Effects of milky disease on hemolymph coagulation and on the number of hemocytes in infected larvae of the Japanese beetle

JOURNAL

OF INVERTEBRATE

PATHOLOGY

12, 288-293

( 1968)

Effects of Milky Disease on Hemdymph Coagulation and on the Number of Hemocytes in Infected Larvae of the Japanese BedIe P. H. SCHWARTZ AND B. G. TOWNSHEND Entomology Research Division, U. S. Department of Agrkulture,

Agricultural Research Service, Moorestown, New Jersey 08057

Received April 22, 1968 Daily

counts

of hemocytes in diseased and healthy larvae of the Japanese beetle, showed a 1.4-fold increase in hemocytes in diseased larvae after 98-day degrees. The average counts/mm3 for infected (24,988) and healthy (25,044) larvae were not significantly different for the test period of Ml-day degrees. Ability of the hemolymph to coagulate was not significantly altered in diseased larvae. The average weight of all larvae was 211+4 mg (standard error with 160 observations), and it did not change significantly during the test.

P~piUia japonica,

MATERIALS

Type A milky disease is a bacterial infection caused by Bacillus popilliue. Signs and symptoms appear when the causal organism is present in such large numbers that the hemolymph becomes turbid. Then turbidity advances with sporulation, the dorsal blood vessel is obscured, and the lower parts of the body become chalky white (Dutky, 1940). The disease has been recognized as a biological control agent for the Japanese beetle, Popilliu japonica, for more than 30 years (Ladd and McCabe, 1967). Attempts to produce spores of milky disease on a synthetic medium have been successful, but these spores have not been infectious by ingestion. This failure has been investigated by studying larval and bacterial factors, but the contribution of the hemocytes has not generally been considered even through Beard (1945) reported that the total hemocyte count of infected larvae increased nearly twofold when the Bacillus was in the sporulation stage. The present study was made to investigate further the contribution of the hemocytes and hemolymph to the development of B. popilliae. 288

AND METHODS

About 250 third-instar larvae collected in the field were placed individually in 95 x 25-mm glass shell vials. Each vial was then filled to within 20 mm of the top with a mixture of 2 parts Michigan peat, 1 part soil (by vol), and redtop-white clover sprouts. (The soil had been air-dried and heated at 93°C for 5 hr before it was mixed with the peat, and the peat-soil mixture was brought to 50% of the “ball point” value of the soil [Dutky, 19421 after the introduction of the seed. ) Then the vials were stoppered and held at 30” + 1°C for 7 days. The rearing medium was replaced each week until the supply of larvae was exhausted. After 7 days of feeding, surviving larvae were divided into two equal groups, one treated with B. popilliue and the other left untreated. Treatment was accomplished by injecting 280,000 spores/larva in 1 ~1 of sterile distilled water into the tergopleural portion of the posterior second or third abdominal terga with a l-ml tuberculin syringe with a 27-gauge needle and a Dutky-Fest microinjector (U. S. patent 2,270,804). The larvae were then heat-fixed for 1 min at 60°C in tap water to prevent coagulation

MILKY

DISEASE

EFFECT3

ON

of the hemolymph. After a leg was snipped off the hemolymph was collected to the I mark in a small Thoma blood diluting pipette (white cell), diluted 1:lO with sterile distilled water, and shaken for 1 min. The first few drops were discarded, and then both sides of a hemacytometer (Spencer Bright Line) were filled. After the hemocytes had settled for 1 min, they were counted at a magnification of 200x by using phase contrast. Eighty ‘/+,-mm _ squares in each chamber were counted for each larva. The degree of coagulation of the hemolymph was determined for the unheated larva by positioning it over four air holes cut in the top of a small plastic box that was connected to a vacuum source. While the larva was held stationary, the tip of a leg was removed, and the stub was immediately inserted into a l-mm diameter capillary tube and held until the column of hemolymph was stationary. The height of the column was recorded to the nearest millimeter. Five larvae from each group were weighed and used immediately in the daily hemocyte or hemolymph determinations for 8 days. RESULTS

AND

DISCUSSION

In a preliminary study, two types of hemocytes were found to predominate in the hemolymph of unfixed larvae. The first type was the spherical cell shown in Fig. 1. It was about 12 p in diameter and was densely granulated; also, the nucleus was obscured or absent in some cells. The second type was oblong, slightly granulated, and contained a distinct nucleus (Fig. 1). This hemocyte too was about I2 X 17 p. A third type that was about 6 p in diameter accounted for a small percentage of the hemocyte count; this cell was devoid of granules but contained a phase dark body along one side of the cell wall (Fig. 1). The remaining cells were composed of fusiform and rapidly disintegrating types

JAPANESE

BEETLE

BLOOD

289

that are not illustrated. The brilliant white objects in Fig. 1 are lipid droplets or fat cells (as determined by Sudan Black B staining techniques) that are apparently circulating in the hemolymph along with the hemocytes. After heat-fixation of the larva, differentiation of the blood cells became more difficult: the relative sizes of the cells remained unchanged, but the fixed cells tended to extrude cytoplasmic inclusions into the surrounding medium (Fig. 2). Observations of the infected and uninfected larvae that were heat-fixed did not indicate any gross changes in the relative proportions of the first two types of cells. However, no fusiform and rapidly disintegrating types were observed, and the proportion of small spherical cells was greatly reduced-to less than lo/o of the total cell count. Because fixation appears to cause significant changes in the differential count in some insects and can greatly alter the appearance of certain hemocytes (Jones, 1962), our observations ‘were confined to total hemocytes counts, During infection by milky disease, the appearance of the hemocytes remained unchanged (Fig. 3)) and we did not observe bacterial phagocytosis by the hemocytes. In a few larvae, the Bacillus rods were observed in great numbers around the hemocytes (Fig. 4), but not all the cells were affected though some were completely surrounded by the rods (Fig. 4). This massing of the rods around the hemocytes did not appear to be an invasion of the cells by B. popilliae and did not result in visual damage to the hemocytes. During the 8 days after infection, the average total hemocyte count/mm” was 24,988 for infected larvae and 25,044 for uninfected larvae. The average count for both groups was 25,016 -+ 914, standard error (SE) for 160 observations. Although differences between the two groups were not significant at the 5% level of confidence, the differences at specific times were signifi-

290

FIG. FIG.

SCHWARTZ

1. 2.

Hemocytes Hemocytes

from from

an unfixed a heat-fixed

larva larva

AND

of of

cant. Beard (1945) obtained a total cell count of 26,296 + 2655 for uninfected larvae which agrees with our figure within 1 sI3. The ! daily counts are presented in Table 1 with (lay degrees used in lieu of days be-

TOWNSHEND

the Japanese the Japanese

beetle. beetle.

450 X . 450 X .

cause the course of the disease is affc:cted by temperature. Dutky (1963) expr day degrees in his equation (1) as time (t-60) = K, where K is day degrees : and t-66 is the number of Fahrenheit de1grees above the lower limit of development. The

MILKY

FIG. 3. Hemocytes from FIG. 4. Clusters of rods Japanese beetle. 1000 X .

DISEASE

EFFECTS

ON

JAPANESE

BEETLE

a larva of the Japanese beetle infected with of Bacillus popilliae around the hemocytes

greatest differences in cell counts occurred after 98 day degrees (Table 1). At this developmental stage, the hemocyte count for infected larvae increased LCfold beyond that of the untreated larvae. After

BLOOD

milky disease. of a diseased

450X. larva of

the

98 day degrees, the hemocyte count dropped back to the level at which it had remained during the period of 651 day degrees. Eighty percent of the larvae examined during the 98 day degree contained rods. How-

292

SCHWARTZ

AND

TABLE EFFECTS

4 25 51 72 98 120 141 161

1

OF M-Y DISEASE ON THE HEMOCYTES AND HEMOLYMPH COAGULATION OF LARVAE OF THE JAPANESE BEETLE Avg no. hemocytes/ mm3 (X1000)

Avg Day degrees after spore injection

TOWNSHEND

weight larvae (md 200 196 207 207 227 220 210 223

of Diseased larvae 20 24 20 31 42 24 18 21

ever, although the hemocytes appeared to increase at about the time the rods were abundant in hemolymph, they decreased in density when spore formation was observed at 120 day degees. Beard ( 1945) observed a similar increase in the total hemocyte count ( 47,648/mm3 compared with our figure of 42,000). However, the increase he observed occurred during the sporulation stage which occurs on the average at 102 to 168 day degrees ( Dutky, 1963). Beard ( 1945) suggested that the increase he reported might have been caused by an artifact in the counting technique, by a modification of the balance between the tissue fluids, or by affecting in some way the cell-plasma ratio of the blood. The possibility that an artifact did exist in the counting technique in both studies is demonstrated by the increased counts that occurred during two different developmental phases of the Bacillus in the two studies. However, we found no increase in hemocytes in the untreated larvae, which suggests that the increase is caused by the presence of the large numbers of rods in the hemolymph. Jones ( 1962) indicated that the sphemle cells greatly increased after injection of certain bacteria and may play an important,

Avg height of hemolymph ( mm ) Healthy larvae 22 20 29 21 29 26 26 27

Diseased larvae 5 11 11 14 20 14 17 9

Healthy larvae 8 12 15 9 11 11 13 11

if unknown, part in the bacterial immunity of the corn borer, Pyrausta nubialis, and the greater wax moth, Galleriu mellonella. We do not know how much the hemocytes contributed to immunity in our study. Perhaps the increased number of hemocytes was caused by a response to offset the drain of hemolymph nutrients or by an attempt to absorb the toxins created by the large numbers of rods in the hemolymph. Some evidence indicates that the pathogen is using nutrients which may be supplied by the hemocyte. Sylvester and Costilow (1964) demonstrated that B. popilliae required biotin, thiamine, and 11 amino acids for growth, and three of these amino acids (glycine, tyrosine, and histidine) were in lower concentration in diseased larval hemolymph than in uninfected larvae (Shotwell et al., 1963, 1965). (Unfortunately, they did not include the hemocytes in their 1965 study because the hemolymph was centrifuged to remove the Bacillus spores; their work in 1963 was performed on larval hemolymph that probably contained hemocytes-they did not report hemolymph centrifugation.) In view of Jones’ (1962) report that hemocytes in some insects contain tyrosine, the decreased level of tyrosine found in diseased larvae by Shotwell et al.

MILKY

DISEASE

EFFECTS

ON

(1965) could be explained by differences in technique. Then, since Sylvester and Costilow (1964) did not attain any degree of sporulation on synthetic media, the role of hemocytes in contributing either an unknown or an increase in a known nutritional factor bears further study because of their significant increase just before sporulation in our study. The role of the hemocytes in removing toxins created by the Bacillus which could inhibit their subsequent sporulation is also worthy of future study. Some evidence exists that toxins are present in cultures of B. popilliae, and Dutky (1963) demonstrated that cell-free filtrates of cultures of B. popilliae were lethal when small amounts were injected into larvae. Additional but indirect evidence of the toxic substance produced by B. popilliae was demonstrated by Haynes and Rhodes (1966) when they obtained sporulation of B. popilliae by shaking a culture in a tryptone-glucose-yeast extract broth with activated carbon present; the carbon may have removed the toxic substance produced by the pathogen. Hemolymph coagulation in the two groups was not significantly different (Table 1) at the 5% level of significance. The height was 12.6 mm for the diseased group and 11.2 mm for the untreated group. The overall average height was 11.9 % 0.7 mm (SE with 80 observations). The results of this study support those of Beard ( 1945)) who failed to find a significant difference in time for coagulation between healthy and diseased hemolymph. The weights of the infected group and the untreated group were not significantly different (Table 1) and averaged 266 and 216 mg, respectively; the overall average for the study was 211 f 34 mg (SE with 160

JAPANESE

BEETLE

293

BLOOD

observations). Thus the weight of the larvae did not appear to increase. By using covariance analysis, we determined that weight did not influence the determinations previously discussed. REFERENCES BEARD, R. L. 1945. Studies on the milky of Japanese beetle larvae. Connecticut Expt. Sta. Bull. No. 491, pp. 506583. DUTKY, S. R. 1940. Two new spore-forming teria causing milky diseases of Japanese larvae. .I. Agr. Res., 61, 57-68.

disease Agr. bacbeetle

DUTKY, S. R. 1942. Method for the preparation of spore-dust mixtures of Type A milky disease of Japanese beetle larvae for field inoculation. U. S. Dept. Agr. Bur. Entomol. Plant Quart. ET-192. 10 pp. DUTKY, S. R. 1963. Pathology, An Steinhaus, ed.), Press, N. Y.

The milky diseases. Advanced Treatise” Vol. 2, pp 75-115.

In “Insect (E. A. Academic

HAYNES, W. C., AND RHODES, L. J. 1966. Spore formation by Bacillus popilliae in liquid medium containing activated carbon. J. Bacteriol., 91, 2270-2274. JONES, J. C. 1962. insect hemocytes. LADD,

Current Am.

concepts concerning Zool., 2, 209-246.

T. L., JR., AND MCCABE, P. J. 1967. Persistence of spores of Bacillus popilliae, the causal organism of Type A milky disease of Japanese beetle larvae, in New Jersey soil. J. Econ. Entomol., 60, 49-95.

SHOTWELL, 0. L., BENNETT, G. A., HALL, H. H., STUBBLEFIELD, R. D., PETERS, J. E., VAN ETTEN, C. H., AND JACKSON, R. W. 1965. Amino acids in the haemolymph of diseased Popilliu japonica (Newman) larvae. J. Insect Physiol., 11, 671-682. SHOTWELL, 0. L., BENNETT, G. A., HALL, H. H., VAN ETTEN, C. H., AND JACKSON, R. W. 1963. Amino acids in the haemolymph of Popillia japonica (Newman) larvae. J. Insect Physiol., 9, 3542. SYLVESTER, C. Nutritional J. Bacterial.,

R. J., AND COSTILOW, requirements of Bacillus 87, 114-119.

N. 1964. popilliae.