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Abundance and distribution of the gut flora of the desert locust, Schistocerca gregaria
JOURNAL
OF INVERTEBRATE
Abundance
PATHOLOGY
38, 378-385 (1981)
and Distribution of the Gut Flora of the Desert Schistocerca gregaria J. HUNT AND A.K. School
of Biological
Sciences,
University
Locust,
CHARNLEY of Bath,
Bath,
BA2 7AY, England
Received October 14, 1980 The abundance of bacteria in the gut of Schistocerca gregaria was determined. A large population of Enterobacteriaceae was found with numbers increasing posteriorly from foregut to rectal sac. In the hindgut, Enterobacter agglomerans was the dominant organism. Streptococci were also present but they were lo- to lOO-fold less numerous than the Enterobacteriaceae. The distribution of the microflora was investigated using light microscopy and scanning electron microscopy. Bacteria in the anterior regions of the gut were restricted to the lumen and inside of the peritrophic membrane. However, in the hindgut, bacteria were also associated with the cuticular lining. KEY WORDS: Schistocerca gregaria; gut flora; bacteria; Enterobacteriaceae; streptococci.
INTRODUCTION
Although locusts have a rich gut bacterial flora (Stevenson, 1966), little is known of the relationship between the microbes and their host. Bacteria have been implicated in the production of the gregarization pheromone in Locusta migrutoria (Nolte, 1977), and recently Strebler (1978) suggested that microbial waste products and toxins may limit carbohydrase activity in the gut of Schistocerca gregaria. However, the evidence for these observations will remain largely circumstantial until the interaction between locusts and their gut flora is studied with germ-free insects. This has been done with a technique for culturing locusts axenically, and the results will form the subject of a further publication. Previous work on the bacterial flora of S. gregaria has been limited to qualitative descriptions of the types present (Stevenson, 1966; Payne and Davidson, 1974). Organisms isolated include Escherichia coli, Enterobacter liquefaciens, Klebsiellu pneumoniue, and Enterobucter cloacue, as well as a number of Gram-positive streptococci (Stevenson, 1966). No cellulolytic bacteria have been found (Payne and Davidson, 1974). This paper is concerned with a preliminary study in which the bacteria were
enumerated determined.
MATERIALS
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
in the gut
AND METHODS
Mature (14 to 21-day-old) adult male S. gregaria were used and the conditions of rearing were similar to those described by Hunter-Jones (1966). Isolation, enumeration, and identification of bacteria. Locusts were kept at 4°C for 10 min prior to handling to prevent regurgitation of gut contents. They were killed by breaking the cervical membrane and thus severing the ventral nerve cord. Animals were surface sterilized in 1% HgCl, in ethanol for 2 min and washed in sterile distilled water. The body cavity was opened by a dorsal longitudinal incision and double ligatures were placed between four regions of the gut (Fig. 1). The different regions were separated by cutting between the ligatures and the volume of each piece determined by displacement in a measuring cylinder containing insect saline (Maddrell and Klunsuwan, 1973). Each region was lightly homogenized in 1 ml of nutrient broth. Ten-fold dilutions were made of each homogenate and inoculated onto one or more of the following media: nutrient agar (Oxoid CM3), all pur378
0022-2011/81/060378-08$01,00/O
and their location
DISTRIBUTION
OF
Schismcerca
pose Tween agar APT (Evans and Niven, 1951), MacConkey agar (Oxoid CM7), or blood agar base (Oxoid CM55) containing 7% defibrinated horse blood (Oxoid SRSO), 1% glucose, and 0.025% sodium azide. The Petri dishes were incubated aerobically for 24 hr at 32”C, the colonies enumerated, and the number of bacterial cells (colony forming units) expressed per milliliter of locust gut region. Representative colonies from nutrient agar were selected on the basis of morphology and recultured. Pure isolates were characterized by the API 20E system (API Laboratory Products, La Balme-lesGrottes, France) for members of the Enterobacteriaceae. Light microscopy. The whole of the alimentary canal was quickly removed and transferred to Schaudinn’s fluid (saturated HgCl, + absolute ethanol, 2: 1, v/v) for 24 hr. The gut was embedded in paraffin wax, serial sections were cut at 5-6 pm, and stained by the Gram Twort method (Ollett, 1947). Scanning electron microscopy (SEM). Whole guts were fixed in Schaudinn’s fluid as above, passed through a graded series of water-acetone solutions to absolute acetone and dried by the critical point method. Dried specimens were split open, mounted on aluminium stubs, coated with gold in a Polaron Argon sputter coater, and examined with a Cambridge S4 Stereoscan SEM. Statistics. Statistical comparisons of data were performed using the sum of squares simultaneous test (Sokal and Rohlf, 1969). TABLE BACTERIAL
Medium
COUNTS
FROM
LOCUST
HINDGUTS
Number of determinations
gregaria
379
GUT FLORA
RESULTS
Density and Distribution
of Gut Bacteria
Table 1 shows bacterial counts from locust hindguts obtained using three media. Large populations of bacteria were found. ca. 10y bacteria/ml, and there were no significant differences between the numbers isolated on nutrient, one-quarter strength nutrient, and MacConkey agars (Table 1). All the isolates from nutrient agar were identified with the Enterobacteriaceae; they were facultative anaerobes (oxidative-fermentative in Hugh and Leifson’s (1953) medium), motile by the hanging drop method, oxidase negative, non-sporeforming, Gram-negative rods of 1 x 0.5 pm (determined by the Gram Twort method of Ollett, 1947). As the numbers and types of organisms recovered on APT medium equaled those on nutrient agar, it was concluded that the nutritionally fastidious Lactobacillus spp. did not occur in large numbers. Bucher and Stephens (1959) showed that bacteria isolated from related species of insects (grasshoppers) on a variety of media all grew on nutrient agar. Therefore nutrient agar was used to determine the numbers of Enterobacteriaceae in various regions of the gut (Figs. 1, 2). A significant increase in the size of the bacterial population occurred posteriorly, in the order foregut < midgut < ileum and colon < rectal sac (Fig. 2. Table 2). However, there was much variation in the numbers of bacteria found, particularly in the ileum and colon where an approximate log 4 1 OBTAINED
BY PLATING
ON THREE
Bacteria/ml (mean x log)
MEDIA
SE (X 1091
MacConkey
4 4
3.34 2.51
NS
1.1 0.45
Nutrient l/4 strength nutrient
5 5
0.77 0.72
NS
0.29 0.25
Nutrient
Note. SE, standard error. NS, no significant difference between means.
380
HUNT
AND
CHARNLEY
TABLE STATISTICAL
FG F
RS
I/C
FIG. 1. The general morphology of the alimentary canal of Schistocerca gregaria. Key: Foregut (F); midgut ceca (MC), anterior lobe (a), posterior lobe (b); peritrophic membrane (PM); midgut (m); ectoperitrophic space (ES); Malpighian tubules (MT); ileum (I); colon (C); rectal sac or rectum (RS); double ligatures tied here (L).
difference existed between the highest and lowest values. This variation was not uniform in the anterior hindgut or rectal sac where bacterial densities followed a skewed distribution. By comparison with the rest of the midgut few bacteria were present in the anterior cecal lobe (182 bacteria/ml) and none were found in the posterior lobe. Isolations on blood agar, containing sodium azide to restrict the growth of the Enterobacteriaceae, revealed a large population of Gram-positive streptococci (Fig. 3). These were not apparent on nutrient, APT, or MacConkey agars where they were out competed by the faster growing Enterobacteriaceae. The numbers of the 10
t. : . ..
. .
: 5.
mean
5.m
L ..
i
i
kN*$Wt
% .3
.
.* .
midput
FG MG I/C RS
RS
X
NS s** s**
X
!3** s**
X
NS
X
2. Streptococci FG MG I/C RS
hS NS NS
X
NS S*
X
NS
X
Note. Bacterial count data from Figs. 2 and 3. FG, foregut; MG, midgut; I/C, ileum/colon; RS, rectal sac. NS, not significant. S*, significant at P < 0.05; S**, significant at P < 0.01.
streptococci tended to increase posteriorly, though the differences between the gut regions were not significant apart from between midgut and rectal sac (Table 2). At the highest densities, in the hindgut, the streptococci were lo- to loo-fold less abundant than the Enterobacteriaceae. The relative status of the Enterobacteriaceae and streptococci populations on grass fed to the locusts was similar to that in the foregut. Isolations using techniques already described revealed average populations of 7.9 +- 2.3 x 106/g of grass for the Entero-
.
z il*“mlcolon
,*clal
*ac tor.~“t
642
I/C
Yi : : i : .
7.6.
7.95
2. The distribution and density of Enterobacteriaceae in the gut determined on nutrient agar plates. Each point represents counts from a different insect. FIG.
MG
COUNTS
1. Enterobacteriaceae
a--
M
2
COMPARISONS OF BACTERIAL IN FOUR REGIONS OF THE GUT
m*sn
5.29
mirlgvt 493
il*“nllCOlo”
,llCtal
6.1,
6.14
*ac
3. The distribution and density of streptococci in the gut determined on blood agar plates. Each point represents counts from a different insect. FIG.
DISTRIBUTION
OF
Schistocerca
CharactPrbation of thr Enterobacteriaceae isolated from the Hindgut
The most abundant bacterial types were characterized. Bacterial cultures (255) isolated on nutrient agar from the hindguts of 50 animals were studied. All isolates were identified with the Enterobacteriaceae (see earlier). They formed colonies within 24 hr at 32°C that were 2-4 mm in diameter, round, and white to cream in color. From the 2.55 bacterial colonies, 79 pure cultures were characterized. Their biochemical characteristics are shown in Table 3. These
H*S
Urea Tryptophane deaminase Indole Vogues-Proskauer Gelatin Acid from: Glucose Mannitol Inositol Sorbitol Rhamnose Sucrose Melibiose Amygdaline Arabinose Oxidase Catalase Identification
3
OF ENTEROBACTERIACEAE
FROM
THE
HINDGUT
Data
_-~~
-
+
+
+
+
+
-
-
-
-
+
-
+
+
+
-
-
-
+
-
+
-(67) -
+ -
+ -
+ -
-
-
-
-
+
-(66) -
+ + -
+ -
+ -
-. t-.
+ + -(W -
+ + + + +
+ + + + +
+ + + + +
+ + +
-(67)
+
+
+
+
+
+ +
+ +
+ +
+ -t
+ (67)
+
+
+
+
+
+
+
.+
+
Enrerabacter agglomerans
No. of isolates
in the Gut
The guts of 50 locusts were examined by light microscopy or SEM. Many different
Characteristic Hydrolysis ONPG Arginine dihydrolase Lysine decarboxylase Omithine decarboxylase Utilization of citrate
381
FLORA
The Location of the Bactrria
TABLE CHARACTERISTICS
GUT
data indicate that the majority of isolates (68) were strains of Entuobacter agglnmerans. In accord with the biogroup designations of Ewing and Fife (1972), 66 of the strains were assigned to anaerogenic biogroup 2 (nitrate positive, indole negative, Voges-Proskauer negative), whereas the other 2 were assigned to biogroup 1 (indole negative, Vogues Proskauer positive). Four other members of the Enterobacteriaceae were also isolated and were identified with Klebsiella oxytoca, Enterobactcjr aerogenes, K. pneumonia, and E. cloacuo.
bacteriaceae and 8.07 + 4.2 X 106/g of grass for the streptococci.
BIOCHEMICAL
gregaria
68
Klebsiella oxytoca 4
E.
E.
K. aerogenes 5
pneumoniae
1
cloacae
1
382
HUNT
AND
morphological types were seen in Gram Twort-stained smears of the foregut and midgut contents. it proved difficult, however, to find bacteria in sections of the lumen of these gut regions. No bacteria were found on either the cuticular lining of the foregut, or on the epithelial linings of the ceca and midgut, using Gram Twortstained sections or SEM. Thus, the contents of the fore and midguts contained a diverse flora but their cuticular and epithelial linings were uncolonized. Cysts of the amoeboid parasite M&ameba locustue were commonly seen in the ectoperitrophic space (Fig. 1). The cephalont stage of the gregarine parasite Gregarina gurnhumi was also prominent both in the ectoperitrophic space of the midgut and on the epithelial lining of the ceca. Both of these parasites are regularly present in laboratory stocks of locusts (Canning, 1956; Henry, 1968; Hunter-Jones, 1966). Microcolonies of rod-shaped bacteria (l-4 x 0.5 pm) were found on the surface of the anterior hindgut cuticle (ileum and colon) (Figs. 4, 5) and on the outside of the peritrophic membrane. Numbers were extremely variable, ranging from a few scattered colonies to many millions of cells. Malumebu cysts were also found alongside the bacteria of the cuticle of the ileum and colon (Fig. 5). Bacteria on the rectal cuticle were similar in morphology and size (2 x 0.4 pm) to those in the ileum and colon. The rod-shaped bacteria were located predominantly in the deep crevices between rectal papillae (Figs. 6, 7) as well as in the ectoperitrophic space. Although both anterior hindgut and rectum contain cuticular spines (Fig. 4), bacteria were not specifically associated with them as they are in cockroaches (Foglesong et al., 1975). Once again it was difficult to find bacteria in sections of the hindgut contents and stained smears were unreliable as indicators of the presence of bacteria in the lumen, due to unavoidable contamination from the hindgut cuticle. However, it is most probable that bacteria were present in the hindgut contents. In conclusion, unlike the foregut
CHARNLEY
and midgut where the bacteria were only found in the lumen, in the hindgut the cuticular lining was also colonized. Many bacteria were found on the outside of fecal pellets confirming the presence of bacteria in the ectoperitrophic space of the hindgut. Cysts of M. locustue were also present on the surface of the fecal pellets from infected animals. In severe infections the cyst population totally obscured any bacteria that might have been present. DISCUSSION
It is apparent that desert locusts have a large microbial gut flora. Organisms able to grow on nutrient agar were more abundant in the hindgut than in the fore or midguts, suggesting that the bacteria were multiplying in the gut. Examination of stained smears of foregut and midgut contents revealed the presence of a diverse bacterial flora, presumably reflecting the heterogeneous nature of the microbial flora on grass (Campbell, 1977; Lynch and Poole, 1979), that constituted the bulk of the locusts’ diet (Hunter-Jones, 1966). The observations of Stevenson (1966) have also shown a diverse flora in the foregut but fewer types in the midgut. Bacteria in the anterior regions of the gut were restricted to the lumen and the inside of the peritrophic membrane, the cuticular lining of the foregut and the epithelium of the midgut being uncolonized. This must preclude the buildup of a stable indigenous bacterial flora in the anterior gut because the food is present there for only a short period of time (total food throughput time is 2 hr, Hunt and Charnley, unpubl.). The hindgut had bacteria intimately associated with the cuticular lining, particularly at the bottom of the folds in the ileum and between adjacent rectal pads. This protected niche, away from the mainstream of the gut, must afford a survival value not available to the microbes in the anterior gut. The SEM study indicated that the bacterial population on the hindgut cuticle was
DISTRIBUTION
OF
Schistocerca
gregario
GUT
FLORA
383
FIG. 4. Scanning electron micrograph of part of the gut wall in the ileum. Note the dense bacterial population made up solely of rod-shaped cells. C, Cysts, of Malamebu hcustae. Scale = 15 pm. FIG. S. Scanning electron micrograph of part of the gut wall in the ileum. Note many rod-shaped bacteria. C, Cysts of Mahmba locusrae; S, small cuticular spines. Scale = 15 pm. FIG. 6. A light micrograph showing a section through two rectal pads. Note bacteria over the surface of the pads and in the crevice between them. B, Bacteria; Cu. cuticle: P. peritrophic membrane with bacteria on the outside; C, cysts of Malameba locustae. Scale = 62.5 pm, FIG. 7. Scanning electron micrograph of adjacent rectal pads showing the opening to the crevice between them. Note many rod-shaped bacteria. R. Rectal pads; Cr. opening of crevice. made up entirely of rod-shaped cells. Isolations confirmed that the majority of bacteria in the hindgut were Gram-negative rods. However. there was also a large Gram-positive coccoid population. Although the latter were IO- to lOO-fold less
abundant than the rods, the difference was insufficient to account for the failure of cocci to appear on scanning electron micrographs of the hindgut. It may be that a significant proportion of the streptococci are in the gut lumen.
384
HUNT
AND
The populations of streptococci and Enterobacteriaceae were of similar size on grass prior to its ingestion, and in the foregut. However, whereas the numbers of streptococci remained relatively constant from foregut to rectum, the numbers of Enterobacteriaceae increased significantly. Part of the reason for this may lie in the ability of the Enterobacteriaceae to colonize the protected niche provided by the cuticle of the hindgut. A number of other factors may also contribute to the large number of bacteria found in the hindgut, in particular a neutral pH (Evans and Payne, 1964) and the presence of organic metabohtes from the Malpighian tubules (Maddrell and Gardiner, 1974). As the peritrophic membrane is known to be an effective barrier to particles the size of bacteria (Schildmacher, 1950; Brandt et al. 1978), the origin of the bacteria in the ectoperitrophic space of the hindgut is not immediately obvious. However, when the food bolus enters the desert locust’s hindgut, enclosed within the peritrophic membrane, it is broken up into discrete pellets by the action of the colon (Goodhue, 1963). Clearly some bacteria may be dislodged during this process and colonize the ectoperitrophic space of the hindgut. Cysts of M. locustae are also found here, carried down in the primary urine from the Malpighian tubules. In addition cysts appear in the ectoperitrophic space of the midgut, due either to antiperistaltic movements of the gut (Baines, 1976) or to being swept forward from the hindgut in an anterior flowing fluid stream within the ectoperitrophic space (Berridge, 1970). As bacteria could also be transported forward, their absence from the ectoperitrophic space of the midgut suggests either that conditions here are unsuitable for bacterial growth or the absence of suitable sites for attachment. Payne and Davidson (1974) reported the presence of an indigenous flora in the midgut ceca. This is not supported by the present study. A few bacteria were found in the anterior lobes but since these lobes are not
CHARNLEY
penetrated by the peritrophic membrane (Goodhue, 1963) their occurrence may be attributed to contamination from the foregut contents during dissection. Only occasionally were bacteria found in the smaller posterior lobes. Enterobacter agglomerans was the most abundant organism found in the hindguts of S. gregaria from our laboratory culture. This is commonly believed to be a plant saprophyte, although it has been isolated from a variety of environmental sources and from men and lower animals (Ewing and Fife, 1972). E. agglomerans is also a prominent member of the gut flora of the wood eating termite Coptotermes formanosanus (Potrikus and Breznak, 1977). The lack of complexity of the hindgut flora of S. gregaria is probably a reflection of the short throughput time and the simple gut structure. Of the five types of Gram-negative bacteria that were isolated from the hindgut, Klebsiella pneumoniae and Enterobacter cloacae were found in locusts examined by Stevenson (1966). However, she also detected Klebsiella oxytoca, Enterobacter aerogenes, Escherichia coli, and Enterobatter liquefaciens. Stevenson (1966) also reported that large populations of cocci were present in Gram-stained smears and considered that many of them were overgrown in culture. This is the case in the present study since Gram-positive cocci were only found when a medium containing sodium azide was used to restrict the growth of the Enterobacteriaceae. The differences between the gut flora of S. gregaria reported here and that found by Stevenson (1966) may be due to variations in the conditions of culture, as it is generally believed that an insect’s gut flora is reflective of its environment (Gilliam and Valentine, 1974; Eutick et al., 1978). ACKNOWLEDGMENTS We would like to thank Dr. R. G. Board for advice and helpful criticisms. The work was supported by an SRC Studentship.
DlSTRlBUTlON
OF
Schisrocerca
REFERENCES D. M. 1976. “Studies on the Structure and Function of the Gut of Locusta migratoria migrarorioides (R. and F.) with Particular Reference to the Midgut and Caeca.” Ph.D. thesis, University of London. BERRIDGE, M. J. 1970. A structural analysis of intestinal absorption. In “Insect Ultrastructure” (A. C. Neville, ed.), pp. 135-151. Blackwell, Oxford. BRANDT, C. R., ADANG, M. J., AND SPENCE, K. D. 1978. The peritrophic membrane: Ultrastructural analysis and function as a mechanical barrier to microbial infection in Orgyia pseudotsugafa. J. Invertebr. Parhol., 32, 12-24. BUCHER. G. E., AND STEPHENS, J. M. 1959. Bacteria of grasshoppers of Western Canada. 1. The Enterobacteriaceae. J. Insect Parhol.. 1, 356-373. CAMPBELL, R. 1977. “Microbial Ecology.” Blackwell, London. CANNING, E. U. 1956. A new eugregarine of locusts. Gregurino garnhami n. sp., parasitic in Schislocercu BAINES,
gregaria.
J. PWt0iOoi.,
3, 50-62.
M. L., O’BRIEN, R. W.. AND SLAYTOR, M. 1978. Bacteria from the gut of Australian termites.
EUTICK,
Appi.
Environ.
Microbid.,
35, 823-828.
EVANS, J. B.. AND NIVEN, C. F. 1951. Nutritionof the heterofermentative lactobaccilli that cause greening of cured meat. J. Bacterial.. 62, 599-603. EVANS, W. A. L.. AND PAYNE. D. W. 1964, Carbohydrases of the alimentary tract of the desert locust Schistocrrcu gregaria. J. Insect Physiol.. 10, 657-674.
EWING. W. H., AND FIFE, M. A. 1972. Enterobacter agglomerans (Beijerinck) comb. nov. (the Herbicola-Lathyri bacteria). Inc. J. Syst. Bacterial.. 22,4-l].
FOGLESONG, M. A., AND MARKOVETZ, phology of some sociated with the teriol.,
WALKER, D. H., PUFFER, J. S., A. J. 1975. Ultrastructural morprocaryotic micro-organisms ashindgut of cockroaches. J. Bac-
123, 336-345.
GILLIAM. M., AND VALENTINE, D. K. 1974. Enterobacteriaceae isolated from foraging honey bees, Apis mellifera. J. Invertebr. Path&., 23, 38-41. Goonuu~, D. 1963. Some differences in the passage of
gregarin
GUT
385
FLORA
food through the intestines of the desert and migratory locusts. Nature (London), 200, 288-289. HENRY, J. E. 1968. Malamoeba locus~ac and its antibiotic control in grasshopper cultures. J. Inverwbr. Pathol.,
11, 224-233.
HUGH, R., AND LEIFSON. E. 1953. The taxonomic significance of fermentative versus oxidative metabolism of carbohydrates by various Gram negative bacteria. J. Bacturiol. 66, 24-26. HUNTER-JONES, P. 1966. “Rearing and Breeding Locusts in the Laboratory.” Centre for Overseas Pest Research Publ., London. LYNCH, J. M.. AND POOLE, N. J. 1979. “Microbial Ecology, A Conceptual Approach.” Blackwell. London. MADDRELL, S. H. P., AND GARDINER, B. 0. C. 1974. The passive permeability of insect Malpighian tubules to organic solutes. J. E+p. Biol.. 60, 641-652. MADDRELL, S. H. P., ANDKLUNSUWAN, S. 1973. Fluid secretion by in vitro preparations of the Malpighian tubules of the desert locust Schistocerru gregariu. J. Insect
Ph.vsiol..
19,
1369-
1376.
NOLTE, D. S. 1977. The action of locustol. J. fnscc/. Physiol.,
23, 899-903.
OLLETT, W. S. 1947. A method for staining both Gram-positive and Gram-negative bacteria in sections. J. Pathol. Bacterial., 59, 357-358. PAYNE, D. W., AND DAVIDSON, L. M. 1974. Cellulose digestion in the locust, Schisrocerca gregaricr. J. Entomol.
Ser. A. 48, 213-215.
POTRIKUS. C. J., AND BREZNAK, J. A. 1977. Nitrogen fixing Enrerubacfer agglomerans isolated from guts of wood-eating termites. Appl. Environ. Microbid.. 33,392-399.
SCHILDMACHER, H. 1950. Darmkanal und Verdauung bei Stechmticken Larven. Bid. Zentralbl.. 69, 390-438.
SOKAL. R. R., AND ROHLF, F. J. 1969. “Biometry, Principles and Practices of Statistics in Biological Research.” Freeman, London. STEVENSON, J. P. 1966. The normal bacterial flora of laboratory stocks of the desert locust, Schistocerca gregario Forskal. J. Inverfebr. Pathol., 8, 205-211. STREBLER, G. 1978. Incidence du jenne et de la presence de microorganismes dans le tube digestif sur I’activite osidasique digestive de Schistocerca gregoricc Forskal. Acriciu, 8. 35-46.
OF INVERTEBRATE
Abundance
PATHOLOGY
38, 378-385 (1981)
and Distribution of the Gut Flora of the Desert Schistocerca gregaria J. HUNT AND A.K. School
of Biological
Sciences,
University
Locust,
CHARNLEY of Bath,
Bath,
BA2 7AY, England
Received October 14, 1980 The abundance of bacteria in the gut of Schistocerca gregaria was determined. A large population of Enterobacteriaceae was found with numbers increasing posteriorly from foregut to rectal sac. In the hindgut, Enterobacter agglomerans was the dominant organism. Streptococci were also present but they were lo- to lOO-fold less numerous than the Enterobacteriaceae. The distribution of the microflora was investigated using light microscopy and scanning electron microscopy. Bacteria in the anterior regions of the gut were restricted to the lumen and inside of the peritrophic membrane. However, in the hindgut, bacteria were also associated with the cuticular lining. KEY WORDS: Schistocerca gregaria; gut flora; bacteria; Enterobacteriaceae; streptococci.
INTRODUCTION
Although locusts have a rich gut bacterial flora (Stevenson, 1966), little is known of the relationship between the microbes and their host. Bacteria have been implicated in the production of the gregarization pheromone in Locusta migrutoria (Nolte, 1977), and recently Strebler (1978) suggested that microbial waste products and toxins may limit carbohydrase activity in the gut of Schistocerca gregaria. However, the evidence for these observations will remain largely circumstantial until the interaction between locusts and their gut flora is studied with germ-free insects. This has been done with a technique for culturing locusts axenically, and the results will form the subject of a further publication. Previous work on the bacterial flora of S. gregaria has been limited to qualitative descriptions of the types present (Stevenson, 1966; Payne and Davidson, 1974). Organisms isolated include Escherichia coli, Enterobacter liquefaciens, Klebsiellu pneumoniue, and Enterobucter cloacue, as well as a number of Gram-positive streptococci (Stevenson, 1966). No cellulolytic bacteria have been found (Payne and Davidson, 1974). This paper is concerned with a preliminary study in which the bacteria were
enumerated determined.
MATERIALS
Copyright 0 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.
in the gut
AND METHODS
Mature (14 to 21-day-old) adult male S. gregaria were used and the conditions of rearing were similar to those described by Hunter-Jones (1966). Isolation, enumeration, and identification of bacteria. Locusts were kept at 4°C for 10 min prior to handling to prevent regurgitation of gut contents. They were killed by breaking the cervical membrane and thus severing the ventral nerve cord. Animals were surface sterilized in 1% HgCl, in ethanol for 2 min and washed in sterile distilled water. The body cavity was opened by a dorsal longitudinal incision and double ligatures were placed between four regions of the gut (Fig. 1). The different regions were separated by cutting between the ligatures and the volume of each piece determined by displacement in a measuring cylinder containing insect saline (Maddrell and Klunsuwan, 1973). Each region was lightly homogenized in 1 ml of nutrient broth. Ten-fold dilutions were made of each homogenate and inoculated onto one or more of the following media: nutrient agar (Oxoid CM3), all pur378
0022-2011/81/060378-08$01,00/O
and their location
DISTRIBUTION
OF
Schismcerca
pose Tween agar APT (Evans and Niven, 1951), MacConkey agar (Oxoid CM7), or blood agar base (Oxoid CM55) containing 7% defibrinated horse blood (Oxoid SRSO), 1% glucose, and 0.025% sodium azide. The Petri dishes were incubated aerobically for 24 hr at 32”C, the colonies enumerated, and the number of bacterial cells (colony forming units) expressed per milliliter of locust gut region. Representative colonies from nutrient agar were selected on the basis of morphology and recultured. Pure isolates were characterized by the API 20E system (API Laboratory Products, La Balme-lesGrottes, France) for members of the Enterobacteriaceae. Light microscopy. The whole of the alimentary canal was quickly removed and transferred to Schaudinn’s fluid (saturated HgCl, + absolute ethanol, 2: 1, v/v) for 24 hr. The gut was embedded in paraffin wax, serial sections were cut at 5-6 pm, and stained by the Gram Twort method (Ollett, 1947). Scanning electron microscopy (SEM). Whole guts were fixed in Schaudinn’s fluid as above, passed through a graded series of water-acetone solutions to absolute acetone and dried by the critical point method. Dried specimens were split open, mounted on aluminium stubs, coated with gold in a Polaron Argon sputter coater, and examined with a Cambridge S4 Stereoscan SEM. Statistics. Statistical comparisons of data were performed using the sum of squares simultaneous test (Sokal and Rohlf, 1969). TABLE BACTERIAL
Medium
COUNTS
FROM
LOCUST
HINDGUTS
Number of determinations
gregaria
379
GUT FLORA
RESULTS
Density and Distribution
of Gut Bacteria
Table 1 shows bacterial counts from locust hindguts obtained using three media. Large populations of bacteria were found. ca. 10y bacteria/ml, and there were no significant differences between the numbers isolated on nutrient, one-quarter strength nutrient, and MacConkey agars (Table 1). All the isolates from nutrient agar were identified with the Enterobacteriaceae; they were facultative anaerobes (oxidative-fermentative in Hugh and Leifson’s (1953) medium), motile by the hanging drop method, oxidase negative, non-sporeforming, Gram-negative rods of 1 x 0.5 pm (determined by the Gram Twort method of Ollett, 1947). As the numbers and types of organisms recovered on APT medium equaled those on nutrient agar, it was concluded that the nutritionally fastidious Lactobacillus spp. did not occur in large numbers. Bucher and Stephens (1959) showed that bacteria isolated from related species of insects (grasshoppers) on a variety of media all grew on nutrient agar. Therefore nutrient agar was used to determine the numbers of Enterobacteriaceae in various regions of the gut (Figs. 1, 2). A significant increase in the size of the bacterial population occurred posteriorly, in the order foregut < midgut < ileum and colon < rectal sac (Fig. 2. Table 2). However, there was much variation in the numbers of bacteria found, particularly in the ileum and colon where an approximate log 4 1 OBTAINED
BY PLATING
ON THREE
Bacteria/ml (mean x log)
MEDIA
SE (X 1091
MacConkey
4 4
3.34 2.51
NS
1.1 0.45
Nutrient l/4 strength nutrient
5 5
0.77 0.72
NS
0.29 0.25
Nutrient
Note. SE, standard error. NS, no significant difference between means.
380
HUNT
AND
CHARNLEY
TABLE STATISTICAL
FG F
RS
I/C
FIG. 1. The general morphology of the alimentary canal of Schistocerca gregaria. Key: Foregut (F); midgut ceca (MC), anterior lobe (a), posterior lobe (b); peritrophic membrane (PM); midgut (m); ectoperitrophic space (ES); Malpighian tubules (MT); ileum (I); colon (C); rectal sac or rectum (RS); double ligatures tied here (L).
difference existed between the highest and lowest values. This variation was not uniform in the anterior hindgut or rectal sac where bacterial densities followed a skewed distribution. By comparison with the rest of the midgut few bacteria were present in the anterior cecal lobe (182 bacteria/ml) and none were found in the posterior lobe. Isolations on blood agar, containing sodium azide to restrict the growth of the Enterobacteriaceae, revealed a large population of Gram-positive streptococci (Fig. 3). These were not apparent on nutrient, APT, or MacConkey agars where they were out competed by the faster growing Enterobacteriaceae. The numbers of the 10
t. : . ..
. .
: 5.
mean
5.m
L ..
i
i
kN*$Wt
% .3
.
.* .
midput
FG MG I/C RS
RS
X
NS s** s**
X
!3** s**
X
NS
X
2. Streptococci FG MG I/C RS
hS NS NS
X
NS S*
X
NS
X
Note. Bacterial count data from Figs. 2 and 3. FG, foregut; MG, midgut; I/C, ileum/colon; RS, rectal sac. NS, not significant. S*, significant at P < 0.05; S**, significant at P < 0.01.
streptococci tended to increase posteriorly, though the differences between the gut regions were not significant apart from between midgut and rectal sac (Table 2). At the highest densities, in the hindgut, the streptococci were lo- to loo-fold less abundant than the Enterobacteriaceae. The relative status of the Enterobacteriaceae and streptococci populations on grass fed to the locusts was similar to that in the foregut. Isolations using techniques already described revealed average populations of 7.9 +- 2.3 x 106/g of grass for the Entero-
.
z il*“mlcolon
,*clal
*ac tor.~“t
642
I/C
Yi : : i : .
7.6.
7.95
2. The distribution and density of Enterobacteriaceae in the gut determined on nutrient agar plates. Each point represents counts from a different insect. FIG.
MG
COUNTS
1. Enterobacteriaceae
a--
M
2
COMPARISONS OF BACTERIAL IN FOUR REGIONS OF THE GUT
m*sn
5.29
mirlgvt 493
il*“nllCOlo”
,llCtal
6.1,
6.14
*ac
3. The distribution and density of streptococci in the gut determined on blood agar plates. Each point represents counts from a different insect. FIG.
DISTRIBUTION
OF
Schistocerca
CharactPrbation of thr Enterobacteriaceae isolated from the Hindgut
The most abundant bacterial types were characterized. Bacterial cultures (255) isolated on nutrient agar from the hindguts of 50 animals were studied. All isolates were identified with the Enterobacteriaceae (see earlier). They formed colonies within 24 hr at 32°C that were 2-4 mm in diameter, round, and white to cream in color. From the 2.55 bacterial colonies, 79 pure cultures were characterized. Their biochemical characteristics are shown in Table 3. These
H*S
Urea Tryptophane deaminase Indole Vogues-Proskauer Gelatin Acid from: Glucose Mannitol Inositol Sorbitol Rhamnose Sucrose Melibiose Amygdaline Arabinose Oxidase Catalase Identification
3
OF ENTEROBACTERIACEAE
FROM
THE
HINDGUT
Data
_-~~
-
+
+
+
+
+
-
-
-
-
+
-
+
+
+
-
-
-
+
-
+
-(67) -
+ -
+ -
+ -
-
-
-
-
+
-(66) -
+ + -
+ -
+ -
-. t-.
+ + -(W -
+ + + + +
+ + + + +
+ + + + +
+ + +
-(67)
+
+
+
+
+
+ +
+ +
+ +
+ -t
+ (67)
+
+
+
+
+
+
+
.+
+
Enrerabacter agglomerans
No. of isolates
in the Gut
The guts of 50 locusts were examined by light microscopy or SEM. Many different
Characteristic Hydrolysis ONPG Arginine dihydrolase Lysine decarboxylase Omithine decarboxylase Utilization of citrate
381
FLORA
The Location of the Bactrria
TABLE CHARACTERISTICS
GUT
data indicate that the majority of isolates (68) were strains of Entuobacter agglnmerans. In accord with the biogroup designations of Ewing and Fife (1972), 66 of the strains were assigned to anaerogenic biogroup 2 (nitrate positive, indole negative, Voges-Proskauer negative), whereas the other 2 were assigned to biogroup 1 (indole negative, Vogues Proskauer positive). Four other members of the Enterobacteriaceae were also isolated and were identified with Klebsiella oxytoca, Enterobactcjr aerogenes, K. pneumonia, and E. cloacuo.
bacteriaceae and 8.07 + 4.2 X 106/g of grass for the streptococci.
BIOCHEMICAL
gregaria
68
Klebsiella oxytoca 4
E.
E.
K. aerogenes 5
pneumoniae
1
cloacae
1
382
HUNT
AND
morphological types were seen in Gram Twort-stained smears of the foregut and midgut contents. it proved difficult, however, to find bacteria in sections of the lumen of these gut regions. No bacteria were found on either the cuticular lining of the foregut, or on the epithelial linings of the ceca and midgut, using Gram Twortstained sections or SEM. Thus, the contents of the fore and midguts contained a diverse flora but their cuticular and epithelial linings were uncolonized. Cysts of the amoeboid parasite M&ameba locustue were commonly seen in the ectoperitrophic space (Fig. 1). The cephalont stage of the gregarine parasite Gregarina gurnhumi was also prominent both in the ectoperitrophic space of the midgut and on the epithelial lining of the ceca. Both of these parasites are regularly present in laboratory stocks of locusts (Canning, 1956; Henry, 1968; Hunter-Jones, 1966). Microcolonies of rod-shaped bacteria (l-4 x 0.5 pm) were found on the surface of the anterior hindgut cuticle (ileum and colon) (Figs. 4, 5) and on the outside of the peritrophic membrane. Numbers were extremely variable, ranging from a few scattered colonies to many millions of cells. Malumebu cysts were also found alongside the bacteria of the cuticle of the ileum and colon (Fig. 5). Bacteria on the rectal cuticle were similar in morphology and size (2 x 0.4 pm) to those in the ileum and colon. The rod-shaped bacteria were located predominantly in the deep crevices between rectal papillae (Figs. 6, 7) as well as in the ectoperitrophic space. Although both anterior hindgut and rectum contain cuticular spines (Fig. 4), bacteria were not specifically associated with them as they are in cockroaches (Foglesong et al., 1975). Once again it was difficult to find bacteria in sections of the hindgut contents and stained smears were unreliable as indicators of the presence of bacteria in the lumen, due to unavoidable contamination from the hindgut cuticle. However, it is most probable that bacteria were present in the hindgut contents. In conclusion, unlike the foregut
CHARNLEY
and midgut where the bacteria were only found in the lumen, in the hindgut the cuticular lining was also colonized. Many bacteria were found on the outside of fecal pellets confirming the presence of bacteria in the ectoperitrophic space of the hindgut. Cysts of M. locustue were also present on the surface of the fecal pellets from infected animals. In severe infections the cyst population totally obscured any bacteria that might have been present. DISCUSSION
It is apparent that desert locusts have a large microbial gut flora. Organisms able to grow on nutrient agar were more abundant in the hindgut than in the fore or midguts, suggesting that the bacteria were multiplying in the gut. Examination of stained smears of foregut and midgut contents revealed the presence of a diverse bacterial flora, presumably reflecting the heterogeneous nature of the microbial flora on grass (Campbell, 1977; Lynch and Poole, 1979), that constituted the bulk of the locusts’ diet (Hunter-Jones, 1966). The observations of Stevenson (1966) have also shown a diverse flora in the foregut but fewer types in the midgut. Bacteria in the anterior regions of the gut were restricted to the lumen and the inside of the peritrophic membrane, the cuticular lining of the foregut and the epithelium of the midgut being uncolonized. This must preclude the buildup of a stable indigenous bacterial flora in the anterior gut because the food is present there for only a short period of time (total food throughput time is 2 hr, Hunt and Charnley, unpubl.). The hindgut had bacteria intimately associated with the cuticular lining, particularly at the bottom of the folds in the ileum and between adjacent rectal pads. This protected niche, away from the mainstream of the gut, must afford a survival value not available to the microbes in the anterior gut. The SEM study indicated that the bacterial population on the hindgut cuticle was
DISTRIBUTION
OF
Schistocerca
gregario
GUT
FLORA
383
FIG. 4. Scanning electron micrograph of part of the gut wall in the ileum. Note the dense bacterial population made up solely of rod-shaped cells. C, Cysts, of Malamebu hcustae. Scale = 15 pm. FIG. S. Scanning electron micrograph of part of the gut wall in the ileum. Note many rod-shaped bacteria. C, Cysts of Mahmba locusrae; S, small cuticular spines. Scale = 15 pm. FIG. 6. A light micrograph showing a section through two rectal pads. Note bacteria over the surface of the pads and in the crevice between them. B, Bacteria; Cu. cuticle: P. peritrophic membrane with bacteria on the outside; C, cysts of Malameba locustae. Scale = 62.5 pm, FIG. 7. Scanning electron micrograph of adjacent rectal pads showing the opening to the crevice between them. Note many rod-shaped bacteria. R. Rectal pads; Cr. opening of crevice. made up entirely of rod-shaped cells. Isolations confirmed that the majority of bacteria in the hindgut were Gram-negative rods. However. there was also a large Gram-positive coccoid population. Although the latter were IO- to lOO-fold less
abundant than the rods, the difference was insufficient to account for the failure of cocci to appear on scanning electron micrographs of the hindgut. It may be that a significant proportion of the streptococci are in the gut lumen.
384
HUNT
AND
The populations of streptococci and Enterobacteriaceae were of similar size on grass prior to its ingestion, and in the foregut. However, whereas the numbers of streptococci remained relatively constant from foregut to rectum, the numbers of Enterobacteriaceae increased significantly. Part of the reason for this may lie in the ability of the Enterobacteriaceae to colonize the protected niche provided by the cuticle of the hindgut. A number of other factors may also contribute to the large number of bacteria found in the hindgut, in particular a neutral pH (Evans and Payne, 1964) and the presence of organic metabohtes from the Malpighian tubules (Maddrell and Gardiner, 1974). As the peritrophic membrane is known to be an effective barrier to particles the size of bacteria (Schildmacher, 1950; Brandt et al. 1978), the origin of the bacteria in the ectoperitrophic space of the hindgut is not immediately obvious. However, when the food bolus enters the desert locust’s hindgut, enclosed within the peritrophic membrane, it is broken up into discrete pellets by the action of the colon (Goodhue, 1963). Clearly some bacteria may be dislodged during this process and colonize the ectoperitrophic space of the hindgut. Cysts of M. locustae are also found here, carried down in the primary urine from the Malpighian tubules. In addition cysts appear in the ectoperitrophic space of the midgut, due either to antiperistaltic movements of the gut (Baines, 1976) or to being swept forward from the hindgut in an anterior flowing fluid stream within the ectoperitrophic space (Berridge, 1970). As bacteria could also be transported forward, their absence from the ectoperitrophic space of the midgut suggests either that conditions here are unsuitable for bacterial growth or the absence of suitable sites for attachment. Payne and Davidson (1974) reported the presence of an indigenous flora in the midgut ceca. This is not supported by the present study. A few bacteria were found in the anterior lobes but since these lobes are not
CHARNLEY
penetrated by the peritrophic membrane (Goodhue, 1963) their occurrence may be attributed to contamination from the foregut contents during dissection. Only occasionally were bacteria found in the smaller posterior lobes. Enterobacter agglomerans was the most abundant organism found in the hindguts of S. gregaria from our laboratory culture. This is commonly believed to be a plant saprophyte, although it has been isolated from a variety of environmental sources and from men and lower animals (Ewing and Fife, 1972). E. agglomerans is also a prominent member of the gut flora of the wood eating termite Coptotermes formanosanus (Potrikus and Breznak, 1977). The lack of complexity of the hindgut flora of S. gregaria is probably a reflection of the short throughput time and the simple gut structure. Of the five types of Gram-negative bacteria that were isolated from the hindgut, Klebsiella pneumoniae and Enterobacter cloacae were found in locusts examined by Stevenson (1966). However, she also detected Klebsiella oxytoca, Enterobacter aerogenes, Escherichia coli, and Enterobatter liquefaciens. Stevenson (1966) also reported that large populations of cocci were present in Gram-stained smears and considered that many of them were overgrown in culture. This is the case in the present study since Gram-positive cocci were only found when a medium containing sodium azide was used to restrict the growth of the Enterobacteriaceae. The differences between the gut flora of S. gregaria reported here and that found by Stevenson (1966) may be due to variations in the conditions of culture, as it is generally believed that an insect’s gut flora is reflective of its environment (Gilliam and Valentine, 1974; Eutick et al., 1978). ACKNOWLEDGMENTS We would like to thank Dr. R. G. Board for advice and helpful criticisms. The work was supported by an SRC Studentship.
DlSTRlBUTlON
OF
Schisrocerca
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EWING. W. H., AND FIFE, M. A. 1972. Enterobacter agglomerans (Beijerinck) comb. nov. (the Herbicola-Lathyri bacteria). Inc. J. Syst. Bacterial.. 22,4-l].
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GILLIAM. M., AND VALENTINE, D. K. 1974. Enterobacteriaceae isolated from foraging honey bees, Apis mellifera. J. Invertebr. Path&., 23, 38-41. Goonuu~, D. 1963. Some differences in the passage of
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SOKAL. R. R., AND ROHLF, F. J. 1969. “Biometry, Principles and Practices of Statistics in Biological Research.” Freeman, London. STEVENSON, J. P. 1966. The normal bacterial flora of laboratory stocks of the desert locust, Schistocerca gregario Forskal. J. Inverfebr. Pathol., 8, 205-211. STREBLER, G. 1978. Incidence du jenne et de la presence de microorganismes dans le tube digestif sur I’activite osidasique digestive de Schistocerca gregoricc Forskal. Acriciu, 8. 35-46.