Ultrastructure of midgut events in the pathogenesis of Bacillus sphaericus strain SSII-1 infections of Culexpipiens quinquefasciatus larvae

ELIZABETH WEST DAVIDSON [ ) r { ~ o ~ . l ~ ~ r o r l (!/'Zoology, A~izotro S~ctlc Utri~.c't:ti/?., T o ~ ~ r p r . A Z , U . S . A . ~YSY5281

Acceptetl November 8, I978

DAVIDSOK, E. W. 1970. U I t r i ~ \ t r ~ ~ c t l ~ r e o f midgut events in the pithogenesis o f Btrc~illr~.~ .sphcrc~r~icrr.s strain SSII- I infections o f L'rtlc.vpil~ic~rr.s c l r r i r r c ~ r ~ c : / i r . c c ~ i t r / r r . s larvae. Can. J. Micro- hiol. 25: 178- 184.

The fate ofBcrcillrrs s~jhcrer~icrr.~ stwin SSII-I cells ingested by Cirlc.vpil~iots q r r i t ~ c l r r c ~ f i t . s c ~ i ~ ~ / ~ ~ . s (= C. pipic,rr.\ ,fir/igcr~r.s, C . fir/igtr~r.s, C . c ~ i r i ~ ~ c ~ r r c ~ f i r . s c ~ i ~ ~ / ~ i . s o f authors; Diptefii: Culiciclae) larvae ancl the cytological events precedingdeath o f the host were observed using electron microscopy. Bnc.illrr.s ,splrcieric.~r.s cells were digested ~xp id ly in the anterior and central midgut. The outer cell wall layel. and cytoplasmic ground sub\tance tlisappeared soori after ingestion. Cytolysosomes beczime prominent i n midgut cells as these cells graclu~illy separated from one another. All bacteri:~, including B. .s~~lrcrc~rierrs, were confined within the peritrophic membrane until after tleathof the host. Digestion by the I;irval host isconfirmed as ;r possible mechanism fo~.rele:~se o f B. .sphtro.ic~r.s toxin from the bacterial cells.

I> ; \v r~)so~. E . W. 1979. U I t r : ~ s t r ~ ~ c t ~ ~ ~ . c o f miclgi~t events in the pathogenesis o f Boc~illrr,~ .tplrtroric~rr.t strain SSII- I infections o f CII/P.B I J ~ ~ ~ C I I . S ( ~ ~ i i ~ r c / ~ r e J i r ~ c ~ i ~ ~ / ~ r ~ I;LI.V:I~. Cali. J. Micro- biol. 25: 178- 184.

Le tlevenir cle Utrc~illrrs .\plrtro.ic~rrs souche SSII-I ingel-6 par dcs I ~ ~ r v e s de C'ctlc,.r pil,ic,rrs clrrbrclrrc~fir.scier/cis (= C'. pi{~ ic~r~.v . f i r~ i~( i~r .c . . C.jir/iii'o~r.s, C. q i i i ~ r t l ~ ~ c : f i r . s c . i ~ r / r ~ s t l ' a ~ ~ t l r s :u~teurc; I l ip- tcra: Culicitlkle) el les changemcntscytologiq~re\ c1ui pl-ecctlent la niort de I'hbte ont dte et~!tliPs en micl-oscopic ilectronique. Les cellules de B. .sphrro.ic~~r.s sont rapidement cligdrecs tliuis I'intestin anterieuret central. L a couche externe de la paroi cellul;~irc ct le miiteriel cytopl:~srnique tle base tlisp>~l.aissent tht apre\ I'ingestion. Lcs cytolysosomes prennent tle 1'import;lnce dans les cellules tle I'intehtin ii mesure q~re ces cellules \e tldt:~chent gracluellcnicnt les unes tlcs ;lutres. Avant la mart de I'hhte toutes le\ b:ictdries. y compris 11. .splrtre,ricos. sont contindes i~ I'interieur de la membrane pi~.itrophe. 11 semble donc que la digestion par la Iarve-hbtc soit un mecanisme pouvant expliquer la secretion de toxine par les cellulcs tle B . .splrtro.ic.rr.s.

[Tl'arluit pal- le journal]

Introduction Bocillrrs splltrericlrs strain SSII-1 kills at least

50% of Clrles pipic1l.s yrii~~cj~refir.scitrt~is (= C. pi- pielzsftrtigtrtl.~, C. Jirtiglrizs, C. yrri~zyucf(lscitrtrrs of authors; Diptera: Culicidae) mosquito larvae in labol'atol-y bioassays at a concentration of about 10Qbucter-ia/niL (Myers and Yousten 1978n). A toxin is responsible for B. .rpl~cle~Yc,~r.c insecticidal activity; this toxin is not released into the culture medium, is intimately involved with the bacterial cell, and is not present in the form of a parasporal crystal (Singel- 1973, 1974; Myers and Yousten 197811; 19780). I t has been PI-oposed that digestion releases toxin from B. sp1zneric.u~ cells (Davidson et (11. 1975). The location of the toxin in the cell, its chemical identity, and its mode of action are un- known.

To understand better the events which lead to death of the host, this ultl-astructural study was undertaken to observe the fate of ingested B. sphtreric,lrs strain SSII-1 cells and pathological changes in lal-val tissues.

Materials and Methods A loopfill o f fresh sl;~nt c u l t ~ ~ r e o f B. .sphcror.ic~~r,s strain SSII- I

was ~ ~ s e d to inoculate 21 Rnsk containing 25 mL o f synthetic bacterial medium (Singer (,I (11. 1966). After shaking for 18-24 h at 150 rpm kind 28"C, I m L o f this cult~rl-e was used to inoc111;lte a second 25 mL. o f l iquid medium, which was gain shaken at 150 rpm and 28°C for 18-24 h. This second culture was ;itltled to the tap water surrounding secontl- or third-instar C. l~il~ic,rr.s yrlitr- qrrqfirscio~rr.~ mosquito larvae to a concentriltion o f about 10' ce l ls lml . Larvae were allowed to feed for 15 min, aftel- which they were transferred to tap water without added bactel.i;r. They were fed T e t ~ i m i n fish food (Tetra Werke, W. Germany) 12 h after B. .splrtro~iclr.s treatment.

At least 10 larvae were fixed for electron microscopy ;it the time o f removal o f larvae from contact withB. .s~~lrtrericrr,s, and at 15 and 30 min, and 1, 2, 3, 4, 5.6, 7, 10, 12. 18. and 24 h later. Larval appearance, behavior, and mortality were noted at each fix;ltion time. Larvae of the same age. but not fedB. splrtro.icrrs. were fixed for control>.

At the time o f fixation, heads and siphons of larvae were removed and bodies were nicked with :I razor blade. Larvae were fixed in ice cold 5% glut;iraldehyde in sodium cacodylnte buffer, p H 7.4. for 12-24 h. washed in buffer, and postfixed overnight in ice cold 1% buffered osnii~lrn tetroxide. After rins- ing. I:irvae were refrigelxted overnight in 0.5% aqueous ur:~nyl acetate solution. then dehydrated end infiltl-ated and imbedded in Spurr resin (Dawes 1971). Thin sections were stained with

0008-4 166/79/020 178-07%0 1 .00/0 0 I979 National Research Council o f C;in;ida/Con\eil nationill de recherche5 du Canada

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u ~ a n y l acetate ancl leacl citrate nncl were viewed ~ ~ s i n g a Philips EM 300 electlun niicl.o\cope.

Results All tissues of B. spl1aeritrr.c-fed larvae were

examined fol evidence of histopathology. Until neat- death of the lal-vae, only midgilt cells exhibited major changes in appeal-ance, and differed from controls. Therefore, only the contents of the mid- gilt and the condition of the midgilt cells are de- 5cribed here.

Briefly, the digestive tract of the mosqirito larva is constructed as follows: the cuticle-lined fol-egut i s joined to the midgut at the stomodeal valve. The midgut is lined with the secreted, acellular peri- trophic membl-nne, which encloses ingested food during passage through the midgut. The midgilt is divided into distinct anterior and postesiol- regions. with a rather sharp centl-al tl-ansition zone. At the PI-octodeal valve, the midgut joins the cuticle-lined hindgut (Richards and Richad\ 1971; Richards 1975).

In contt-ol larvae fed no B. .\pltae/.it.rrs, the bacte- rial flora within the midgilt were located in the peripheral area within the pesitrophic membrane (Fig. 1). The center of the lumen contained lamellar material. possibly a degsadation product of the per-itrophic membl-ane (Richards and Richku-ds 1971). but few bacteria. In control larvae, or- ganisms with GI-am-positive cell wall arrangement wel-e curved in longitudin:~l outline and possessed only single-layered cell walls, features distin- guishing these 'normal flora' 01-ganisms from B. .cpl~aericrr.s. Anterior midgut cells were distin- guished by numerous microvilli and the very com- plex infolding of the basal plasma membrane, termed the basal labyrinth (Richards 1975). Cells of the thinner posteriol- midgut epithelium bore more spasse micl-ovilli and a less extensive basal labysinth than anterior cells (Fig. 3). Along the entire midgut, intel.cellular junctions were closely joined throughout most of their lengths. Small membrane-bounded bodies containing whorls of lamellae and dense granules, similar to organelles desct-ibed by Smith (1968) and Richat-ds (1975) as cytolysosomes, wer'e often seen (Figs. 2 and 3).

In larvae which had been fixed immediately after feeding on B. sphrr~r.ic.us, large numbers of B. ~plzarricrrs cells were found in the stomodeal valve and anterior midgut (Fig. 4). These cells bore the general cell wall profile described by Holt et (11. (1975) and by Myers and Yousten (19786), i.e. a thick outer layer, an unstained intermediate layer, and a thin inner peptidoglycan layer. The outer cell

wall layer has been called the "T" or tetragonally arranged layer by Howat-d and Tipper (1973) and others. This term is not used in the present st~ldy as the outer cell wall layer of strain SSII-1 is not tetrn- gonally arranged (Myers and Yousten 1978h). The bacteria seen entering mosquito larval midguts were 0.5-0.6pm in diameter and contained dense cytoplasmic ground substance; some contained a prominent nuclear region (Fig. 5). No similar bac- terial cells were seen in control larvae.

In lasvae fixed immediately aftel- feeding on B. .splrlre/.ic~trs, progressive loss of bacterial structure was noted through the anterior and central midgut regions. The outer cell wall layer disappeared first; cells appeased to be otherwise unaltered (Fig.6). 111 the centsal midgut (Fig. 7), most B. .spl~rr~/~icrrscells contained whorled laminar internal membranes, ruffled cell membranes. and cytoplasm of low elec- tron density sun-ounded by only the inner pep- tidoglycan cell wall layer. A few smaller normal flora bacteria were also present. In the most pos- terior region of the midgut, near the pt-octodeal valve (Fig. 8). a large number of bacterial cells with dense cytoplasm were seen. These cells wer-e iden- tical with B. splirrericrr.s in cell diameter and straight I-od-like shape, but lacked the outer cell wall layer. No similal- cells were seen in control larvae. The hindgut contained only a few scattesed intact bac- tel-ial cells.

Thirty minutes after feeding on B. splllcc~ricrrs, some bactet-ial cells with dense cytoplasm were still present within the anterior midgut near the stomodeal valve. Most cells in this region boreonly the peptidoglycan cell wall layer, though adhering fragments of the outer layer were present on a few cells. In the centl-al midgut region (Fig. 911 and h ) , nearly all bacterial cells wet-e devoid of cytoplasmic gl-ound substance and possessed single-layered cell walls. some of which wese broken, enclosing whorled and ruffled internal membranes. Some bactesia with dense cytoplasm still remained in the posterior region of the midgut.

One hour after feeding on B. .splllrt~/.icrrs, very few B. splllrrricrrs cells were found in the midgut and hindgut. Presumably defecation of the ingested meal of bacteria had occuri.ed (Davidson et (11. 1975). Some smaller bacteria with electron-dense cytoplasm and irregular cell outlines, probably cells of nol-mal flora, were present in the peripheral iu-ea of the gut contents within the peritl-ophic membrane (Fig. 10).

Between 1 and 7 h after feeding, the numbers of bacteria within the pel-itrophic membl-ane had in- creased markedly (compare Figs. 10 and 11). Most

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180 CAN. J . MICROBIOL. VOL. 2 5 . 1979

of these bacteria were probably nol-nial flora, but some were similar in appearance to B. .splzetet.icrr.s, bearing the characteristic triple cell wall arrange- ment (Fig. I I).

Around 10 to 12 h, external gross symptoms of intoxication were observed. Larvae were sluggish. The gut contents within the peritrophic membrane were thrown into zigzag folds, d ~ ~ e to swelling of the midgut. Swelling progressed until the m i d g ~ ~ t wall came to lie against the outer body wall, eliminating most of the heniocoelic space (see Fig. 13). No striking histological changes in midg~lt cells were observed ~lntil 10 h after feeding on B. splllrcric.rrs. By 10 h, cytolysosomes in miclg~lt cells were larger and more numerous (Fig. 12; compare with Fig. 2). As the midgut swelled, the midgut cells became separated from one another at the bases (Fig. 12). Mitochondria and microvilli appeared unaffected.

At 12 H, histological effects varied widely in se- verity. In some larvae, midgut cells resembled those at 1Oh. In others (Fig. 13), large cytolyso- somes and loss of microvilli were seen in anterior niidgut cells, with cytolysis and sloughing of post- erior midgut cells occasionally occurring. Base- ment membranes remained intact. Bacteria were confined to the peritrophic membrane.

All larvae were moribund or dead by 24h. In moribund larvae, the peritrophic membrane was filled with bacteria, sonie of which resembled B. splzcrer~iccts. No bacteria were seen outside the peritrophic membrane. Very large cytolysosomes containing a variety of membranes and granules were prominent in all midgut cells. Mitochondria were swollen, causing cristae to be disorganized. Cells were widely separated at the bases, and most of the micl-ovilli were missing.

Discussion The rapid loss of cellular integrity in ingested B.

spl7net.ic.rrs cells indicates that these bacteria are digested by the mosquito larvae. Early in the diges- tion of B. spl7cter.ictrs the outer layer of the cell wall and part of the cytoplasmic ground substance dis- appear rapidly; these cellular components are largely 01- totally proteinaceous (Howard and Tip- per 1973; Mizushimi et 01. 1966). The outer cell wall layer and some cytoplasmic structures were simi- la-ly removed by treatment with the protein ex- tractant, 8 M urea (Myers and Yousten 19780). Dadd (1975) has shown that proteases are probably most active in the anterior midgut region of C. pipic.rzs larvae, while amylases are probably most active in the posteriorregion. Indeed, in the present study digestion of the outer cell wall layer and cytoplasm occurred in the anterior and central mid- gut regions. This evidence agrees with results of an earlier study (Davidson ct crl. 1975), in which B. sphcterictts strain SSII-1 numbers were found to drop sharply during the 1st hour aftel- feeding, and before defecation. Therefore, digestion is probabl y the mechanism for release of B. sp1icrericlr.s strain SSII-1 toxin from the bacterial cells. Stl-uctures which are visibly removed during early digestion may or may not be the location of the toxin. Re- search is curl-ently in progress seeking to locate the toxin in the B. sphcrericus cell.

Changes in fine structure of midgut cells occur- ring during B. sphaericus strain SSII-I infection resemble those seen during some other toxin- mediated bacterial and fungal infections of insects (e.g. Ebersold et crl. 1977; Zacharuk 1971). Kellen pt rrl. (1965) described the histopathology induced by another, less insecticidal strain ofB. sphcrpricus

FIG. I. Control. Bacteria contained in anterior pel-itrophic membrane (PM). FIG. 2. Control. Anterior midgut cell with nucle~ls (N), microvilli (MV). extensive basal labyrinth (BL), cytolysosome (CL). and intercellularjunction (arrows). F I G . 3. Control. Posterior midgut cell demonstl-sting intercellula~- junction (arrows), cytolysosome (CL). microvilli (MV). less extensive basal labyrinth (BL) than anterior midgut. and basement membrane (BM). FIG. 4. Fixed immediately after feeding on B. .spllrrerictrs. Bacteria near foregut cells of the stomodeal valve (FG).

FIG. 5. Fixed immediately after feeding on B. sp11oericrr.s; stomodeal valve area. Brrcillrrs .sphrrrricirs cell withoutercell wall layer (0). ~lnstained intermediate cell wall layer ( I ) , inner peptidoglycan cell wall layer (P), cytoplasmic membrane (CM), nuclear region (NR), and dense cytoplasmic ground substance (CG). F I G . 6. Brrcillrrs spl~lrericrrs in anterior midgut. fixed immediately after feeding. Outer cell wall layer ( 0 ) is missing in al-ens (arrows); inner peptidoglycan cell wall layer (P) is intact; cytoplasmic ground substance (CG) is still dense. FIG. 7. Fixed immediately after feeding on B. sphrrericrrs: central midgut. Most B. .sphrrericirs cells exhibit greatly reduced cytoplasmic density, whorled internal membranes, rumed cyto- plasmic membranes. and only one cell wall layer. A few smaller organisms of normal flora (NF) are present. FIG. 8. Fixed immedizttely after feeding on B. sphrrc,ricus; posterior midgut near proctodeal valve. Large proportion of cells exhibit dense cytoplasm, though only one cell wall layer.

FIG. 9. Fixed 30min after-feeding on B. sphnericrrs; cen~l-al midgut region. Cells with cytoplasmic ground substance nearly absent, whorled internal membranes (W). ruffled cytoplasmic membranes(CM), and only the peptidoglycan cell wall layer(P) present. In Fig. 919, this layer has broken (arrows). FIG. 10. Fixed I h after feeding on B. sphcrericus. Contents of anterior peritrophic membrane (PM). Electron-dense bacteria resemble cells of normal flora. FIG. 11. Fixed 7 h after feeding on B. ~phnericr ts ; contents of anterior midgut. Large numbers of bacteria are enclosed in the peritrophic membrane (PM); some bacteria resemble B. sphrrericrrs (arrows). FIG. 12. Fixed I0 h after feeding on B. spl~ciericrrs. Anterior midgut cells showing separation ofcells at bases(arr0ws)and increase in number and size ofcytolysosomes (CL). Mitochondria (MI) and microvilli (MV) appear normal.

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DAVIDSON 181

Figs. 1-4

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182 CAN. J MICROBIOL VOL 2 5 . 1979

Figs. 5-8

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Figs. 90-12.

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184 CAN. J . MICKOE llOL. VOL. 2 5 . 1979

FIG. 13. Fixed 12 h after feeding on B. .sp/rtrc,r.ic~~s: posterior midgut cells. Lower cell (star) has lysed. releasing cell contents. Upper cell contains large cytolysosomes (CL): microvilli (MV) are sloughing. Basement ~nembranes (BM) are intact. Gut has expanded against the outer body wall (BW).

using light microscopy. In this infection symptoms did not appear until 3 days, and bacteria invaded the hemocoel late in the infection. Vac~~olation and sloughing of posterior midgut cells were seen. Though changes in midgut cells were buperficially similar to those seen in infections produced by strain SSII-I, in SSII-1 infections pathological changes appear much more rapidly and there is no evidence of hemocoelic growth of bacteria.

In the present study, a refractory period of about 7-10 h was found between ingestion of B. sphrre~.icrrs and the appearance of major histologi- cal changes in the midgut. It cannot be determined from this study whet her the increase in numbers of bacteria in the pel-itrophic membrane noted at 7-10 h causes the visible histological changes, or whether these bacteria multiply as a result of host changes which provide a more favorable environ- ment in the gut. A similar increase in both B.

.2

sphae~.icus and normal flora numbers at 7- 10 h was found by Davidson et crl. (1975). Two studies have shown that multiplication of B. splzcre~.icus in the larval gilt or hemocoel is not necessary in the pathogenesis (Davidson et n l . 1975; Myers and Yousten 1978tr). Current research utilizing axenic

mosquito larvae should clarify the [-ole of multipli- cation of normal l3ol.a bacteria in the pathogenesis of B. sphrr~r icrrs strain SSII-1.

Acknowledgements I am grateful to the Division of Agriculture, the

Department of Zoology, and the Electron Micro- scopy Laboratory of the Department of Botany and Microbiology, Arizona State University, for the use of I-eseal-ch space and equipment. This research was supported in part by NSF Grant No. BMS-72- 1954-A01, and in part by an Arizona State Univer- sity Faculty Grant-in-aid.

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