Immune reactions of Chironomus larvae (Insecta: Diptera) against bacteria

J. Inwct Physioi. Vol. 33, No. 12, pp. 993-1004, Printed

in

0022-1910187$3.00 + 0.00 Copyright 0 1987 Pergamon Journals Ltd

1987

Great Britain. All rights reserved

IMMUNE REACTIONS OF CHIRONOMUS LARVAE (INSECTA: DIPTERA) AGAINST BACTERIA PETER~TZ*, GISELAENDERLEINand INGE ROET~GEN Institute for General Zoology, Free University of Berlin, Kiinigin-Luise-Stral 1-3, 1000 Berlin 33 (West), Federal Republic of Germany (Received 24 March 1987; revised 21 May 1987)

Abstract-Humoral encapsulation (“melanization”) represents the predominant defence reaction of Chironomus larvae against injected bacteria. Only low levels of phagocytic activity were observed; cellular encapsulation and nodule formation were completely missing due to low numbers of haemocytes. No other humoral antibacterial activity was detected in normal Chironomus haemolymph and even haemolymph of preinjected (“immunized”) Chironomus larvae showed little inhibition of bacterial growth on agar test plates. / Low cellular and lytic activity of Chironomus haemolymph against bacteria is well compensated for by its fast and efficient capacity of humoral encapsulation. Within 5-10 min, even high numbers of injected bacteria (up to lo5 per larva) were surrounded by capsular material. Within this range of injection dose, the fates of pathogenic and non-pathogenic strains were identical, and bacteria which are highly pathogenic for many other insects, e.g. larvae of GaNeriu meiloneffu, proved to be harmless to Chironomus larvae. The rapidity of humoral encapsulation may prevent the release of toxins or enzymes by which pathogenic bacteria normally damage its host and weaken its immune system. Key Word Index: Chironomus, insect immunity, phagocytosis, immunization, bacterial pathogenicity, phenoloxidase activation

INTRODUCTION Bacteria as pathogens of insects

Infections with bacteria are rather common in insects. There are some well-known bacterial diseases such as foul brood (Bacillus larvae, Streptococcus pluton) in honey bees or milky disease (B. popilliue) of the Japanese beetle, Popillia japonica. Strains of Serratia marcescens are frequently detected in diseased insects of laboratory cultures. The most famous insect pathogenic bacterium, B. thuringiensis, has successfully been used as biological control agent against a large variety of insect pests, especially Lepidoptera, Diptera and Coleoptera. The importance of bacteria as biotic factor for insect populations was also demonstrated by Bucher (1960) who found 70 different strains of bacteria as pathogens of grasshoppers, and who defined insect pathogenic bacteria as “those bacteria capable of multiplying in the hemocoel of insects and producing a lethal septicemia from small inocula, usually much less than 10,000 cells per insect”. Bacteria are not able to actively penetrate the cuticle or peritrophic membrane and gut wall of insects; this capacity is the privilege of other pathogens or parasites, e.g. fungi, microsporida, nematodes or parasitic insects. Under natural conditions bacterial cells reach the insect haemocoel only through injuries or lesions in the cuticle and the epithelium of the midgut (as occurring, e.g. during a moult). Furthermore, invasion of bacteria may occur in connection with parasite invasion or in the course of

*To whom correspondence should be addressed.

nodule formation,

encapsulation,

oviposition by parasitoid insects. For successful development in insects, bacteria need specific capabilities. They should be able to (1) withstand antibacterial conditions within the gut (pH, digestive enzymes, lysozyme); (2) produce toxins which destroy the gut epithelium; (3) release enzymes and toxins which damage insect haemocytes thus reducing or preventing the effect of phagocytosis and cellular encapsulation; (4) inhibit or destroy humoral antibacterial factors of the host haemolymph; (5) assimilate host-like surface molecules and deceive the recognition mechanisms of the host; (6) digest tissues and organs of the insect body and multiply with high growth rate in insect haemolymph. For most of these points listed above evidence has already been found. In B. thuringiensis a parasporal crystal and further toxins present in the spore or released from vegetative cells destroy the gut epithelium thus opening the way for invasion of the haemocoel. Active inhibition of antibacterial activity has been demonstrated for B. thuringiensis (Dalhammar and Steiner, 1983) and S. murcescens (Flyg et al., 1980; Flyg and Xanthopoulos, 1983). Growing cells of 5’. marcescens were found to release a chitinase and proteolytic enzymes (Grimont et al., 1979). The insect pathogenic bacterium Xenorhabdus nematophilus uses nematodes of the genus Neoaplectana as transport vehicle (vector); the bacteria are released after the nematodes have invaded the haemocoel of an insect. Within the insect, the bacteria multiply and lyse the host tissue thus furnishing adequate food for a subsequent mass reproduction of the nematodes (Poinar, 1979). The nematodes in turn produce an immune inhibitor which destroys the bacteriolytic activity of immune haemolymph (GGtz et al., 1981). 993

Considering the powerful equipment of insect pathogenic bacteria, it seems surprising that no bacterial infections have so far been reported for Chironomus. Chironomus larvae, especially those of the common species C. riparius (formerly called C. thum mi), may occur in dense populations in polluted water and therefore are likely to suffer from epizootics. In fact, chironomids are occasionally infected with other pathogens such as viruses, rickettsia, fungi, microsporida and nematodes, but bacterial infections are extremely rare. Investigating large numbers of chironomid larvae, we met only one case of bacterial infection which was caused by a rodlike spore-forming bacterium (Gotz, unpublished). We therefore decided to investigate the defence reactions of Chironomus larvae against bacteria and to compare them with the antibacterial capacities of Gulleriu mellonella, an insect whose immune reactions have already been studied in detail (Hoffmann et al., 1981; Chain and Anderson, 1982; Walters and Ratcliffe, 1983). Types of immune reactions of insects

Insects react against pathogens and invading parasites with cellular and humoral responses (Ratcliffe and Rowley, 1979; Rowley and Ratcliffe, 1981; Gotz and Boman, 1985). Among cellular reactions, phagocytosis (i) occurs against small numbers of particles such as bacteria or fungal spores. Larger numbers of bacteria and spores elicit nodule formation (ii) where disintegrating granular haemocytes together with trapped bacterial aggregates are surrounded by concentric layers of flattened plasmatocytes (Ratcliffe and Gagen, 1977). The success of such cellular antibacterial responses depends greatly upon the strain of bacteria involved. Nonpathogenic bacteria are killed within phagocytes or nodules and acid phosphatase can be demonstrated within phagocytic cells as evidence for lysosomal activity. By contrast, pathogenic bacteria, though eliciting even faster and more efficient phagocytosis or nodule formation are able to develop and multiply within the host cells or nodules finally leading to lethal bactetiaemia. Phagocytes containing such pathogenic bacteria showed only little lysosomal activity (Walters and Ratcliffe, 1983). From 11 bacteria1 strains exposed to Blaberus cruniifer 6 were killed while Pseudomonas aeruginosa and 4 other strains remained unaffected within the phagocytic haemocytes (Anderson et al., 1973). Parasites as well as implanted foreign tissues or pieces of inert materials provoke cellular encap sulation (iii) which involves the formation of a multicellular envelope containing melanizing granular cells in the centre and extremely flattened plasmatocytes on the periphery. Humoral encapsulation (iv) has only been found as defence reaction of certain Diptera against parasitic nematodes (Bronskill, 1962; Esslinger, 1962; G&z, 1969, 1986a) and fungi (GBtz and Vey, 1974). The deposition of capsule material occurs within minutes after invasion or injection of foreign organisms. Humoral encapsulation can also be provoked by certain nonliving materials such as cellulose, polystyrene, agar etc. (Wilke 1979; Giitz 1986b). Hmnoral antibacterial activity (v) can be induced in certain insects by injecting nonpathogenic or inac-

tivated pathogenic bacteria. The possibility of immunizing insects has already been reported by Glaser (1918) and explored in more detail by Briggs (1958) and Stephens (1959). Part of the bacteriolytic and bactericidal activity comes from lysozyme (Mohrig and Messner, 1968), which, however, acts only upon Gram-positive bacteria. A completely new group of antibacterial proteins was discovered by Boman and co-workers (Faye et al.. 1975) using diapausing pupae of saturniid moths, especially H_valophora cecropia. Referred to as cecropins and attacins, these highly potent bacteriolytic compounds have meanwhile intensively been investigated. Their amino acid composition was analyzed and an artificial cecropin synthesized (Steiner et al., 1981; Hultmark et al., 1983; Boman et al., 1986). Cecropins and attacins are formed by the fat body and act upon both Grampositive and Gram-negative bacteria. They were also discovered in other Lepidoptera, e.g. Galleria mellonella (Hoffmann ef al., 1981). Recently, antibacterial peptides were isolated from certain Diptera, e.g. from Calliphora erythrocephala (Reinicke, 1982), Sarcophuga peregrina (Okada and Natori, l985), Drosophila melanogaster (Robertson and Postlethwait, 1986) and Phormia terranovae (Keppi et al., 1986). In the present investigation we intended to analyze the efficiency of humoral encapsulation of bacteria in Chironomus larvae and to search for cecropin-like antibacterial proteins in their haemolymph. MATERIALS

Chironomus

AND METHODS

and Galleria larvae

Chironomus larvae were either field-collected (Chironomus luridus, C. plumosus, C. annuiarius) or

laboratory-grown starting from egg masses of C. tentans (courtesy of Dr H. Tichy, Max Planck Institute for Biology, Tiibingen, West Germany) or from larvae sold in pet shops as fish food (C. riparius syn. C. thummi). Chironomus larvae were cultured in aerated tap water (5cm deep) in plastic vessels or glass tanks (40 x 20 x 25 cm). The larvae (about 300 per vessel) were furnished with pieces of paper tissue as a substrate for tube construction. As food we offered powdered leaves of stinging nettles (Pofiae urticae), but ground dog-food pellets or soy bean flour was also used. The food was added 3 times per week in a quantity of 0.34.5 g/l culture water (w/v). For mating, adults of C. riparius need a cage of about IOOcm height; copulation occurs most likely when swarms of 30 or more midges are present. In C. tentans, individual males and females mate on the water surface and therefore breeding of this species is even possible in small vessels. Galleria mellonella were grown in glass vessels at 30°C on an artificial diet consisting of crushed corn (22%), crushed wheat (1 l%), wheat flour (1 1 %), dry milk (II%), dry yeast (5.5X), wax (17.5%), honey (11%) and glycerol (11%) [Haydek, 19361. Bacteria

The strains of bacteria tested represent different groups (Table I). All bacteria of group highly pathogenic for many insects. Group composed of bacteria which occurred together

four A are B is with

Immune

reactions

of Chironomusagainst

bacteria

Table I. List of bacterial strains used for experiments with Chironomus and Origin of material

Strain-designation A. Insect pathogenic Bacillus

thuringiensis

Bacillus

rhurin~iensis

Bacillus

rhuringiensis

Bacillus

cereus

bacteria

popilliae subrilis

Serraria

marcescens

+ + + +

++ ++ ++ + +/++ +/++ +++ ++ ++

+ + + + + + + + + + +

nematophilus

Pseudomonas

aeruginosa

Pseudomonas

aeruginosa

-

+ _ + _

+ + + + + + +

e e

_ _

+I+I+i--

+ + +

d d

+ _ -

+I_ +i-

+ + + + +

+I+ _ _ _ _

Xn2l OT

91

B. Bacteria isolated from Chironomus outdoor pool water tank, I water tank, II dead larva of C. fenfans gut of C. tentans psychrophilic bacterium ( 18°C) psychrophilic bacterium (I 0°C)

habitats:

C. Mutants of E. coli: Escherichia coli K12 D21 Escherichia co/i K12 D21 f2 Escherichia coli K12 D3 I

_ _ _ -

e

D. Other bacteria: Enterobacter Staphylococcus Vibrio

cloacae

fi I2

aureus

extorquens

Mycobacterium Klebsiella

in

Chironomus

:

var. alesti var. dendrolensis

Xenorhabdus

Humoral encapsulation

Pathogenicity for Galleria

Bs 1 I BlZlO marcescens Dbl I

Serratia

larvae

Galleria

Gram +I-

I5G I

Bacillus BaciNw

995

lacticola

sp.

a: Biologische Bundesanstalt Darmstadt, W. Germany; courtesy of Dr A. Krieg. b: INRA, St Christol-les-Al&, France; courtesy of Dr. A. Vey. c: ETH Ziirich, Switzerland; courtesy of Professor Liichty, Dr H. Ebersold. d: Dept Microbiology, University Freiburg, W. Germany; courtesy of Dr Reichenbach. e: Dept Microbiology, University Stockholm, Sweden; courtesy of Professor Boman. f: Institute of Limnology, Konstanz, W. Germany; courtesy of Dr Reichert. g: Inst. Zool., Free University Berlin, W. Germany; isolated by G. Enderlein. Pathogenic effects on larvae of G. mellonella and successful humoral encapsulation haemolymph are indicated by one or several + or - symbols.

Chironomus larvae. They have been isolated from outdoor waterbasins, from Chironomus culture vessels or directly from the haemocoel or gut of dead Chironomus larvae. Some isolations were performed at 18 and 10°C respectively to favour psychrophilic strains which are adapted to lower temperatures. Group (C) represents mutants of E. cofi K12 with defined characteristics such as resistance to certain antibiotics or different lipopolysaccharide coating. Group (D) are various bacteria furnished from microbiological departments to increase the diversity of organisms tested. Preparation of bacterial suspensions Insect Ringer. 7.5 g NaCl; 0.35 g KCl; 0.21 g CaCl,, and 1000 ml distilled water (w/v). 0.02% KCl; Grellets’s solution. 0.09% NaCl; 0.02% MgCl,; 0.1% glucose in distilled water (w/v), adjusted with NaHCO, to pH 6.8. During a first set of experiments, bacteria were collected with a loop from agar plate cultures of varying ages. The bacteria were suspended in insect Ringer solution, counted in a haemocytometer (Neubauer)and diluted to 1 x 10s cells/ml. Chironomus larvae received lo5 bacterial cells per larva by injection of 1~1 of this suspension into the hind proleg. For all further experiments, bacteria were collected from liquid growth cultures at log phase. The bacteria

in

Chironomus

(except the psychrophilic strains) were grown at 36°C on a waterbath shaker with nutrient broth (Standard I, Merck) as culture medium. Bacteria were harvested when the growth culture reached an optical density of 0.6 (Eppendorf photometer at 546 nm), which represents l-5 x 10’ bacterial cells/ml. After harvesting, all bacterial suspensions were cooled on ice to stop further reproduction. The bacteria were centrifuged at 2000 t-pm and resuspended in insect Ringer solution. For establishing the final dilution, the bacterial concentration was calculated using a haemocytometer (Buerker). The actual concentration of the injected suspension was finally checked by viable counts spreading appropriate dilutions of the injection fluid onto nutrient agar plates (Standard I, Merck). Formalin-killed bacteria. Log-phase Xenorhabdus nematophilus (strain Xn21) were incubated in 3% formalin for 3 min, then washed 5 times by centrifugation (2000 rpm) and resuspended in insect Ringer solution. The supernatant of the last wash was injected into a control group of C. tentans larvae to test for toxicity. In vitro encapsulation Humoral encapsulation of bacteria can easily be observed microscopically in isolated haemolymph of Chironomus larvae. For preparation of sterile microchambers 4 strips of Vaseline (using a syringe as

PETER GdTZ

996

TOP

detail the methods used for injection encapsulation.

VIEW

\\ cover slip

Vaseline rim

hemolymph drop

/

/

1 SIDE VIEW 1 Fig. 1. Top and side view of sterile micro-chamber for in vitro observation of humoral encapsulation of bacteria in isolated haemolymph of Chironomus larvae. dispenser) were arranged to form the sides (each about 1.5 cm long) of a square on a sterile glass slide (Fig. 1). For bleeding, Chironomus larvae, which have been kept overnight in sterile tap water containing antibiotics, were dried on sterilized paper tissue. With a fine pair of iridectomy scissors one of the larval hind prolegs was cut off to obtain haemolymph which was placed in the centre of the micro-chamber. If contamination of the haemolymph with gut contents or tissue fragments extruding from the wound occurred, the preparation was discarded. A tiny volume of bacterial suspension was quickly added to the haemolymph (the dilution of the haemolymph should not be higher than 1:2) and a sterile cover slip (22 x 22 mm) was placed over the square of Vaseline strips. Soft pressure was exerted to lower the cover slip until its centre touched the haemolymph droplet. Now the haemolymph formed a column between two horizontal glass planes which offered better conditions for light microscopy than the lens-like form of a hanging or sitting drop (see Fig. 1). Care must be taken that the haemolymph remains in the centre of the micro-chamber without touching the Vaseline rims. The Vaseline frame seals the chamber against desiccation and prevents contamination of the haemolymph from the outside. Haemolymph preparations of this type can be kept for several days offering a convenient way to follow humoral encapsulation under in vitro conditions with phase contrast and high magnifications (400 x ). Injection of bacteria into larvae of Chironomus and Galleria mellonella

and in vivo

Injection of Chironomus Iarvae through hind prolegs. Injections of insect larvae were performed under semi-sterile conditions. Previous to injection, Chironomus larvae (full-grown 4th~larval instar) were kept

\ glass slide

el d

spp.

Chironomus larvae are rather small and delicate to handle. It therefore seems necessary to describe in

for several hours in sterilized tap water with antibiotics (a mixed solution of streptomycin, penicillin and tetracycline). Larvae were fixed on sterilized glass slides with sterilized, moistened paper tissue and placed under a stereo microscope. A loop of nylon thread of hair was prepared around one of the hind prolegs (Fig. 3f). The injection apparatus consisted of a glass micropipette filled with the bacterial suspension and connected via silicone tubing with a syringe. The plunger of the syringe could be advanced with the help of an electric motor operated by foot. The apparatus was adjusted to inject a volume of 1 PI/S which was the usual amount per Chironomus larva. After retracting the injection needle, the wound was first squeezed with a pair of forceps and then closed by tightening the loop (Fig. 3f). Injection of Chironomus larvae through the abdominal cuticle. Using finely drawn capillary tubes (Haematocrit) prepared with a pipette puller (David KopfInstruments, Vertical), injection of Chironomus larvae became possible without the need for subsequent woundsealing. The puncture hole in the integument was closed within a few seconds by blood clotting. To avoid additional stress by exposure to antibiotics, Chironomus larvae (full-grown 4th instar) were only washed several times with clean tap water. Using this injection method, mortality of controls (Chironomus larvae injected with I ~1 of insect Ringer solution) could be lowered to an average of 3% (until day 3 after injection). Larvae bleeding because of wound rupture, which occasionally occurred, were withdrawn from the experiment. An analytical balance was used for calibration of capillary tubes: 2 ~1 injection fluid corresponded to a meniscus displacement of 3 mm. The capillary containing the bacterial suspension was connected to a plastic tube and a syringe both completely filled with paraffin oil. After inserting the tip of the micropipette into one of the anterior segments of the larva (Fig. 2), the plunger of the syringe was carefully moved by hand until the borderline between paraffin oil and bacterial suspension had moved forward over a distance of 3 mm. The capillary was left in the insect larva for several more seconds to make sure that the bacteria were plastic tubing connectedyringe

Fig. 2. Method for injecting Chironomus through dorsal cuticle.

Fig, 3. a-g: Humoral encapsulation of B. rhuringiensis in haemolymph of Chironomus larvae. a<: In vitro encapsulation in isolated haemolymph of C. kntnn~ 5,6 and 10 mitt after incubation of the bacteria. Note progressing deposition of capsular material on the surface of the bacteria (arrow) and-to a lesser extent-on the bottom of the glass slide. d, e: B. thuringiensis (vegetative and sporulated cells). d: before incubation e: after heavy encapsulation (17 h in isolated haemolymph of C. riparius larvae). f, g: In uiuo encapsulation f: Abdominal end of C. riparius showing its hind prolegs, anal papillae and abdominal tubuli. The larvae have been injected through one of the prolegs and the wound was closed afterwards by tightening a ligature made of a nylon thread (arrow). g: Cross section through abdominal region of C. riparius 1 h after injection of B. /huringiensk Aggregations of encapsulated bacteria adhere to body tissues.

997

Fig. 4a-h: Histology and ultrastructure of humoral encapsulation in larvae of C. ripari~s. a: phagocytic tissue (stationary haemocytes) filtering the blood stream in the abdominal end of C. riparius larva. b: Bacteria (B. thuringiensis) entrapped within the phagocytic tissue I min after injection. Humoral encapsulation most often occurs before phagocytosis is achieved. c, d: Humoral encapsulation of P. aeruginosa. C. riparius larvae were prepared for electron microscopy 3 min (c) and 1 h (d) after injection of the bacteria. e, f: B. thuringirnsis 10 min (e) and 2 days (f) after injection. Note the large amount of capsule material around the bacterium deposited within 10 min and the aggregation of several bacteria after a longer period of time within the haemocoel. g: S. marcescens 1 day after injection. h: S. murw.vu’n,c, 10 days after injection four encapsulated bacteria have been ingested by a plasmatocyte. Note evidence for decomposition of the capsule material. 998

999

Immune reactions of Chironomus against bacteria

distributed by the blood stream, as revealed by blending of the different colours of haemolymph and injection fluid. After injection, Chironomus larvae were kept in groups of 20 in small Petri dishes (diameter 9 cm) at 18°C with powdered stingingnettle leaves as food. Chironomus larvae from those experiments where psychrophilic (adapted to low temperature) bacteria had been injected, were stored at IO or 18°C. Injection of Galleria larvae. The injection method for Galleria larvae was similar to the procedure described above (b). However, the cuticle of the larvae was cleaned with ethanol (70%) and the larvae were not placed under a microscope, but were held gently between the fingers during injection. After injection, the larvae were kept at room temperature (22°C) in Petri dishes and supplied with food. Electron bacteria

microscopy

of humoral

encapsulation

of

To analyse the events during humoral encapsulation and to elucidate possible participation of haemocytes, Chironomus larvae were fixed and prepared for electron microscopy at different times after bacterial injection. The times chosen were I, 3, 5, IO and 30min, I, 2, 6 and 13 h, I, 2, 3, 5, 9, 10, 11 and 12 days. For electron microscopy Chironomus larvae were dissected in 6% glutaraldehydre in 0.067 M cacodylate buffer (pH 7.4) at &2”C. Only the 9th abdominal segment (together with the hind prolegs) and the 8th segment (with the tubuli) were used for electron microscopy. These segments were incubated in glutaraldehyde for 90 min, washed in cacodylate buffer for 30 min and postfixed for another 90 min in cacodylate buffered 1% 0~0,. After several washings in cacodylate buffer and dehydration in ethanol and isopropanol (lOO%), the segments were transferred into Epon 8 12 using propylene oxide as intermediate. Ultrathin sections (OM U2 of Reichart-Sitte) were stained with alcoholic uranium acetate and electron micrographs taken with a Zeiss EM 9s.

5 X 10’ cells/ml. For each 8 cm Petri dish, 5 ml of this agar was used. Wells of 3 mm dia were punched into the solidified agar and filled with 4~1 of the haemolymph samples to be tested. If antibacterial substances are present in the haemolymph they will diffuse into the agar and prevent the growth of bacteria in a zone around the wells. As noted by Mohrig and Messner (1968), the diameter of such clear zones is logarithmically proportional to the concentration of antibacterial substances in the samples. For such plate tests, haemolymph was used from untreated larvae and from larvae injected 1, 2 or 4 days before with IO4log-phase Enterobacter cioacae (strain bl2). In several groups of insects E. cloacae proved to be suitable for inducing neosynthesis of peptides with antibacterial activity (Hultmark et al., 1980; Hoffmann et al., 1981). For controls we used haemolymph from Hyalophora cecropia pupae which had been immunized 4 days before bleeding by injecting 106cells of E. cloacae. Such H. cecropia immune haemolymph produces lysis zones with a diameter of 12-I 5 mm on E. coli test plates. RESULTS Humoral encapsulation

of bacteria in vitro

Humoral encapsulation was observed in isolated haemolymph in microchambers with phase-contrast microscopy. Within 2-4 min after introducing bacteria into freshly drawn haemolymph of Chironomus larvae, clouds of fine granules appeared around the bacteria. Then, fast-growing droplets of capsular material were deposited on the surface of the bacteria (Fig. 3a-c). After 5-10 min, most bacteria were completely covered by a thin layer of capsular material which was at first colourless and highly refractive. Within the following hours the capsule increased in thickness and changed to a transparent brown colour (or orange, under phase-contrast conditions) [Fig. 3d, e]. Groups of bacteria lying close together were frequently included in one common batch of capsular material (see Fig. 4f, g). Encapsulation after a primary injection of bacteria Such in vitro encapsulation has been observed in all (Immunization) strains of bacteria (Table 1). Differences in intensity We wanted to determine whether preinjection or speed of encapsulation according to the type of influences the efficiency of humoral encapsulation. bacteria (Gram-positive, Gram-negative, pathogenic For the first injection, 10410g-phase cells of E. cofi or nonpathogenic strains) have not been found. How(strain K12 D21) were used for each 4th-instar larva ever, after introducing high numbers of bacteria, in of C. tentans. Challenge injections with IO6log-phase vitro encapsulation was incomplete and some of the cells of D21 were given on days I, 2 and 3 after the bacteria multiplied causing heavy bacterial growth in first injection. Mortality of the different larval groups the haemolymph drop. In isolated haemolymph, deposition of capsular material is not restricted to was determined on the third day after the challenge injection; a control group which had only received the bacterial surfaces only. Other foreign substances, first injection was observed for mortality on day 6 such as glass surfaces or the contact zones of haeafter the first injection. molymph with air will also provoke the formation of a granular deposition. This reaction is not as heavy Control for normal and inducible antibacterial activity as the reaction against bacteria (or fungi, nematodes in Chironomus haemolymph; E. Coli plate test and certain foreign materials introduced), but weakA plate test with E. coli D31 as test organism was ens the defensive power and leads, within about used to check Chironomus haemolymph for normal 30 min, to a complete exhaustion of the encapsulation or inducible antibacterial activity. A suspension was capacity of isolated haemolymph. Such haemolymph prepared of 1% bacteriological agar (Oxoid Nr. 1, is no longer capable of encapsulating newly introDifco) in nutrient broth (Standard 1, Merck). After duced bacteria. the agar had been autoclaved and had cooled to 47°C the following additions were made: streptomycin to Humoral encapsulation of bacteria in vivo and log-phase cells of D31 to The fate of bacteria after injection into Chironomus 100 PI/ml

1000

PETER

GijTZ

larvae was also followed in uiuo. The last abdominal segments of Chironomus larvae and their appendages are free from fat body and therefore translucent enough for microscopic observation (Fig. 3f). Immediately after injection, the bacteria could be seen circulating within the blood stream. Minutes later, they became attached to tissues and membranes bordering the haemocoel (Fig. 3 g). Many bacteria adhered to the epidermis and to the network of cells of the phagocytic tissue (Lange, 1932) in the abdominal segments 8 and 9 (Fig. 4a, b). AS during in vitro encapsulation, deposition of capsule material around the bacteria progressed rapidly but was even more efficient than under in vitro conditions (Fig. 3g). Ultrastructure of humoral encapsulation of bacteria

The events connected with humoral encapsulation are documented by electron micrographs (Fig. 4c-h). Granular electron-dense material was already observed within 1 min after injecting bacteria and attachment of capsular material was noted 3-5 min after injection. At 10 min after injection, the majority of bacteria was completely enclosed and the capsule, at this time, presented granular and fibrillar structures of medium electron density (Fig. 4e). Besides heavily encapsulated bacteria, we discovered a few which were not encapsulated. We do not know whether these were dead bacterial cells which so far evoked no or only weak defence reactions. Thirteen hours after injection, all bacteria were encapsulated. No evidence was found that blood cells participated directly in the formation of capsules. The early deposits around bacteria were free from blood cells or recognizable blood-cell debris (Fig. 4e). Phagocytosis

Phagocytosis of bacteria was not observed during the first minutes or hours after injection. Only during the following days we found an increasing number of blood cells containing encapsulated bacteria (Fig. 4h). This observation affirms that encapsulation is the primary defence reaction which precedes phagocytosis. However, phagocytosis may still be important to clear the haemocoel of encapsulated bacte-

et 01.

ria. Some evidence for decomposition material within the blood cells (Fig. 4h). Mortality of Chironomus bacteria

of the capsular was observed

larvae after injections of

Mortality of injected larvae of Chironomus tentans and Galleria mellonella was counted until day 3 after injection. To evaluate their resistance to different bacterial strains, a period of 3 days has been chosen to allow even slow-growing bacteria to multiply. Periods of more than 3 days proved to be less suitable for measuring bacterial susceptibility since other adverse factors such as culture conditions and pupation lead to a general increase in mortality among both the test animals and the controls. We can see that the effect of high doses of injected bacteria (1 x 106,4 x 106) compared to lower doses and controls was most obvious around day 3 after injection (Fig. 5). The results of injection experiments with larvae of Chironomus and Galleria are represented in Table 2. Mortality of Chironomus larvae after injection of up to 1 x 10’ bacteria was between 0% and 20% (6.3% on the average). With injection doses between 2 x lo6 and 1 x 10’ bacteria the mortality of Chironomus larvae varied between 5 and lOO%, and an average of 56% of them died until day 3. Control larvae, which have been sham injected with insect Ringer solution or nutrient broth showed an average mortality of 2.6% (until day 3 after injection) with a maximal mortality of 5%. Following the definition of Bucher (1960), none of the bacterial strains injected was pathogenic for the Chironomus larvae used. Increased mortality of Chironomus larvae was caused only by more than 106 bacterial cells per larva and only now, typical insect pathogens such as B. thuringiensis, S. marcescens, P. aeruginosa and X. nematophilus were more effective than nonpathogenic strains. Mortality of Galleria larvae after injection of bacteria

In Galleria, the effect of bacterial injections was significantly correlated with the type of bacteria used (Table 2). Those bacteria which are known to be 0

4.106

0 1.106

8 4.102 A 1’104

Time

Fig. 5. Mortality (in %) of

C. tentans

(days)

after

.

untreated

l

medium injected

injection

larvae after injection of various doses of B. thuringiensis903. The

bacterial suspensions were prepared from growth cultures at log phase and injections performed with finely drawn capillary tubes through the dorsal cuticle of one of the anterior segments of the insect larvae. Injectiondose per lamue: 4 x 102, 10’. 106 log-phase cells of B. thurtngtensts.Controls: untreated larvae and larvae injected with growth culture medium (Standard I, Merck) without bacteria. Group size per injection: 20 fourth-instar larvae of C. tentmu.

Immune reactions of Chironomusagainst bacteria

1001

Table 2. Mortality (in %) of C. tentans and G. melonella larvae until day 3 after injection of different doses (IO’-IO6 loa-ohase cells ner larva) of various bacterial strains C. tentans

Bacterial strain

102

(A) Insect pathogenic bacteria 8. thuringiensis B. thuringiensir 903 S. marcescens Dbl I P. aeruginosa OT91 X. nematophilus Xn21

IO

IO’

(B) Bacteria from Chironomus habitats Water tank I Water tank II Outdoor pool From dead C. wttans Psychrophilic isolate 18” Psychrophilic isolate IO”

G. mellonella

IO’

10s

>I06

IO 0

20

0 5

0 0 5

80 100 85 60 90

IO 0 15 IO 0 5

20 5 10 5 0 5

85 30 55 55 40 5

IO 5 0 15

60 55 IO 40

(C) Laboratory strains E. coli D2l E. coli D2l f2 E. coli D31 E. cloacae B I2

IO’

102

103

80

80

100 loo 100

100 100 IO0

104

IO’

> 106

80

100

100 100

60 0 100 0 0 0

IO0 0 100 60 0 0

0 0 0 0

0 0 0 70

100 100

20 0 100 0 0 0

Group size per injection: 20 larvae of C. tetttons, 5 larvae of G. mellonella. The average weight of fullgrown Chironomw larvae is 25 mg, that of Galleria larvae 250 mg. Controls: Sham injections with Ringer solution or nutrient broth produced an average mortality of 2.6% (until day 3 after injection).

pathogenic to many insects (group A) had a strong effect on Galferiu larvae killing 80-100% of their hosts within 3 days even after injecting such low doses as 10’ (X. ptematophilus) or lo2 (B. thuringiensis, S. marcescens, P. aeruginosa). Among the bacteria which were isolated from Chironomus habitats (group B) two strains were pathogenic for Galleria (between 20 and 100% mortality after injection of 2 x lo4 bacterial cells). Strains of E. coli (group C) did not induce mortality even at doses of up to lo6 cells per Gaileria larva (5th instar).

relation between injection dose and resulting mortality and in most cases, the data available were not homogeneously distributed over the range of O-100%. Therefore, the LD,, values presented in Table 3 cannot be considered as sound figures, but they are suited to illustrate the different effects of humoral encapsulation in C. tentuns and cellular defence in G. mellonella. All estimated LD, values for C. tentuns are higher than 5 x lo’, but range from 10’ to 10’ for G. mellonella, depending upon the type of bacteria injected.

Estimation of LD,,

Injection of dead bacteria

The experiments summarized in Table 2 allow a rough estimation of the LD,, vaiues which represent here the dose of bacteria causing within 3 days the death of 50% of the injected larvae. For some bacteria, we had only limited observations for the

The death of Chironomus larvae after injecting high doses of bacteria (e.g. 10’ per larva) might be caused partly by the mechanical burden resulting from so many encapsulated particles which obstruct the circulatory system or overload the phagocytic cells.

Table 3. Rough estimates of bacterial doses causing 50% mortality (“LD,“) within 3 days after injection into larvae of C. remans and G. mellonella LD, for c. tentans

Bacterial strain Iniected (A) Insect pathogenic bacteria X. nematophilus Xn2l

LD, for G. mellonella

6x 6x 6x 2x 2x

10’ IO’ IO’ 106 I06

1 x IO’

(B) Bacteria isolated from Chironomus habitats Water tank I Water tank II Outdoor pool From dead C. tentans Psychrophilic isolate 18” Psychrophilic isolate 10”

Ix 3x 2x 9x 3x 4x

106 106 106 10s 106 106

Ix Ix 5x 8x Ix 5x

(C) Laboratory strains E. cloacae 8 I2 E. coli D21 t2 E roli -. __..D21 --. E. coli D31

3x 4x 5x 7x

I06 106 106 I+

5x 106

B. thuringiensis B. thuringiensis

903

S. marcescens

Dbl I

P. aeruginosa

OT97

I x 102 I x I02 I x 10’

10s IO’ IO’ 10s IO’ IO’

1 x IO’ I x IO’

1x 106

%TER

1002

GiiTZ

et al.

Table 4. Mortality (in %) of C. lenfuns larvae until day 3 after injection living or formalin killed cells of X. nemo~hilus Injection dose (number of bacteria injected)

X. nematophiius (living cells)

IO’

Number of c. fenInns larvae tested --R = 20

X. nemamphilus (formalin fixed)

IO’

n = 20

25

0

n = 20

5

Material Injected

Suaernatant (control)

of IO’

Mortality (in %) until day 3 post Injection 100

The control group of Chironomus larvae was injected with an equal volume the last supernatant of the formalin treated bacteria.

On the other hand, too many bacteria could in fact exhaust the encapsulation capacity and enable unencapsulated bacteria to grow. To obtain information about the impairment resulting from high numbers of encapsulated particles we compared the mortality after injecting living and formalin-killed X. nematophifus (Table 4). Injection of 10’ living bacteria caused 100% mortality, the same number of dead bacteria 25%, whereas mortality after sham injections with saline solution was only 5%. We conclude from these results that the burden resulting from high numbers of particles affects the host and increases mortality; it becomes totally fatal if pathogenic bacteria are involved. Searching for normal and inducible antibacterial activ ity in the haemolymph of C. tentans using plate assays

Haemolymph of untreated larvae of C. tentans did not significantly inhibit the growth of the test bacterium (Table 5). Small inhibition zones (up to 4.5 mm dia, which means a 0.75 mm zone of reduced bacterial growth around the 3mm well) were obtained with haemolymph of immunized C. tentans larvae. Compared to inhibition zones of 1l-l 3 mm for Cecropia immune haemolymph, this indicates an extremely low level of antibacterial activity in “immunized” Chironomus larvae. Table 5. Antibacterial activity (expressed as diameter of inhibition zones on E. co/i D31 agar plates) of haemolymph from C. wnfan~ and H. cecropia Diameter Time after injection Day Day Day Day

I 2 3 4

(mm) of inhibition

Normal c. lentans

Immunized c. lenIans

3* (n 3’ (n 3.5 (n 3.5 (n

4.5 4.5 4.0 4.5

= = = =

2) 2) 3) 3)

(n (n (n (n

= = = =

6) 5) 4) 5)

zones Immunized H. cecropia

l2(n = 14)

3* = diameter of well; no inhibition zone. For immunization, C. feltfans larvae have been injected with IO4 log phase cells of E. cloacae B12 and haemolymph samples were taken 1,2,3 and 4 days after injection. Pupae of H. cecropia were immunized with IO6 cells of E. cloncae p I2 and haemolymph was taken at day 4 after injection.

DISCUSSION

Humoral encapsulation tions

versus cellular immune reac -

Injection experiments with bacteria of 26 different strains revealed that humoral encapsulation of Chironomus larvae represents an extremely successful de-

--

of

fence reaction against bacterial disease. The bacteria belong to various systematic groups representing 6 different families (Krieg and Holt, 1984). None of the bacterial strains tested caused a significant increase in mortality when up to 10’ cells per Chironomus larva were injected. Only injections of higher doses of bacteria resulted in increased mortality and only then differences between pathogenic and non-pathogenic strains of bacteria became evident. Clearance of Chironomus haemolymph from injected bacteria by humoral encapsulation occurred rapidly. Within minutes after introduction into the haemocoel, the bacteria were covered with a solidifying capsule which was deposited without visible participation of blood cells. The coated bacteria agglutinated and stuck to various surfaces within the haemocoel. The deposition of capsular material continued for several hours until the capsules around the bacteria were several microns thick. Phagocytosis, occurring late and with little frequency, played only a minor role in bacterial defence of Chironomus larvae. Nodule formation was not at all observed and a prominent antibacterial activity, based on the presence or de novo synthesis of substances such as Iysozyme, cecropins and attacins (Hultmark et al., 1983), was not detectable in Chironomus larvae. Completely different results were obtained after injection of the same bacterial strains into Galleria larvae which reacted by cellular defence mechanisms (phagocytosis and nodule formation). Bacterial strains known to be pathogenic for insects caused high mortality of Galleria larvae even if injected in low numbers but nonpathogenic strains were harmless also after injection of high doses. The differences in the results obtained with Chironomus and with Galleria demonstrate that humoral encapsulation is suited to prevent even highly pathogenic bacteria from developing their damaging activity. Humoral encapsulation of bacteria may be more efficient than cellular defence due to the different kinetics of the two types of reactions. In humoral encapsulation, deposition of capsular material occurs within a few minutes after entrance of bacteria into the haemocoel. Phagocytosis and nodule formation, however, are more time-consuming reactions which depend on the contact of bacteria with the appropriate type of blood cells. After contact, attachment, internalization and killing by the phagocytic cells must occur. In the case of nodule formation, degranulation of granular cells and attraction of plasmatocytes forming the multicellular envelopes are

Immune reactions of Chironomus against bacteria involved. It is true that a first reaction of granular cells with injected bacteria happens within minutes (Ratcliffe and Gagen, 1977), but complete phagocytosis or nodule formation takes hours and does not necessarily result in a definite inactivation of the pathogens (Anderson et al., 1977; Vey and Giitz, 1986). Thus, the sequence of events which are necessary for successful cellular defence against bacteria offers ample opportunity for bacterial pathogens to release toxins or inhibitors interfering with the defence system of their hosts. Biochemistry of humoral prophenoloxidase-activating

encapsulation cascade

and

the

Evidence for the melanin nature of the capsular material produced during humoral encapsulation and the involvement of phenoloxidase was first presented on the basis of histochemical tests and inhibition experiments (Poinar and Leutenegger, 1971; Vey and Gijtz, 1975). Investigations of haemolymph from freshwater crayfish Astacus astacus and the silk moth Bombyx mori have shown that activation of prophenoloxidase follows a sophisticated biochemical pathway (Ashida and Siiderhlll, 1984; Siiderhgll and Smith, 1986). This prophenoloxidase-activating cascade is triggered by non-self molecules such as bacterial lipopolysaccharides or /I-1,3-glucans, which are typical constituents of fungal cell walls (Smith and Siiderhill, 1986). Several serine proteases are involved in the activating process, one of them, the proper phenoloxidase-activating enzyme, requires limited proteolysis for its own activation. At least one step of the cascade is Ca*+ dependent. Receptor molecules binding /I - 1,3-glucans and peptidoglycans have recently been identified by Ashida et al. (1986). There is increasing evidence that the role of phenoloxidase in arthropod haemolymph is not restricted to those reactions where visible melanization occurs. The prophenoloxidase-activating system seems to act as a genera1 recognition mechanism and, therefore, is responsible for initiating all type of cellular defence reactions including phagocytosis (SBderhHll, 1982). Recent investigation showed that humoral encapsulation in Chironomus haemolymph is controlled by a similar activating cascade as known from A. astacus and B. mori. Prophenoloxidase in Chironomus haemolymph can be activated by /3-1,3-glucans (zymoSan). Addition of EDTA or serine protease inhibitor (ST1 = soy bean trypsin inhibitor) interrupts activation of Chironomus prophenoloxidase (Harmstorf and G&z, 1986). Humoral encapsulation occurs in two steps, first binding of capsular material (probably consisting of activated phenoloxidase and further proteins) on the challenging surface and second solidifying and tanning of the capsular material by quinone sclerotization. This second step is Ca*+ dependent, consumes tyrosine and can be inhibited by phenylthiourea and only this second process produces the brown colour, electron-dense structure and tough consistency of the definite capsule (Wilke, 1979; Giitz, 1986a, 1986b). Acknowledgements-We

would like to thank Mrs S. Jaweed for excellent assistance and Professor E. L. Cooper for his help in reviewing the manuscript. The work was supported by grant GO 165/10-13 from the Deutsche Forschungsgemeinschaft.

1003

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