eight or more spores and because we have seen sporulation on occa sion in late instal' female larvae in other species. The thin membrane observed by Sen is probably the hypertrophied wall of the oenocyte hosting the infection. The narrow width of the spore given in his measurements and the illustrated spore showing a large vacuole are also characteristic of spores in adult females. Additional material must be studied, especially spores from male larvae, before we can be certain that this species belongs in Pa1'a thelohania. These studies could be easily conducted by egging indi vidual engorged adult females for a supply of infected larvae and subsequent microscopic examination of the female after oviposition. By this method the spores in larvae can be associated with those in the adult female (Hazard and Weiser 1968).

Parathelohania 1:ndica (Kudo 1929)

Thelohania indica Kudo, 1929, Arch. Protistenk. 67: 3; Sen, 1941, J. Malar. Inst. India 4: 258; Weiser, 1947, PI'. Moravske Pi'ir. Spol. 18: 38; Thomson, 1960, J. Insect Pathol. 2: 359. Thelohania obesa Weiser (in part), 1961, Monogr. Angew. Entomol. 17: Ill. New synonymy. REDESCRIPTION OF PARATHELOHANIA 19

Host.-An01Jheles hyrcanus (Pallas). Locality.-India. Tissue.-Adipose tissue of larvae. Spore size.-4.0-5.2X2.4-2.8 fLm (fixed from larvae).

Kudo did not examine adult females, and nothing is known con cerning the transovarial stages. This microsporidium has not been studied since it was originally described in 1929; therefore, nothing is known of the ultrastructure of spores from larvae.

Parathelohania obscura (Kudo 1929)

Thelohania obscum Kudo, 1929, Arch. Protistenk. 67: 4; Weiser, 1947, Pl'. Moravske PHr. Spo!. 18: 30; Thomson, 1960, J. Insect Patho!. 2: 360. Thelohania obesa Weiser (in part), 1961, :'lonogr. Angew. Entomo!. 17: 11l. New synonymy.

Host.-Anopheles vanma Iyengar. Locality.-India. Tissue.-Not given by Kudo. Spore size.-4.5-5.0X3.0-3.5 I~m (fixed from larvae) .

Nothing is known about the spores or the transovarial nature of this species since Kudo did not examine adult ~emales. Nothing is 1.110"'11 concerning the ultrastructure of spores from larvae, because it has not been studied since it was origin,:lly described in 1929.

Parathelohania periculosa (Kellen and Wills 1962)

Thelohania periculosa Kellen and Wills, 1962, J. Insect Pathol. 4: 54. Nosema chapmani Kellen et aL, 1967, J. Invertebr. Pathol. 9: 20.

Host.-Anopheles f1'Clnciscam ,s McCracken. Locality.-California, U.S.A. Tissue.-Oenocytes and adipose tissue of male larvae; oenocytes of adult females. Spore size.-4.71X2.62 11m (fresh) :lnd 3.81x2.44 Jlm (fixed) from male larvae; 5.54X 1. 73 11m (fresh) and 5.78 X 1.45 I,m (fixed) from adult females.

This microsporidium has been adequately described in both larvae and adults by Kellen and Wills (1962) and Kellen et al. (1967). The spores in larvae, however, should be studied in electron photomicro ~ graphs. 20 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE Parathelohania africanus sp. n. Host.-Anopheles gambiae Giles. Locality.-Nigeria. Tissue.-Adipose tissue and oenocytes in males; oenocytes and ovaries in females. Spore size.-3.7X2.3 p'm (fresh from male larvae); 4.8X2.0 {Lm estimated (fresh from adult females) . The spores in larvae are small and oval. Their constricted and rounded posterior ends, with several short, indistinct ridges, dis tinguish them from spores of other speCIes of Parathelohania (figs. le and '3). The polar filament has six coils in the spore, three of which are made by the basal end (fig. 9) . Meronts and sporonts are similar to those of P. an01Jhelis. Although the spores in larvae approach the size of those of P. obesa, the spores in the adult female are smaller, elongate, slightly bent, an.d narrowed at one end, ~,nd they are found inside the ovaries

PN-390B FraURE 8.-Scanning electron photomicrograph of P. a{1'icantts sp. n. spores. X 15,000. CPE, constricted posterior end. ~

~ I'"d

~ :J>

I) PN-3909 FIGURE 9.-Transmission electron photomicrograph of P. africanus sp. n. spore. X 32,250. CPE, constricted posterior end; P, l:I:) polaroplast; PF, polar filament. I-' 22 TECHNICAL BULLETIN 1505, U.s. DEPT. OF AGRICULTURE

in small groups. The spores in adults are no more than 5 f.tm long and often have a vacuole in the narrow end. Little is known about the sporonts or their development in the females.

Parathelohania octolagenella sp. n. Host.-Anopheles 1J1"eto1'iensis (Theobald) . Locality.-Nigeria. Tissue.-Adipose tissue and oenocytes in males; oenocytes and ovaries in females. Spore size.-6.0X2.6 Jim (fresh from male larvae); 5.5X1.5 p'm (fresh from adult females). This l'pecies has relatively large spores in larvae. The spores are pointed at the posterior end (fig. 1D). Prominent ridges run nearly thefull length of the spore and terminate in [.. pointat the constricted end (fig. 10). The long polar filament makes eight coils inside the spore, the first three formed b:r the broad basal end (fig. 11). l\fer onts and sporonts are similar to those of P. ano7Jhelis.

PN-3910 FIGURE 10.-Scanning electron photomicrograph of P. octoZagenella sp. n. spores. X 15,000. CPE, constricted posterior end. ~

~ § ;j @

~ I'd

~r o

~ >

PN-3911 FIGURE H.-Transmission electron photomicrograph of P. octoiagenoUa sp. n. spore. X 24,510. OPE, constricted posterior end; I\:) Pli', polar filament. ~ 24 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE

The spores in adult females are different from those in other spe cies in being clearly elongate, attenuated to a very narrow anterior end, curved (sometime nearly U-shaped), and having no visible vacuole in fresh preparations. These spores are not found in the ovaries, but can be sean in blood cells (oenocytes) scattered through out the body of the adult host. Nothing i.s known about the sporonts or their development in females.

LITERATURE CITED Anderson, J. F. 1968. Microsporidia parasitizing mosquitoes collected in Connecticut. J. Invertebr. Patho!. 11: 440-445. \ Canning, E. U. 1957. On the occurrence of Plistophora culicis Weiser in Anopheles gambiae. Rev. Malario!. 36: 39-50. Canning, E. U., and Hulls, R. H. 1970. A microsporidian infection of )!nopheles gambiae Giles, from Tanzania. Interpretation of its mode of transmission and notes on Nosema infections in mosquitoes. J. Protozool. 17: 531-539. Chapman, H. C.; Woodard, D. B.; Kellen, W. R.; and Clark, T. B. 1966. Host parasite relationships of Thelohania associated with mos quitoes in Louisiana (Nosematidae: Microsporidia). J. Invertebr. Patho!. 8: 452-456. Codreanu, R. 1966. On the occurrence of spore or sporont appendages in the Micro sporida and their taxonomic significance. In A. Corradetti (ed.), Proc. 1st Int. Congr. Parasito!., Roma (1964) 1: 602-603. Per gamon Press, New York. Fox, R. M., and Weiser, J. 1959. A microsporidian parasite of Anopheles gambiae in Liberia. J. Parasito!. 45 : 21-30. Grassi, B. 1901. Studi di uno zoologico sulla malaria. Mem. Cl. Sci. Fis., Mat., Nat., R. Accad. N az. Lincei 3: 299-505. Gurley, R. 1893. On the classification of the MyxClsporidia, a group of protozoan parasites infecting fishes. Bull. U.S. Fish Comm. 9: 407-420. Hazard, E. 1. 1970. Microsporidian diseases in mosquito colonies: Nosema in two Anopheles colonies. Proc. 4th Int. Colloq. Insect Pathol., College Park, Md., pp. 267-271. Hazard, E. 1., and Weiser, J. 1968. Spores of Thelohania in adult female Anopheles: Development and transovarial transmission, and redescriptions of T. legeri Hesse and T. obesa Kudo. J. Protozool.15: 817-823. Henneguy, G., and Thelohan, P. 1892. Myxosporidies parasites des muscles chez quelques crustaces de capodes. Ann. Microgr. 4: 617-641. REDESCRIPTION OF PARATHELOHANIA 25

Hesse, E. 1904a. Thelohania lege1'i n. sp., microsporidie nouvelle, parasite des larves d'Anopheles maculipennis Meig. C. R. Soc. BioI. 57: 570 571. 1904b. Stir Ie developpement de Thelohania lege1·i Hesse. C. R. Soc. BioI. 57: 571-572. Kellen, ""V. R.; Clark, T. B.; and Lindegren, J. E. 1967. Two previously undescribed Nosema from mosquitoes of Cali fornia. J. Invertebr. Pathol. 9: 19-25. Kellen, W. R., and Wills, W. 1962. New Thelohania from California mosquitoes (Nosematidae: Microsporidia). J. Insect Pathol. 4: 41-56. Kudo, R. R. 1921. Studies on Microsporidia with special reference to those parasitic in mosquitoes. J. Morphol. 35: 153-192. 1922. Studies on Microsporidia parasitic in mosquitoes. II. On the effect of the parasites upon the host body. J. ParasitoI. 8: 70-77. 1924a. Studies on Microsporidia parasitic in mosquitoes. III. On Thelo hania lege1·i Hesse 1904 (= T. illinoisensis Kudo 1921). Arch. Protistenk. 49: 147-162. 1924b. A biologic and itaxonomic study of the Microsporidia. Ill. BioI. Monogr., vol. 9, Nos. 2-3, 268 pp. 1925. Studies on Microsporidia parasitic in mosquitoes. IV. Observa tions upon the Microsporidia found in the mosquitoes of Georgia. Zentralbl. Bakteriol. Parasitenk. Infektionskr. Hyg. Abt. 1. Orig. 96: 428-440. 1929. Studies on Microsporidia parasitic in mosquitoes. VII. Notes on Microsporidia of some Indian mosquitoes. Arch. Protistenk. 67: 1-10. 1962. Microsporidia in Southern Illinois mosquitoes. J. Invertebr. Pathol. 4: 353-356. Missiroli, A. 1929. Sui Microsporidie parasite dell' Anopheles maculipennis. Rev. Malariol. 8: 393-400. Mollenhauer, H. H. 1964. Plastic mixtures for use in electron microscopy. Stain Technol. 39: 111-114. Sen, P. 1941. On some Microsporidia including a new form from anopheline larvae. J. Malar. Inst. India 4: 257-261. Stone, A.; Knight, K. j and Starcke, H. U161. A synoptic catalog of the mosquitoes of the world, supplement I (Diptera: Culicidae). Proc. Entomol. Soc. Wash. 6: 30. Thomson, H. M. 1960. A list and brief description of the Microsporidia infecting insects. J. Insect Pathol. 2: 346-385. Tour, S.; Rioux, J. A.j and Croset H. 1971. Systematique et ecologie des microsporidies (Microsporidia Nosematidae) parasites de larves de culicides (Diptera-Culi cidae). Ann. Parisitol. (Paris) 46: 205-223. 26 TECHNICAL BULLETIN 1505, U.S. DEPT. OF AGRICULTURE

Vavra, J., and Undeen, A. 1970. Nosema algerae n. sp. (Cnidospora, Microsporida), a pathogen in a laboratory colony of Anopheles stephensi Liston (Diptera, Culicidaq. J. ProtozooI.17: 240-249. Venable, J. M., and Coggeshall, R. 1965. A simplified lead citrate stain for use in electron microscopy. J. Cell BioI. 25: 407-408. Weiser, J. 1947. Klie k. Ureovani Mikrosporidii. Pro Moravske Pfir. Spol. 18: 1 64. 1961. Die Mikrosporidien aID Parasiten der Insekten. Monogr. Angew. Entomol.17, 149 pp. 1963. Diseases of insects of medical importance in Europe. Bull. W.H.O. 28: 121-127. Wills, W., and Beaudoin, R. 1965. Microsporidia in Pennsylvania mosquitoes. J. Invertebr. Pathol. 7: 10-14.

ACKNOWLEDGMENTS

We want to thank Susan W. Crosby, biological laboratory techni cian, Insects Affecting Man Research Laboratory, Agricultural Re search Service, Gainesville, Fla., for her assistance with the prep arations for light, scanning, and transmission electron microscopy. Special recognition is given to Sherleen W. Oldacre, research associ ate, Department of Entomology, Institute of Food and Agricultural Sciences, University of Florida, Gainesville, Fla., for her assistance in the preparation of this manuscript and for her assistance in the preparation of the photomicrographs. We also thank Harold C. Chapman, research leader, Gulf Coast Mosquito Research Labora tory, Lake Charles, La., who supplied us with some of the infected mosquitoes used in preparations for electron microscopy, and the World Health Organization, which made it possible for the senior author to study microsporidian infections in anopheline mosquitoes in Kaduna, Nigeria.

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