Some Observations on the Distribution of Gut Flora in the American Cockroach, Periplaneta americana

JOURNAL OF INVERTEBRATE PATHOLOGY 29, 338-343 (1977)

Some Observations on the Distribution of Gut Flora in the American Cockroach, Periplaneta americana D. E. BIGNELL1 Department of Biology, York University, Downsview, Ontario, Canada M3J IP3 Received May 2, 1976 Viable bacteria counts, corrected for differences in volume, were made by plating samples from each section of the alimentary canal. The density of organisms capable of growth on nutrient and blood agars increased successively in the foregut, anterior midgut, posterior midgut, and anterior colon. Lower densities occurred in the posterior colon and rectum. It is proposed that the high bacterial density in the anterior colon may be correlated with the complex filling mechanism of this section of the gut.

INTRODUCTION

The gut floras of cockroaches have been the subject of a number of investigations showing the viability of human pathogens within the alimentary canal and the potential of the insects as vectors of disease, e.g., Morrell (1911), Read (1933), Wedburg et al. (1949), Janssen and Wedburg (1952). In addition to pathogens, Bitter and Williams (1949) and others have identified less harmful enteric bacteria from the hindgut of Periplaneta americana, including species of the genera Proteus, Alcaligenes, Pseudomonas, and Escherichia. Despite the wide variety of microorganisms isolated from cockroach guts (for review, see Roth and Willis, 1960), no study has offered quantitative data, nor has any attempt been made to analyze the distribution of the organisms within the alimentary canal. Gier (1947) and House (1949) reared nymphs of P. americana and Blattella germanica under aseptic conditions that prevented the establishment of gut flora. This treatment reduced the growth rates of the insects, suggesting that organisms normally found in the digestive tract supply essential dietary constituents or assist development in some other way. While the contribution I Present address: Department of Zoology, The University of Alberta, Edmonton, Canada, T6G 2E9.

of the gut flora to nutrition remains to be specified, knowledge of the distribution and density of microorganisms in the gut may aid in an assessment of their role. MATERIAL AND METHODS

Adult females of the American cockroach, Periplaneta americana, were taken from stock cultures maintained on Purina brand dog chow. Insects were anesthetized with CO2 and were dissected under sterile Ringer's solution (Griffiths and Tauber, 1940). The gut was partitioned in situ into five sections by means of double ligatures placed between the crop and anterior midgut, the anterior midgut and posterior midgut, the posterior midgut and colon, and the colon and rectum, respectively. Additional single ligatures were placed around the pharynx and between the rectum and anus. For the purposes of this study, the anterior midgut was defined as that part of the midgut from the proventriculus to a point immediately posterior to and including the caeca. The posterior midgut was defined as the remaining portion of the midgut, the Malpighian tubules, and the ileum. With the ligatures ensuring that none of the contents was spilled, each section of the gut was separated and washed in four changes of sterile Ringer's solution. This 338

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COCKROACH GUT FLORA

procedure removed contaminating microorganisms from the hemolymph side of the preparation. Using a glass homogenizer with a Teflon pestle, each gut section was then lightly homogenized in 1 ml of sterile Ringer's solution. Homogenization was necessary to achieve the complete disruption of the preparation, ensuring that the gut contents were released into suspension and did not adhere to the lining cuticle or peritrophic membrane. Serial dilutions were prepared from the homogenates, and aliquots were plated on nutrient, 5% horse blood and Endo agars (Grand Island Biological Company, New York). The plates were incubated for 3 days at 25°C. Additional incubations were carried out anaerobically on nutrient agar containing GIBCO fluid thioglycollate medium (20% by volume). In this case, the plates were incubated in sealed vessels containing a saturated solution of pyrogallol in 4 Y NaOH, for 3 days at 30°C. Bacteria were characterized by colonial size and shape and by cell shape and Gram-staining reaction. To determine bacterial density, it was necessary to estimate the mean volume of each gut section. Thus, the foregut was considered a cone (volume given by ~37r r 2 1), the anterior midgut (without caeca), the caeca, the posterior midgut and the colon were considered as cylinders (volume given by 7r r 2 1), and the rectum was considered as two basally opposed cones (volume given by 2/3 7r r 2 1). Measurements of length and diameter were made on each of the gut sections of 20 insects selected at random from the stock cultures. For sections considered as cylinders, measurements of diameter were made at four evenly spaced points and were averaged. The mean volumes (+ SD) in microliters were: foregut, 90 _+ 54; anterior midgut, 22 _+ 0.3; anterior midgut without caeca, 8 _+ 0.2; posterior midgut, 11_+1; colon, 56_+5; rectum, 6_+0.5. An experimental diet was prepared to examine the movement of food materials in the hindgut. The diet contained casein

protein (21% by weight), dextrin (51%), yeast extract (8%), Hawk-Oser salt mixture (3%), cellulose powder (16%), and trypan blue (1%).

RESULTS

Distribution and Density of Gut Bacteria Bacterial counts obtained by plating on nutrient agar are presented in Figure 1. In mature adults, the largest numbers of organisms were found in the colon, followed in descending numerical order by the posterior midgut, rectum, foregut, and anterior midgut. The greatest individual variations were found in the foregut and the anterior midgut, where the largest populations counted exceeded the smallest by factors of 120 and 990, respectively. The least variation occured in the colon, where the extremes were separated by a factor of 5. In newly molted insects, the bacterial populations were uniformly low in the foregut, anterior midgut, and posterior midgut, approached those of mature adults in the colon, and equaled those of mature adults in the rectum. Figure 1 also shows the same data expressed as bacterial densities. In the posterior midgut, colon, and rectum, similar densities were found (7.0-9.6 x 107 organisms/ml in mature adults). The average bacterial density of the anterior midgut was 6.7 x 106 organisms/ml, a value comparable with that of the foregut (3.6 x 106 organisms/ ml). It is noticeable during dissection that solid food particles are not found in the caeca; therefore, if the caecal volume is excluded from the calculation of bacterial density in the anterior midgut, a revised value of 1.8 x 107 organisms/ml is obtained for mature adults. This is intermediate between the mean densities of the foregut and the posterior midgut. The actual population density within the caeca was not determined, but it is evidently much lower than that of the posterior midgut. This observation lends support to the statement of Forbes (1892, cited by Steinhaus, 1946) that

340

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F]o. 1. The distribution and density of gut bacteria determined on nutrient agar plates. (0) Insects 2 months after adult emergence; (©) insects not more than 1 hr after adult emergence. Each point represents counts from a different insect. The presence of an asterisk denotes the bacterial density of the anterior midgut with caecal volume included in the calculation.

the gastric pouches of cockroaches do not usually contain bacteria. The distribution of bacteria within the colons of mature adults is shown in Figure 2. The anterior colon of each animal contained from three to eight times the number of organisms found in the posterior half. The average bacterial density of the anterior colon was 2.9 × 108 organisms/ml. Additional plating was carried out on blood, Endo, and nutrient-thioglycollate agars (Table 1). The numbers of organisms viable on blood agar were similar to those obtained from nutrient agar. Hemolytic species were not observed. Plating on Endo agar revealed that lactose fermenters were relatively scarce in the foregut, anterior midgut, posterior midgut, and rectum, but more abundant in the colon. Bacteria able

to grow under anaerobic culture conditions were relatively abundant in the foregut but less common in posterior sections of the gut. Plating on potato-dextrose agar revealed the presence of hypha-forming fungi, but, in each gut section, the numbers were less than 1% of the numbers of bacteria obtained with nutrient agar. Yeast colonies were occasionally observed on nutrient and blood agar plates.

Characterization of Colony and Cell Types Twenty distinct colony types were seen on nutrient and blood agars. The most common cell types were Gram- cocobacilli, Gram- rods, and Gram + cocci. Gram + rods, Gram-variable filamentous rods, and Gramcocci occurred infrequently.

C O C K R O A C H GUT F L O R A

341

Examination of Food Movement in the Colon

i COLON

Close examination of the movement of food material from the ileum into the colon showed that, when the muscular valve separating these two sections opened, the diet was carried from a third to half the way along the colon, still enclosed in the peritrophic membrane, before the membrane became dissociated and its contents spilled out (Fig. 3). Thus, the relative age of food material in the colon is not necessarily given by its proximity to the rectum.

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FIG. 2. The distribution of bacteria in the colon of seven insects 2 months after adult emergence, determined with nutrient agar.

Samples of dog chow from stock culture cages were homogenized with sterile Ringer's solution, and aliquots were plated on nutrient agar. The total viable count averaged 1.0 × 106 organisms/g and consisted almost exclusively of Gram- coccobacilli. The colony types closely resembled those formed by the Gram-coccobacilli isolated from the alimentary canal.

Since the bacterial counts obtained by plating are limited by the selectivity of the media employed, the densities quoted in this study are not necessarily estimates of the total count. Nevertheless, the data show that organisms growing on nutrient and blood agars are more abundant in the posterior midgut and hindgut than in the foregut or anterior midgut, and are most abundant in the anterior colon. In addition to the errors introduced by the selectivity of the media, further uncertainty arises from the volume determinations. Davey and Treherne (1963) argue that the crop volume of P. americana remains relatively constant, the space not occupied by food being filled with air. In the present

TABLE1 BACTERIAL COUNTS OBTAINED BY PLATING ON FOUR MEDIA (MATURE ADULTS)

Loglo organisms; mean (range)

Gut section

Number of insects

Foregut Anterior midgut Posterior midgut Colon Rectum

12 11 12 11 6

Nutrient agar a 5.4 5.1 6.0 6.7 5.6

(4.1-6.2) (2.7-5.7) (4.3-6.3) (6.3-7.0) (5.0-5.9)

Blood agar 4.6 4.7 6.5 6.8 5.5

" Graphed in Figure 1. b Where the range includes zero, a mean is not presented. ~ N = Not detected.

(3.7-5.2) (2.4-5.4) (4.3-6.9) (6.2-7.1) (5.0-5.7)

Nutrientthioglycollate agar 5.3 3.6 5.3 5.5 4.7

(4.8-5.4) (3.2-3.9) (3.5-5.8) (4.8-5.8) (3.7-5.1)

Endo agar (lac +) b --5.9 --

(N_4.7)c (N-3.6) (N-5.2) (3.8-6.7) (N-4.4)

342

D.E. BIGNELL

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FIG. 3. Diagrammatic representation of the path taken by an experimental meal on entering the colon from the ileum. AC, anterior colon; IL, ileum; MT, Malpighian tubule; PC, posterior colon; PM, peritrophic membrahe; R, rectum.

study, the mean volume of the foregut, calculated from length and width, showed a large standard deviation. This variation may indicate that the cone formula is inappropriate or may reflect real changes taking place in response to the state of filling or emptying. Changes in the weight and linear dimensions of the colon have been studied by Hominick and Davey (1975). They found that, during a period of imposed starvation following a large meal, the dry weight of the colon increased by more than 25% in the first 3 days, declining thereafter to a constant level. An increase in fluid content also accompanied the passage of food materials through the colon, and, in association with this, the mean widths of the anterior and median and median portions of the colon increased. On the basis of the cylinder formula used in the present study, the changes in linear dimensions observed by Hominick and Davey represent a maximum increase of 60% in the volume of the whole colon during food passage. Since no prescribed regimen of feeding nor any period of starvation was imposed on the insects used in the present study, it is reasonable to assume that the mean colon volume given and, in consequence, the bacterial densities have an associated error of ___30%. In any section of the gut, the bacterial population can be recruited from indigenous organisms and from organisms which enter with the food material. Organisms will be

lost to the population by lysis, predation, and by the displacement of food materials toward the anus (Brock, 1971). In seeking to explain the abundance of organisms in the anterior colon, attention must therefore be given to the pattern of food movement within it. The present study shows that solid food particles entering the colon within the peritrophic envelope are first carried nearly to the midpoint of the section before they are dispersed to fill it. Food materials may therefore be accumulated or retained within the anterior colon while peristalsis continues in the midgut, posterior colon, and rectum. Such a filling mechanism would require anti-peristaltic contractions of the hindgut, which have been observed in the colon of Leucophaea maderae (Cook and Reinecke, 1973). It should be added that the secretions of the Malpighian tubules contribute to the fluid phases of the colon contents. Maddrell and Gardiner (1974) have shown that these secretions may contain organic molecules from the hemolymph, which are presumably available as substrates to the hindgut bacteria.

ACKNOWLEDGMENTS I wish to thank Dr. E. Nestmann for his critical review of the manuscript. The work was funded by the National Research Council of Canada.

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organisms from the American cockroach. J. Infect. Dis., 85, 87-90.

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