Journal of Invertebrate Pathology 90 (2005) 24–31

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The nature of Thelohania solenopsae (Microsporidia) cysts in abdomens of red imported Wre ants, Solenopsis invicta

Y.Y. Sokolova a,¤, J.R. Fuxa a, O.N. Borkhsenious b

a Department of Entomology, Louisiana State University AgCenter, Baton Rouge, LA 70803, USAb Department of Comparative Biomedical Sciences, School of Veterinary Medicine, Louisiana State University, Baton Rouge, LA 70803, USA

Received 4 February 2005; accepted 30 March 2005Available online 13 May 2005

Abstract

Sixty four percent of Solenopsis invicta workers infected with Thelohania solenopsis contained 1–6 “cysts” ranging from 70 to260 �m in diameter. Light and electron microscope analyses showed that cysts are hypertrophied adipocytes transformed by the par-asites, each cyst presumably forming from a single cell. In the Wrst step of the pathogenesis, Nosema-like spores functioning in auto-infection are produced; a diplokaryotic sequence leading to their formation causes fat body hypertrophy. When meiosis occurs, itswitches parasite development to production of octospores and/or megaspores. Adipocytes become 2–4 £ larger than normal in con-junction with intensive parasite multiplication and octospore maturation. Infected cells eventually lose their cellular organizationand are converted into reservoirs for spores. There were no manifestations of cellular immunity, such as encapsulation or nodule for-mation. Similarly, there were no signs of specialized host–parasite interaction that might be interpreted as xenoma-like complexes.The role of the cysts in the parasite’s life cycle is unclear. They may represent a defensive reaction of the host sacriWcing the infectedcells to segregate the infection. Alternatively, the cyst may help protect spores from environmental hazards and provide a concen-trated infectious dose to aid horizontal transmission of the microsporidium. We propose to refer to hypertrophied adipocytes Wlledwith T. solenospsae spores as “sporocytosacs,” not “cysts.” 2005 Elsevier Inc. All rights reserved.

Keywords: Microsporidia; Thelohania solenopsae; Pathogenesis; Life cycle; Sporocytosac; Cyst; Host–parasite relationships

1. Introduction origin,” sometimes called “cysts” or “sporocysts,” located

A common feature in microsporidian sporogony is theproduction of assemblages of spores enclosed in enve-lopes of either host, parasite, or mixed origin (Becnel andAndreadis, 1999; Canning and Vavra, 2000; Spragueet al., 1992; Vavra and Larsson, 1999). At the end of spo-rogony of some species, spores accumulate in parasitoph-orous vacuoles, in sporophorous and polysporophorousvesicles, or in “thick-walled vesicles of host–parasite

* Corresponding author. Present address: Laboratory of Cytology ofUnicellular Organisms, Institute of Cytology, Russian Academy of Sci-ences, Tikhoretsky Aveune, 4 194064, St. Petersburg, Russia. Fax: +225578 1643, +011 7 812 247 03 41.

E-mail addresses: jsokolova@lsu.edu, jumicro@yahoo.com (Y.Y.Sokolova).

0022-2011/$ - see front matter 2005 Elsevier Inc. All rights reserved.doi:10.1016/j.jip.2005.03.006

inside the infected cell. In all of these cases, the host andparasite interact on a subcellular level, without modiWca-tion of the host cell surface and a subsequent immuneresponse of the host organism. The host cell loaded withparasites preserves its integrity within the tissue until thevery Wnal steps of destruction by the parasite.

In several insect hosts, microsporidia develop inblood cells (mainly oenocytes) where they cause signiW-cant hypertrophy. These cells packed with sporonts ormature spores disperse the infection throughout theorganism or deliver infective spores to the target tissue,such as fat body, gut, or ovaries (Andreadis and Hall,1979; Becnel and Andreadis, 1999; Becnel et al., 1986).Granulocytes phagocytizing spores liberated from thedestroyed fat body cells appear similar to infected

Y.Y. Sokolova et al. / Journal of Invertebrate Pathology 90 (2005) 24–31 25

haemocytes and also probably spread the infection(Nassonova et al., 2001; Sokolova et al., 2000).

Several species of microsporidia cause more conspicu-ous alterations in host cell physiology, resulting in prom-inent hypertrophy, nuclear transformation, isolationfrom other cells of the tissue, and Wnal modiWcation ofthe parasitized cell or group of cells into a refuge forspore maturation and maintenance. The complex ofhost–parasite relationships associated with this phenom-enon, which involves the response of the whole hostorganism, is called xenoma (Sprague et al., 1992). Severaltypes of xenomas are known, such as neoplastic xeno-mas, syncytial xenomas, xenocytes, Glugea-cysts (Weiser,1976), xenoma-like cysts (Freeman et al., 2003), or sim-ply “cysts” (Rodriguez-Tovar et al., 2002; Weiser, 1976).Xenomas are mostly associated with microsporidiosis inWsh (Canning and Lom, 1986), but a few xenomas havebeen described in insects (Becnel and Andreadis, 1999;Lange et al., 1996; Weiser, 1976).

Thelohania solenopsae, a microsporidium parasitizingWre ants, Solenopsis invicta, produces at least four types ofspores in Wre ants, S. invicta: Thelohania-like octospores,Nosema-like spores (Knell et al., 1977), megaspores(Sokolova and Fuxa, 2001; Sokolova et al., 2004b), anddiplokaryotic spores with large posterior vacuoles devel-oping exclusively in brood (Shapiro et al., 2003). Milkywhite oval bodies often are found in ant abdomens dur-ing dissections. These bodies are about 100–200 �m insize and contain thousands of sporophorous vesicles withoctospores. The Wrst description of T. solenopsae inS. invictae included the statement, “Heavily infected fatbody cells undergo hypertrophy, forming “cysts” whichemerge from severed gasters” (Knell et al., 1977). Thesebodies have been referred to as “cysts” ever since (Moser,1995). Nothing is known about the nature, origin, or roleof cysts in the T. solenopsae life cycle. The purpose of thisstudy was to determine these characteristics.

2. Materials and methods

A colony of S. invicta infected with T. solenopsae wascollected near Rosepine, Louisiana, a site with natural T.solenopsae infections (Sokolova et al., 2004a). Five eachof major workers, minor workers, alate males, and alatefemales were sampled on ten diVerent days (10 replica-tions). In addition, 25 queens (dealate females layingeggs) were removed from 10 infected colonies collectednear Rosepine, Clinton, and St. Joseph, Louisiana, andreared in the laboratory from 2001 to 2004. Workerswere considered to be minors if their head capsules mea-sured less than 550 �m across, and majors if the headswere more than 700 �m. Head capsule width in workerants in the experiment ranged from 432 to 550 �m(516.6 § 3.68�m, n D 50) in minors and from 703 to1016 �m (783.0 § 10.80 �m, n D 50) in majors.

Infection of the colonies by T. solenopsae was con-Wrmed by modiWed Trichrome staining (Weber et al.,1992) and by PCR (Fuxa et al., 2005; Milks et al., 2004).Infection of individual ants was detected by direct obser-vation of fresh smears under phase contrast optics and indoubtful cases by Trichrome stain. General morphologyand localization of cysts were determined under a LeicaMC125 dissecting microscope equipped with a digitalSPOT Insight camera, coupled with Spot Software (SpotInsight, Version 3.4. 2001, Diagnostic Instruments, Ster-ling Heights, MI), and under a Nikon Eclipse E-600Microscope equipped with a Metaview digital cameraand software (MetaView. 1998, Meta Imaging Series 4.5.Universal Imaging Corporation, West Chester, PA).

For further study, the ant’s abdomen was excisedfrom the body at petiole, and the intestines were care-fully removed. The abdomen was then opened in a dropof water for light microscopy or in Wxative for electronmicroscopy (Becnel, 1997; Sokolova et al., 2003). For thelatter procedure, samples were Wxed in a mixture of 2%paraformaldehyde and 1.25% glutaraldehyde in sodiumcocadylate buVer, pH 7.4, post-Wxed in 1% OsO4 in thesame buVer, incubated overnight in 0.5% uranyl acetate,dehydrated in a descending ethanol series, inWltrated,and embedded in epon-araldite resin. All reagents werefrom EMS Chemicals (Fort Washington, PA). Thin(80 nm) sections were cut on a MT-XL ultratome (RMCProduct, Tucson, AZ) and examined with a Zeiss 10 elec-tron microscope at 80 kV.

STATISTICA for Windows software, version 6.0(Anonymous, 1995) was used for all statistical analyses.Three analyses were done by one-way ANOVA with theTukey HSD test among means for groups of 10 workers:(1) prevalence of infection in minor versus major workers;(2) frequency of “cyst” formation in minor versus majorworkers; and (3) condition of fat body (absent, normal, orhypertrophied; see Section 3 for explanation) in infectedversus uninfected ants. For the latter analysis, there were10 replications with Wve minor and Wve major workers ineach; the percentage of workers with hypertrophied fatbodies was calculated and compared in infected and unin-fected insects. Correlation analysis was used to assess therelationship between the number of workers displayingfat body hypertrophy and percentage of workers withcysts in the same 10 groups of infected workers, irrespec-tive of ant size. The percentages were transformed to arc-sine square root before analyses (Zar, 1999).

3. Results and discussion

3.1. Occurrence of “cysts” in Wre ant castes

Minor and major workers regularly contained cysts.Overall, 39% of the ants examined contained T. solenop-sae spores. The prevalence of infection was 44% in

26 Y.Y. Sokolova et al. / Journal of Invertebrate Pathology 90 (2005) 24–31

majors and 34% in minors, a diVerence which was notsigniWcant (F D 0.19; df D 1, 18; P D 0.667). Overall, 64%of the infected workers had cysts. The prevalence of cystswas 46% in minor workers (mean of nine replications,one was uninfected) and 81% in majors (mean of sevenreplications with three uninfected), a diVerence whichagain was not signiWcant (F D 3.09; df D 1, 14; P D 0.101).The number of cysts per infected ant averaged 1.5 § 0.27and ranged from 0 to 6. Cysts were not observed in 36%of infected ants, 26% of them contained one cyst, 15%-two, 10%-three, 5%-four, 3%-Wve, and 5%-six cysts. Insamples from other mounds we have observed as manyas eight cysts in one worker.

Observations of alate ants indicate that a certainamount of time is required for spore production and cystformation. We did not discover T. solenopsae spores inalate males and females in the sampled colony within 4–6 weeks after it was brought into the laboratory for rear-ing, and consequently not a single alate (n D 100) con-tained a cyst. On the other hand, alates of both sexesfrom other colonies occasionally contained cysts, if theywere reared in the laboratory for more than 3 months.Nine of the 25 queens (dealate females laying eggs)examined were infected with T. solenopsae. Two of themcontained one or two cysts inside their abdomens; theother seven infected females did not have cysts, but theydid contain conglomerates of megaspores in tissues adja-cent to ovaries (Sokolova et al., 2004b).

3.2. Formation of the “cysts” and hyperplasia of the fat body

Cysts and fat body hyperplasia (or increase in thenumber of cells in the tissue (Anonymous, 1981)) werethe only signs of disease in T. solenopsae-infected ants,and both were recognizable only upon dissection of theinsect (Fig. 1). Adipose tissue in ants is normally com-posed of well distinguished adipocytes tightly packedwith fat granules (Figs. 1A, B, and D; Fig. 2A). Hemo-cytes were often seen adjacent to adipocytes (Fig. 2A).The workers examined had three distinctly diVerent con-ditions of fat body: (1) fat body was absent—no fat bodycells were seen inside the abdomen; (2) fat body was nor-mal—several well distinguished ribbons of fat body cells(adipocytes) were concentrated in the anterior part ofthe abdomen; (3) fat body exhibited hyperplasia—largelobes of fat body spilled from abdomens upon dissection(Fig. 2A). Fat body hyperplasia was observed in 74.2%(mean of 10 replications, irrespective of worker size) ofinfected workers and in 21.1% (mean of 10 replications,irrespective of size) of uninfected ones, which was a sig-niWcant diVerence (F D 8.48; df D 1,18; P D 0.009). Inthese same groups of ants, the percentage of infectedworkers with cysts was not correlated with the numberof infected workers with hypertrophied fat body(r2 D 0.013; P D 0.757). Thus, in the colony examined, the

development of cysts was not directly dependent on fatbody hyperplasia caused by increase in numbers of adi-pocytes. Cyst formation may be a manifestation of thelate stage of microsporidiosis, while fat body hyperplasiarepresents a general host response to microsporidiosiswhich is not unusual in insects (Becnel and Andreadis,1999).

3.3. General morphology and ultrastructure

Cysts usually appeared as roundish or oval bodieswith diameters ranging from 70 to 263 �m (mean164.8 § 8.60 �m, n D 25). In comparison, uninfected fatbody cells (adipocytes) ranged from 39 to 65 �m (mean56.3 § 1.58�m, n D 21) (Figs. 1B and D). Cysts remainedconnected with the fat body tracheal system until thevery Wnal stage of pathogenesis (Fig. 1C), suggesting thatthe parasite’s development and multiplication depend onaerobic metabolism of the host. Cysts were observed indissected ants among ribbons of adipocytes at the ante-rior end of abdomens, behind the petiole, where the mostvoluminous portion of the abdominal fat body waslocated (Figs. 1B–D). In some infected workers with nor-mal or hypertrophied fat body, adipocytes of normalsize and appearance contained numerous Nosema-likespores (Fig. 2B). In the same individuals, some adipo-cytes were slightly enlarged, lacked lipid granules (Fig.1D, arrowhead) and were Wlled with spores of either theNosema type (Fig. 2C) or, at a more advanced stage ofinfection, with megaspores and octospores (Figs. 2D andE). These adipocytes also contained numerous sporo-blasts (Figs. 2D and F). Mature cysts were assemblagesof tightly packed octets of octopores, sometimes mixedwith zones of megaspores (Figs. 1E–G; Figs. 2E–H).Usually such cysts were milky white due to the presenceof masses of mature spores, and they were fragile, disin-tegrating at the slightest touch and thereby liberatingnumerous sporophorous vesicles with octospores (Fig.1E; Fig. 2E). A few cysts were melanized, brownish incolor, and relatively solid (not fragile) (Figs. 1F and G).In the abdomens of one major worker and two alatefemales, we discovered partially transparent, immaturecysts, 50–100 �m in diameter with thin non-melanizedwalls, which preserved their integrity during Wxation forelectron microscopy (Fig. 1D).

Electron microscopy of infected fat body conWrmedthe results of light microscopy. The Wne morphology offat body cells containing stages of the Nosema-like dip-lokaryotic sequence was not signiWcantly diVerent fromthat of uninfected cells (Figs. 3A–E). Subsequently,multiplication of parasites and formation of Nosema-like spores caused depletion of lipid and protein gran-ules, increase in host-cell size, enhanced invagination ofthe nuclear envelope, and isolation of the infected cellfrom the attached hemocytes and eventually from thenearby adipocytes (Figs. 3B–H). Mitochondria from

Y.Y. Sokolova et al. / Journal of Invertebrate Pathology 90 (2005) 24–31 27

non-infected adipocytes tended to concentrate in thezone adjacent to cells infected with meronts (Fig. 3F).This suggests that the parasites may secrete intermedi-ates or end products of their anaerobic carbohydratemetabolism (e.g., pyruvate, lactate, or glycerol), thatmight be utilized by host mitohondria (Dolgikh et al.,1996; Weidner et al., 1999). Cysts (Fig. 3G–K) containednumerous T. solenopsae spores and prespore stages, aswell as host cell nuclei and, occasionally, mitochondriagrouped around the parasites. Organization of the sur-face of the cyst and the sub-membrane layer of cyto-plasm (Fig. 3I), as well as nucleus morphology (Figs. 3Gand H), resembled the same structures in the intact adi-pocytes (Fig. 3A). Sporoblasts and octospores develop-ing inside sporophorous vesicles usually occupied thecentral portion of the cyst. Free megaspores occasionallyoccurred inside the cysts among the sporophorous vesi-

cles with octospores (Figs. 3J and K). Most Nosema-likespores were “empty” envelopes left after sporoplasm dis-charge; these were concentrated on the periphery ofimmature cysts (Sokolova et al., 2003; Figs. 2D and L).

Thus, light and electron microscopy of immaturecysts indicated that these structures are enlarged andtransformed adipocytes. In the Wrst step of pathogenesis,Nosema-like spores are produced for autoinfection; thediplokaryotic sequence leading to their formation causesfat body hyperplasia. When meiosis begins, the parasiteswitches to production of octospores and/or megaspores(Sokolova et al., 2004b). Infected adipocytes then enlarge2–4-times in conjunction with parasite multiplicationand octospore maturation. Eventually, the infected cellslose their cellular organization and are converted intoreservoirs for spores. Thus, this cyst formation in antsrepresents “false hypertrophy” (Brooks, 1974), in which

Fig. 1. Cysts produced from adipocytes in abdomens of S. invicta workers infected with T. solenopsae. (A) Dissected ant abdomen displaying hyper-trophied fat body. (B) Ant abdomen and ribbons of adipocytes. Arrow indicates an enlarged cell Wlled with spores, i.e., a cyst; asterisk, petiole. (C)Cysts (black arrows) develop in connection with the tracheal system of the host; white arrow indicates tracheole entering an immature cyst. (D) Cystsat diVerent stages of maturation; black arrowhead indicates slightly enlarged adipocyte lacking refractive lipid granules, presumably containing par-asites undergoing Nosema-like sequence of the life cycle; white arrow points to a large cyst, presumably containing prespore stages of the octosporesequence; black arrows indicate cysts largely Wlled with spores. (E) Mature cyst liberating sporophorous vesicles. (F) Melanized mature cyst. (G)Same cyst as “F,” but at higher magniWcation, Wlled with numerous octospores and a few megaspores (black arrow). A, adipocytes; FB, fat body; S,spores; SVs, sporophorous vesicles; and Tr, trachea. Scale bars: (A and B) 500 �m; (C, D, and E) 100 �m; (F) 50 �m; (G) 10 �m.

28 Y.Y. Sokolova et al. / Journal of Invertebrate Pathology 90 (2005) 24–31

the parasitized cell increases in size due to multiplicationof parasites, but not in order to “meet a demand forincreased functional activity” (Brooks, 1974).

The role of the cyst in the T. solenopsae life cycle isunclear, as is the role of octospores (Sokolova et al.,2004b). The cysts, as agglomerates of large numbers ofspores, may aid horizontal transmission by concentrat-ing infectious dosages. Alternatively, the production ofcysts might be interpreted as a defensive reaction of a

host organism sacriWcing the infected cells to segregatethe infection, although melanization of cysts, whichwould support this hypothesis, was rare, and other cellu-lar immune responses, such as encapsulation or noduleformation, were not observed at all. There were no signsof complicated and specialized host–parasite interaction,such as syncytia formation, or enclosure of infectedhypertrophic cells into an envelope consisting of hemo-cytes or other tissue, that might be interpreted as

Fig. 2. Development of T. solenopsae inside cysts under light microscopy. (A) Uninfected adipocyte with numerous refractive lipid granules (aster-isks) and an attached hemocyte. (B) Adipocyte infected with Nosema-like spores (black arrow heads) the cell still contains lipid granules (asterisks)and is still associated with a hemocyte. (C) The next stage of cyst development characterized by enlargement of the cell and nearly complete disap-pearance of lipid granules. Nosema-like intact spores are indicated by black arrowheads, germinated spores (looking dark under phase contrastoptics) by white arrowheads. Asterisks mark sporonts, which will give rise to the next generation of spores (octospores and megaspores). (D) Theimmature cyst Wlled with Wred Nosema-like spores (white arrowheads), octospores (black arrows), megaspores (white arrows) and sporoblasts. (E)Disintegrated cyst containing octospores (black arrows), and Nosema-like spores (arrowheads). (F) The same cyst (E) at a higher magniWcation,showing a sporont containing developing sporoblasts of the octospore sequence. Black arrowheads indicate Nosema-like spores. (G) Another regionof the same cyst (E) with octospores (black arrow), megaspores (white arrow), and a sporoblast of the megaspore sequence. (H) Octospores (blackarrows) and megaspores (white arrows) being freed from a mature cyst. A, uninfected adipocyte; H, hemocyte; N, nucleus; Sp, sporont; and Spb, spo-roblast. Scale bars: (A–H) 10 �m.

Y.Y. Sokolova et al. / Journal of Invertebrate Pathology 90 (2005) 24–31 29

Fig. 3. Development of T. solenopsae inside cysts under electron microscopy. (A) Uninfected adipocyte. Note invaginated nucleus (N) andcharacteristic structure of the cell surface (insert, arrow). (B) Adipocyte lightly infected with T. solenopsae meronts of the Nosema-like sequence(arrows). (C) Adipocyte Wne structure has not changed noticeably during the Nosema-like sporogony. Arrow points to a sporoblast. (D) Moderateinfection of adipocytes with developmental stages and spores. The numbers of lipid and protein granules are reduced, but the surface structure aswell as the adipocyte-adipocyte and adipocyte–hemocyte contacts do not appear to be noticeably altered. (E) Mass production of Nosema-like sporesleads to disappearance of lipid and protein granules, increase of cell volume, and enhancement of nucleus invaginations. (F) Zone of contact of anuninfected adipocyte (A) and an infected cell Wlled with meronts of the octospore/megaspore sequence. Mitochondria of the uninfected cell haveaccumulated at its periphery in the region of contact with the infected cells. (G–K) Sections through mature cysts. (G) Meront/sporont transitionalstage undergoing meiosis prophase; arrows indicate synaptonemal complexes. (H) Sporophorous vesicles with octospores and a degrading invagi-nated nucleus. (I) Surface of a cyst (arrow) similar to that of an uninfected adipocyte. (J and K) Octospores and megaspores inside a cyst. DK, diplo-karyon; L, lipid granules; Me, meronts; Mit, mitochondria; MS, megaspores; N, nucleus; P, protein granules; S, octospores; and SV, sporophorousvesicles. bars: (A, C, D, E, and H) 5 �m; (B and J) 10 �m; (F, G, I, and K) 1 �m.

30 Y.Y. Sokolova et al. / Journal of Invertebrate Pathology 90 (2005) 24–31

xenoma-like complexes (Canning and Lom, 1986; Langeet al., 1996; Weiser, 1976).

3.4. Terminology of “cyst”

The term “cyst” in the context of the described phe-nomena is inappropriate. In protistology, “cyst” isdeWned as the stage of the protist life cycle characterizedby a robust envelope, such as the cysts of Amoebae(Bovee, 1991) or Infusoria (Lynn and Corliss, 1991), theoocysts and sporocysts of Coccidia (Perkins, 1991), andthe cysts and sporocysts of Microsporidia (Becnel andAndreadis, 1999; Comtet et al., 2003). Vavra and Spra-gue (1976) deWne a “cyst” as any bladder-like envelope;they deWne a “sporocyst” as an envelope or cyst con-taining spores, or as a sporophorous vesicle. They desig-nate “sporogony cysts,” “fragile cysts” and “durablecysts” as thick walled or thin walled cysts produced bythe sporogonial plasmodium. The term “Glugea-typecyst” is used as a synonym of a Glugea-type xenoma(Canning and Lom, 1986). Weiser (1976) distinguishedbetween “xenomas,” which are multicellular from thevery beginning of their development and result fromhyperplasia or syncytium, and “Glugea–cysts,” whichare initially produced from a single infected cell. Thelatter is true of T. solenopsae, but what we observe inants can hardly be considered a Glugea-type xeno-para-sitic complex. In view of diYculties in applying any ofthese terms to “cysts” in S. invicta, we propose to usethe term “sporocytosac” for the conglomerate of sporesenclosed by the membrane of the T. solenopsae-destroyed adipocyte produced in Wre ant abdomens.The term “sporocytosac” implies only a sac with spores;it avoids confusion with previous terminology, and it isconsistent with the absence of a specialized wall orenvelope, which is an essential character in protistologi-cal deWnitions of cysts.

Acknowledgments

We thank Arthur Richter (Department of Entomol-ogy, Louisisana State University Agcenter) for assis-tance in laboratory and Weld work and Igor Sokolov forhis help with statistical analysis. We acknowledge theuse of the Socolofsky Microscopy Center, Departmentof Biological Sciences, and Laboratory of ElectronMicroscopy of Department of Comparative BiomedicalSciences, School of Veterinary Medicine, LouisianaState University, Baton Rouge. This research was sup-ported by the Louisiana Fire Ant Research and Man-agement Legislative Grant and the Texas Fire AntResearch and Management Project. The paper wasapproved for publication by the Director of the Louisi-ana Agricultural Experiment station as manuscript No.05-26-0143.

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