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Biochemistry of mosquito infection: Preliminary studies of biochemical change in Culex pipiens quinquefasciatus following infection with Lagenidium giganteum
JOURNAL
OF INVERTEBRATE
Biochemistry Change
PATHOLOGY
24, 293-304 (1974)
of Mosquito Infection: Preliminary in Culex pipiens quinquefasciatus I.agenidium giganteum’ A. DOMNA~, Biochemistry
P. E. GIEBEL,
Laboratory, Chapel
Botany Hill, Received
Studies Following
AND T. M. MCINNIS,
Department, North Carolina March
University of
North
of Biochemical Infection with JR.~ Carolina,
27514
12, 1974
The effect of infection of larvae of the mosquito Culex pipiens quinquefasciatus III? the fungus Lagenidium gigantelcm has been studied from a biochemical standpoint. Methods were developed to analyze larval extracts that were essentially free of parasitic material. Biochemical parameters invest,igated on a per larva basis were protein, and the enzymes o-diphenol oxidase, glutamate transaminase, alkaline phosphatase, trehalase, and chitobiase. The behavior of the total amino acid and sugar pools were also examined. In general it was found that the infected larvae exhibited a significant decrease in rate of synthesis of most of the parameters studied and that t.he rates eventually fell to zero when compared to healthy control organisms. The progress of mycosis was correlated with visual evidence and it was concluded that the larvae were dying from starvation as a consequence of the utilization of their endogenous reserves by the parasite. Death of the larvae occurred as a general rule, in laboratory conditions, some 48 hr after initiation of infection by the fungus.
An aquatic phycomycete was isolated from dead larvae of C&xc sp. by Umphlett (personal communication) in 1968 and subsequently identified as Lagenidium giganteum (Umphlett, 1973). This organism is a facultative parasite and consequently can and has been cultured on a defined medium in the laboratory. If appropriate age mosquito larvae are present, the zoospores will infect and kill the larvae. The parasitic potential of the fungus has been tested by Umphlett and Huang (1972) and their results indicate that L. giganteunt could funct,ion as an effective biological control agent ‘This work was supported by a United States Army Medical Research Developoment Grant DADA 17-72 C-2168. ’ Requests for reprints should be addressed to A. Domnas. 3 Present address, Department of Botany, Clemson University, Clemson, South Carolina 29631.
against mosquitoes. We have undertaken to examine the physiology and biochemistry of both the host and parasite in order to provide insight into the initiation and development of a host-parasite symbiosis, from the viewpoint’s of biological control studies and t.he physiopathology of disease. The problem was divided into three parts: (1) Biochemical and physiological studies on pure cultures of L. giganteum, (2) biochemical and developmental patterns of the host; and (3) biochemical and physiological interactions of the host and the parasite. The last aspect is stressed in this report, but, of necessity, information of the other two aspects is also presented. To evaluate the contribution of each organism during infection, new techniques were devised for the separation of the two organisms. Because of the complexity of the life systems involved, we have restricted ourselves in this initial report to analyses of
293 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in sny form reserved.
294
DObiN&:.
the basic parameters ble to measurement.
most readily
GIEBEL
acceesi-
AND
.MCINKIS
into small portions and placed into the test pans. Infection levels were always greater tl1an 90%.
MATERIALS
Fungus
Growth
AND
METHOD
and Cell-free
~losyuito growth and preparations Preparation
Our isolate of L. gigunteum n-as obtained from Prof. C. J. Umphlett, Clemson University, and routinely maintained on Cantino Peptone-Yeast Extract-Glucose (PYG) agar and in PYG liquid broth. Maximal yields of mycelium were obtained in 5 days from PYG broth or on the defined medium of Gleason (1968). The fungus was harvested, washed with water, suspended in 0.05 M phosphate buffer, pH 6.0, and then disrupted by an lMSK Braun cell disruptor or by a French pressure cell. The preparation was centrifuged at 12,000g to remove major cellular debris, and the extract tested for various enzymes and other substances. Zoospore production. Zoospores were obtained by the split hemp-seed technique or by the whole hemp-seed extract procedure. Whole hemp seeds were split in half, placed on agar, and inoculated with 1 ml of washed 5-day-old culture of the fungus. After several days, when the hemp seed was overgrown with fungus, the halves were placed into CaCl, (10-3M) for 12 hr and then transferred to pans containing larvae. The whole hemp-seed extract was an easier and much more productive method of obtaining zoospores. Whole hemp seed (5 g) was ground in 100 ml 0.05 M phosphate buffer, pH 7.0. The suspension was stirred for several hours until mcst of the fibrous material of the seed was suspended. The suspensionwas filtered through cheesecloth and the extract was incorporated into agar at the level of 1 mg protein per ml. The plate was inoculated with 1 ml of a j-dayold culture and allowed to grow for 5 days. The plates were placed in a dessicator (CaCI,) under low vacuum. The cultures were kept in the dessicator for 5 days, removed, flooded with 1O-3M CaCl,, and allowed to stand overnight. The agnr was rut
Growth ar2d preparation. Larvae of Culex pipiem quinyuefasciatus were used in this study. The mosquitoes were obtained from U.S. Public Health Service Disease Control Laboratory in Savannah, Georgia and maintained by Dr. S. Romney of this department. Czlle.2larvae were grown in quasisynchronous culture at 28OC in pans containing about 2.5 liters of water with a population density of about 500 larvae per pan; nutrient source was a pellet of Ken-L-Ration dog meal. Uninfected larvae, designated as control populations, were collected at specific time intervals, washed thoroughly with cold distilled water, counted, suspended in pH 7.0, 0.05 M phosphate buffer, and disrupted in a precooled glass homogenizer. (All subsequent operations were conducted at 2-5°C unless otherwise indicated.) The homogenate was centrifuged at 15009 for several minutes and the supernatant volume measured and retained for analysis. Infection studies. Zoospores were obtained as previously described and the detection of infection and monitoring of the rate was done by microscopical examination. The infected populations were sampled, washed, and counted as for control populations, but the infected larvae were disrupted in a Parr nitrogen bomb at 500 psi nitrogen pressure. The intact fungal cells and larval debris (Fig. 2C) were centrifuged at 15009 for several minutes to obtain a cell-free infected larval preparation. The cell-free extracts were assayed for ,&glucosidase and glucose-6-phosphate dehydrogenase, because these are marker enzymes for the fungus. If the fungus had been disrupted to any great extent these activities would be demonstrable (Table 2). Control experiments with uninfected and infected lnrvac indicated clcnrly that, no
BIOCHEMISTRT
OF
3IOSQUITO
TABLE EFFECTS
OF
Control
Infected a Abbreviations: (2-acetamido-2-deoxy-6-D
TECHNIQUES
I~ISKUPTION
Age
Protein fLg/larva
Ilays 7
H 4.8
10
17.8
17.1
4.x
4.5
70
11.0
14.6
(Glucose-B-Phosphate glucosidase); H (glass
Enzyme
(trivial
cu-galactosidase &galactosidase a-glucosidase fl-glucosidase N-acetyl-p-1%glucosaminidase Glucose-B-phosphate dehydrogenase Trehalase L-asparaginase L-sspartic acid transcarbamylase “Glutamate-oxalacetate transaminase “Glutamate-pyruvate aminase
name)
OF ENZYMKS Culex Lagenidium (PYG)a -
+ -
tr +++ -
+ + +
+++ +++ +++
tr + + + -
+
+
tr
+++
tr
-
Lhnv.4~:~ GlcNAc mu/larva
I-I 0
0
H 1.7
C 1.3
‘2 1
2.2
2.1
8 *5
8.1
0
0
0
0
1.7
1.3
2.6
2.1
1.4
2.1
7.4
8.X
II 0
c 0
‘2 2
c
(B-Dglucosidase); cavitation).
GlcNAc
2
IN LagenidiwrL giganteum pipiens quinque.fasciatus Culex
OF Cu/ex
p-Glc mIJ/larva
dehydrogenase); o-Glc homogenized) ; C (Nitrogen
TABLE DISTRIBUTION
E'RI:P.UL\TIONS
G-g-Pas, mU/larvn
7
G-6-Pase
I
ON CELL-FILI:E
c 4 .i
39.5
IKFECTION
Enzyme
AND IN UNINFECTED
(trivial
name) .____~ transcarbamylase
L-ornithine L-glutaminase o-diphenol oxidase Lipase (non-specific) Acid phosphatase Alkaline phosphatase Protease Isocitrate dehydrogenase Lactic Acid dehydrogenase Urease Uricase
Lagenidium +++ ++ ++ +++ ++ + +++ ++
Culex + tr +++ ++ -t+ ? -
trans-
a PYG grown; there are changes - (not present); + (low); + +
in transaminase (average); +++
significant difference in protein or enzyme occurred when either the homogenization or the cavitation technique was employed (Table 1). Thus, in the interest of ease, control larvae were always homogenized, whereas infected larvae were disrupted by cavit.ation. This latter process ensured us that no material from the fungus leaked into the preparation (Fig. 2C). Pnlameters
measured
Insect enzym,es. o-diphenol: oxygen oxidoretluctase (E.C. 1.10.3.1, trivial name
when grown in NH*+ salt (very active); tr (trace).
medium.
o-diphenol oxidase) was assayed at 30% by t’he method of Fling et al. (1963) using L-DOPA as the substrate. One unit of enzyme produces a change in absorbance of 1.35/min. This enzyme was selected because it is a typical insect enzyme and because of its importance in the deferrye mechanism of the larvae. Aspartate: 2-oxyglutarate aminotransfcrase (E.C. 2.6.1.1, aspartate aminotransferase or glutamate-osalacetate transaminase) was assayed with Statzyme GOT (Worthington Biochemical Corporation). One enzyme unit is equal to a decrease in
296
DOMNAS,
GIEBEL
absorbance of O.OOl/min at 30%. This enzyme was selected for study because it would indicate whether or not the amino acids were being depleted. Orthophosphoric monocstcr phosphohydrolase (E.C. 3..1.3.1, alkaline phosphatase) was assayed with p-nitrophenylphosphate in 0.1 M Trizma buffer, pH 9.0. The reaction was stopped with 0.5 M KaOH and the absorbance of p-nitrophenolatc ion read at 420 nm. One enzyme unit is equal to 1 pmole of p-nitrophenolate produced in 30 min at 30°C. The study of the behavior of this enzyme would indicate whether the host was using up or mobilizing its phosphate reserves. (E.C. a7 a’-Glucoside-1-glucohydrolase 3.3.1.28, trehalase) was assayed with glucose oxidase (Worthington Biochemical Corporation) (McInnis and Domnas, 1973). One enzyme unit is equal to 1 pmole of glucose produced in 30 min at 30%. This enzyme was studied because of its importance to larval carbohydrate metabolism. Chitobiose acetamidodeoxyglucohydrolase (E.C. 3.2.1.29, chitobiaee) was assayed with p-nitrophenol p-D-glucoseaminidc. The reaction was stopped with 0 5 di NaOH and the absorbance of p-nitrophenolate read at 420 nm. One unit enzyme is equal to 1 pmole of y-nitrophenolate produced in 30 min at 30%. This enzyme was investigated because of its role in chitin metabolism. A connection between infection through the exoskeleton and chitobinsc activity was thought likely. All measurements were mado with a Hitachi Perkin-Elmer Model 139 spect,rophotometer equipped with a constant tcmperature cell attachment set for 30GC. The pH of optimum activity and cold :tability for the enzymes described above were determined, and as a general rule, all enzyme assays were performed as soon as possible after obtaining the cell-free preparation. Other prrrametem. (1) Protein was measured by the Lowry et al. (1851) procedure using crystalline bovine albumin (Sigma) R’: the standard.
AND
MCINNIS
(2) Total free amino acids were determined by two methods and both procedures gave similar results. The cell-free extract from the larvae was treated with an equal volume of cold 95% alcohol and allowed to stand for 7 days at 5°C. At the end of this time, precipitation was complete and aliquots of protein-free extract were analyzed by the procedure of Rosen (1957), using leucine as n standard. hlt.crnatively, a counted number of uninfected larvae were homogenized in a given volume of 95% cold ethanol and allowed to stand for 7 days in the cold. After centrifugation, infected larval aliquots were tested as before by the Rosen (1957) procedure. The last procedure was not employed for infected larvae. (3) Total sugar was assayed by the phenol-sulfuric acid method of Dubois et al. (1956) on alcohol extracts prepared by the first amino acid procedure described above. The study of the behavior of the amino acids and sugars was of importance as a general criterion of response to the pathogen by the host. Thin layer chromatography of glucose rind trehalose. Larval alcohol extracts were chromatographed one dimensionally on ;:ilica gel G plates with butanol:ethanol: acetone :water (5 :4 :3 : 21 or formic acid : butanone : t-butanol :water (3 : 6 :8 :3) solvent systems (Procaro, 1963). The sugars were visualized by an anisaldehyde :sulfuric acid spray (Lewis and Smith, 1969). Where possible, the accumulated data arc expressed on a per larva basis and each parameter measured was repeated on five different infected populat,ions. The data shown herein represent typical values ohtnincd from the experiments. RESULTS
Microscopical
Observations
Stage I infection (Figs. 1A and B). The earliest indication of infection was the presence of a “bore hole” (Fig. IB, arrow) produced by the hyphal initial penetrating
BIOCHEMISTRY
FIG. 1. (A) Stage II infection
Stage (x20)
OF
RIOSQUITO
I infection, cephalic region ; (D) Stage III infection
(x20); (x12).
‘97
INFECTION
(B)
“borehole”
mow
(x52);
CC)
through the integument, and is often de- some 24-32 hr after initiation of infection tectablc by melanin deposition (Fig. 3). with spores (Fig. 1B). The melanin reaction has been frequently Singe II infection (Fig. 1C). An extenobserved in similar pathologies (Madelin, sive ramification of hyphae in the cephalic 1963; David, 1968; Sanassi and Oliver, region and some hyphae have entered the 1971). The initial lesions nearly always approthorax (arrows). The larvae were still peared in the posterior cephalic segment vigorous and alive in this stage.
298
IIOMNAS,
FIG. 2. (‘4) Sporangium sporangia (arrow) ; (C) gium on hemp seed half
GIEBEL
AND
with spores (Nomarski cavitated larval preparation (x34).
&aye III infectiolz (Fig. 1D). Hyphal segmentation in the thorax and anterior abdominal segments generally was evident. Each hyphal segment represents a potential
.\ICINISlS
intprfcrrnce, x213) ; (B) d~wl Inrv:~ with with intact fungi (X213) ; (I>) spornn-
sporangium vae were still unresponsive Stage T1’
(Willoughby, 1989). The laralive, hut were lethargic ancl to meclianival stimuli. irzfection (Figs. 2X and B‘I.
BIOCHEMISTRY
FIG. 3. Melanin
clcposition
OF
after
JIOSQUITO
pcnetrntion
Larval death and sporulation of the fungus. The larvae generally die soon after the cephalic, thoracic, and upper abdominal segments are filled with the fungus. Eight to 20 hr fo1lowin.g death, some of the hyphal segments function as sporangia (Fig. 2A) and release zoospores. The spores that are released can infect healthy larvae. BIOCHEMICAL
STUDIES
The results of an enzyme survey of both host and parasite are listed in Table 2. It was found that four enzymes, trehalase, L-aspartic acid transcarbamylase, glutamate-oxalacetate transaminase, and lipase (nonspecific) demonstrate comparable activity in both the host and the parasite. r,-ornithine transcarbamylase was present in
299
INFECTION
of hyphnl
initial
(x2.50).
both, but was of very low activity in the mosquito larva. Trehalase and lipase were found to be stable after storage at -1O’C for 7 days. Other enzymes, such as o-diphenol oxidase, were found to be unstable, with loss of activity after 7-9 days. Although Trebatoski and Haynes (1969) reported the presence of both acid and alkaline phosphat’es in Culex pipiens, only the alka,line phosphatase was found in our species. Comparison of the developmental data for total protein (Fig. 4)) free amino acids (Fig. 5), and total sugars (Fig. 6) for infccted versus uninfected populations indicates a similar generalized pattern for all t’hree parameters ; a period of increase or stasis followed by a sharp decline of the
300
DOMNAS,
GIEREL
parameters in the infected populations. The rates of increase of these parameters in the infected populations were demonstrably less than the rates for uninfected populations.
2ool-----1 I .H
-
3 120._;u _ .g s E
P
/ I /
so_ 40-
, ,a”
oc** 0
’
’ 40
’
’ SO
’
’ I20
’
’ 160
’
’ 200
’
Hours FIG.
4. Development
in healthy populations.
(O-----O)
of total protein per larvae and infected (e-----e)
FIG. 5. Effect of Lagenidium free amino acids in C&x ~~yn:;l populations; l -----a
infection on total larvae (O-----O, infected popula-
FIG. 6. Effect of Lagenidiunn total free sugars in Culex populations normal; @-----a, infected larvae).
mycosis (O-----O,
on
AND
MCINNIS
Total protein (Fig. 4) increased from 40 pg/larva at 110 hr to 170 pg/larvae at 180 hr. In contrast, total protein in the infected larvae demonstrated an increase durin.g the first 50 hr of infection, at a rate less than that for t,he infected populations, followed by a decline during the remainder of the infection period. Total free amino acid concentration in uninfected populations showed a net increase from about 30 X 1O-3 @lole/larva (as leucine) at 110 hr to 90 X lo-” pmolc/ larva at 170 hr, followed by a drop at 190 hr. In the infected populations, however, only a slight increase or a stasis period occurred during the first 50 hr of infection, followed by a sharp decrease during the next 10 hr, with concentration remaining constant during the remainder of the infection test period. The total sugar of control population larvae increased from 2 pg/larva at 110 hr to 9 pg/larva at 155 hr, followed by a decline to less than 4 pg at 170 hr. In infected populations, an increase in sugar concentration was observed during the first 15 hr, followed by a 15hr stasis period, after which another increase in concentration occurred which was followed by a sharp decline from 7 p&/larva at 150 hr to less than 1 pg/larvae at 160 hr (Fig. 3). The enzymes chitobiase, trehalase, alkaglutamate-oxalacctate line phosphatase, transaminasc, and o-diphenol oxidase (Figs. 7-11) demonstrated similar development patterns in uninfected populations: an increase in activity with an increase in larval age. Glutamate-oxalacetate transaminase demonstrated a similar pattern, but a decrease was observed in the later stages of development, at about 180 hr (Fig. 10). In infected populations, chitobiase, trehalase, and alkaline phosphatase exhibit,ed an increase in activity during the first. 15 hr of infection, with a decline occurring after this period (Figs. 7-9). The enzymes o-diphenol oxidase and glutamateoxalacetate transaminase, however, demonstrated different patterns in infected popu-
BIOCHEMISTRY
50-
0
2400
/
,= b 30 x fn C
J’ /
J’ /
<20z Ii
/ d m---o
.
IO iA,0
140
160
160
200
HOUCS
Fro. 7. Time-course development in normally developing Culex larvae and infected C&x larvae (O-----e).
MOSQUITO
301
IXFECTIOIi
zymes, but the increase lasted through the first 50 hr of infection at a rate equal to that of the uninfected population. At the end of this period, the enzyme activity in t,he infect,4 populations dropped sharply, whereas the activity in the uninfected populations increased (Fig. 10). The enzyme o-diphenol oxidase (Fig. 11) exhibited an immediate decline in activity observed 30 hr after the initiation of infection. The behavior of t,his enzyme was of interest since it tended to disappear on infection. When t,hese data were plotted as specific activity (Fig. 12)) it was noted that there was a considerable stimulation of the enzyme in the infected relative t.o the control followed by a rapid decline of enzyme. Specific activity plots of the data simply show a general decrease of the enzyme in infected
-
/
ti
: 0
OF
of chitobiasc (O-----O)
‘““I’
_;,
1
h Fro. S. Effect of trchalase development and (O-----O) (O-----O).
Lagenicliwn in norma! infected
infection on C&x larvae CUkX larvae
,o 3 E
OL 100
’
I
I
I
120
I
140
I
,
I
160
,
180
_ 200
Hours
FIG. 10. iransaminase and infected
Responses of glutamate-oxalacetate in normai Culex larvae (0 -----0) larvae (a-----@).
Q / :
-20-o FIG.
normal fected
9. Behavior Culex larvae by Lagenidium
:' P Hours
of alkaline phosphatnse (O-----O) and larvae spores (O-----a).
/' in in-
lations. The transaminase increased in the infected larvae as did the other three en-
p-0' ,+A I , 120
/' I
,
140
I
I_
160
180
I 200
HOUB FIG.
normal infected
11. Behavior of o-diphenol Culex larvae (O-----O) with Lagenidiun~ (a-----
oxidase in and larvae 0).
302
DOMNAS,
I
1
,
,
,
I
,
GIEBEL
,
0.6 -
FIG. 12. Specific activities of o-diphenol in normal and infected Culez larvae.
oxidase
when compared to normal larvae. As explained by Cant.ino (1961), it is best to use a natural biological unit, like the larva, since it shows the amount of enzyme in the organism. DISCUSSION
One of the major problems which confronted us was a method of separating the fungus from the infected larva in order to assay various parameters. In our studies on the fungus, we observed that mature cultures could be subjected to pressure of 2000 psi in the Parr Nitrogen Disruption Bomb without cell breakage occurring. Uninfected larvae of Mea, however, could be disrupted at pressure between 500-1000 psi. Thus, when infected larvae (late Stage II, Stage III, and Stage IV) were subjected to pressures of 500 psi in the Parr disruption bomb, the larvae were completely disrupted but the fungal subthalli remained intact (Fig. 2C). Stage I and early Stage II infectons, however, could not be separated because the mycelial matter was very fragile and ruptured under 500 psi pressure, but it is very unlikely that significant amounts of parasitic protein are mixed with host material. The cell-free larval extract was assayed for the fungal enzymes p-glucosidase and glucose-Bphosphate tlrhyclro-
ANDMCINNIS
gcnase (Lang and Stephan, 1967) and no significant. activity was found (Table 1). It would appear from these controls that little fungnl material was present in the larval extract. Another problem of concern to us was the synchrony of infection. Were the larvae being infected in a short time period, or were the infections occurring over a broad time span? Larvae at the same stage of infecti;n were harvested and their protein concentration compared with that of another infected population whose larvae were randomly sampled. The protein profiles of two experiments were found to be quite clot, and it was concluded that the infection was occurring in a short time period. Infection of the larvae occurs via spores which pen&ate through the integument (Fig. 3). Similar observations have been reported with other fungi (Benz, 1963), but the mechanism of penetration is not known. Some investigators have speculated on the role of chitinase (Benz, 1963), but the presence of this enzyme has not been demonstrated in a11cxscs. A chitinase was not detccted in I,. giganteum, and growth of the fungus on chitin and glucosamine was poor to zero. Our data, however, does not specifically exclude the possibility that the spore may have a chitinase system, since we have not yet, been able to investigate its physiology and biochemistry. Penetration of the integument could conceivably occur via proteolytic and lipolytic activity, with the hyphal initial pushing through weaker parts of the integument or amorphous regions of the chitin layer via mechanical action ; a nonspecific lipase and a proteases have been detect’ed in L. giganteum, with both enzyme systems having relatively high activity (Table 2). Once initial infection had occurred. Ilowever, rapid growth of the fungal parasite ensued, with larval death 60-72 hr later. Protein synthesis in infected larvae slowed down almost from the first day of infection, continued to decelerate, and wcntunlly ccnscd. The data shown here
BIOCHEWSTRY
OF
MOSQUITO
were obtained from still living organisms, yet, “dead” larvae possessed measurable protein and residual enzymic activities. ,4t death, trehalase and glutamate-oxaloacet,ate transminase were still detectable; the free amino acids were very low, and chromatograms of extracts from dead larvae showed that trehalose was absent and glucose was very low. The loss of necessary amino acids would of necessity stop protein synthesis even though the protein synthesizing systems were still present. The results, in effect, seemed to indicate that the organisms were dying of starvation, a conelusion also reached in the case of the Japanese beetle milky disease. (Bulla and St.. *Julian, 1972). One parameter presented aspects anomalous to the overall general pathological picture. The enzyme, o-diphenol oxidase, disappeared in extracts of infected larvae in about 40 hr. The behavior of this enzyme was interesting because of its association with clit,icle sclerotization (Hackman, 1971). Since t’he concentration of enzyme/larva dropped after penetration of the integument, i.e., Stage I, it is unlikely that the enzyme was being inactivated because of a reaction initiated by spore penetration. It would appear more likely that inactivation could be a supression of a defense mechanism. The disappearance of o-diphenol oxidase could indicate an inhibiCon at the biosynthetic or even at the subunit assembly stage (Jolley and Mason, 1965). Further work should help to clarify this point, particularly with regard to tyrosine levels and other aromatic amino acids. REFERENCES BENZ,
G. 1963. “Insect Pathology” (E. A. Steinhnus, ed.) Vol. 1, pp. 229-338. Academic Press, New York. BULLA, L. A., AND ST. JULIAN. G. 1972. Biorhemistry of milk disease: Radiorespirometry of ppruvate. acetate, succinate, and glutamate oxidation by healthy and diseased Japanese beetle larvae. J. Znvertebr. Pathol., 19, 12&124. CANTINO, E. C. 1961. The relationship between
INFECTION
303
biochemical and morphological differentiation in nonfilamentous aquatic fungi. Symp. Sot Geta. Microbial., 11, 243-271. DAVID, W. A. L. 1968. “Insects and Physiology” (J. W. L. Beament and J. E. Treheme, Eds.). 1st ed., 17-35. American Elsevier, New York. DUBOIS, M., GILLES, K. A., HAMILTON. J. K., REBERS, P. A. ASD SMITH, F. 1956. Colorimetric method for determination of sugars and related substan:*es. A&. Chem., 28, 350-356. FLING. M., HOROWITZ, r\T. H., AND HIGINEMAN, S. F. 1963. The isolation and properties of crystalline tyrosinase of Neurospora. J. Biol. Chem., 238, 2045-2053. GLEASON, F. H. 1968. Nutrional comparisons in the Leptomitales. Am. J. Botany, 55, 1003-1010. HACKMAN, R. H. 1971. The integument of the arthropoda. In “Chemical Zoology, VI. Arthropoda, Part B” (M. Florkin and B. T. Scheer, Eds.). Academic Press, New York. JOLLEY, R. L., AND MASON, H. S. 1965. The multiple forms of mushroom tyrosinase. J. Biol. Chem., 240, 1489-1490. LANG, E. A., AND STEPHAS, J. K. 1967. Nicotinamide-adenine dinucleotide phosphate enzymes in the mosquito during growth and aging. Biochem. J., 102, 331-337. LEWIS, B. A. AND SMITH, F. 1969. Sugars and derivatives. In “Thin Layer Chromatography”. (E. Stahl, ed.), pp. 507-837. Springer-Verlag, ?;cw York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. 1951. Protein measurement with Folin phenol reagents. J. Biol. Chem., 193, 323-335. MADELIN, M. F. 1963. Insect Pathology, (E. A. Steinhaus, Ed.), Vol. 2, pp. 233-271. Academic Press, New York. MCINNIS, T. M., AKD DOMSAS, A. 1973. The properties of trehalase from the mosquito parasitizing mater-mold, Lngenidium sp. J. Invertebr. Pathol., 22, 313-320. PROCARO, C. A. 1963. The deteztion of trehalose in the larval hemolymph of Tororhynchitis tntilis Coker. MS. thesis, Department of of North Carolina, Zoology, University Chapel Hill, XC. ROSEN, H. 1957. A modified ninhydrin calorimetric analysis for amino acids. Arch. Biochem. Biophys., 67, 10-15. S.~NASSI, 8., AND OLIVER, J. H. 1971. Integument of the velvet mite, Dinothrombeum giganteum and histopathological changes caused by the fungus Aspergil1lL.s flavus. J. Invertebr. Pathol., 17, 354-365. TREBATOSKI, A. M., AND HAYNES, J. F. 1969. Comparison of enzymes of twelve specirs of
304
DOMNAS,
GIEBEL
mosquitos. Ann. Entomol. Sot. Amer., 62, 327-335. UMPHLETT, C. J. 1973. A note to identify a certain isolate of Lagenidium which kills mosquito larvae. Myeologia, 65, 970-972. UMPHLETT, C. J., AND HUANG, C. 1972. Experi-
AND
MCINNIS
mental infection of mosquito larvae by speties of the aquatic fungus Lagenidium. J. Invertebr. Pathol., 20, 321326. WILLOUGHBY, L. G. 1969. Pure culture studies on the aquatic phycomycete Lagenidium giganteum. Trau-. &it. Mvcol. Sot., 52, 393-410.
OF INVERTEBRATE
Biochemistry Change
PATHOLOGY
24, 293-304 (1974)
of Mosquito Infection: Preliminary in Culex pipiens quinquefasciatus I.agenidium giganteum’ A. DOMNA~, Biochemistry
P. E. GIEBEL,
Laboratory, Chapel
Botany Hill, Received
Studies Following
AND T. M. MCINNIS,
Department, North Carolina March
University of
North
of Biochemical Infection with JR.~ Carolina,
27514
12, 1974
The effect of infection of larvae of the mosquito Culex pipiens quinquefasciatus III? the fungus Lagenidium gigantelcm has been studied from a biochemical standpoint. Methods were developed to analyze larval extracts that were essentially free of parasitic material. Biochemical parameters invest,igated on a per larva basis were protein, and the enzymes o-diphenol oxidase, glutamate transaminase, alkaline phosphatase, trehalase, and chitobiase. The behavior of the total amino acid and sugar pools were also examined. In general it was found that the infected larvae exhibited a significant decrease in rate of synthesis of most of the parameters studied and that t.he rates eventually fell to zero when compared to healthy control organisms. The progress of mycosis was correlated with visual evidence and it was concluded that the larvae were dying from starvation as a consequence of the utilization of their endogenous reserves by the parasite. Death of the larvae occurred as a general rule, in laboratory conditions, some 48 hr after initiation of infection by the fungus.
An aquatic phycomycete was isolated from dead larvae of C&xc sp. by Umphlett (personal communication) in 1968 and subsequently identified as Lagenidium giganteum (Umphlett, 1973). This organism is a facultative parasite and consequently can and has been cultured on a defined medium in the laboratory. If appropriate age mosquito larvae are present, the zoospores will infect and kill the larvae. The parasitic potential of the fungus has been tested by Umphlett and Huang (1972) and their results indicate that L. giganteunt could funct,ion as an effective biological control agent ‘This work was supported by a United States Army Medical Research Developoment Grant DADA 17-72 C-2168. ’ Requests for reprints should be addressed to A. Domnas. 3 Present address, Department of Botany, Clemson University, Clemson, South Carolina 29631.
against mosquitoes. We have undertaken to examine the physiology and biochemistry of both the host and parasite in order to provide insight into the initiation and development of a host-parasite symbiosis, from the viewpoint’s of biological control studies and t.he physiopathology of disease. The problem was divided into three parts: (1) Biochemical and physiological studies on pure cultures of L. giganteum, (2) biochemical and developmental patterns of the host; and (3) biochemical and physiological interactions of the host and the parasite. The last aspect is stressed in this report, but, of necessity, information of the other two aspects is also presented. To evaluate the contribution of each organism during infection, new techniques were devised for the separation of the two organisms. Because of the complexity of the life systems involved, we have restricted ourselves in this initial report to analyses of
293 Copyright All rights
@ 1974 by Academic Press, Inc. of reproduction in sny form reserved.
294
DObiN&:.
the basic parameters ble to measurement.
most readily
GIEBEL
acceesi-
AND
.MCINKIS
into small portions and placed into the test pans. Infection levels were always greater tl1an 90%.
MATERIALS
Fungus
Growth
AND
METHOD
and Cell-free
~losyuito growth and preparations Preparation
Our isolate of L. gigunteum n-as obtained from Prof. C. J. Umphlett, Clemson University, and routinely maintained on Cantino Peptone-Yeast Extract-Glucose (PYG) agar and in PYG liquid broth. Maximal yields of mycelium were obtained in 5 days from PYG broth or on the defined medium of Gleason (1968). The fungus was harvested, washed with water, suspended in 0.05 M phosphate buffer, pH 6.0, and then disrupted by an lMSK Braun cell disruptor or by a French pressure cell. The preparation was centrifuged at 12,000g to remove major cellular debris, and the extract tested for various enzymes and other substances. Zoospore production. Zoospores were obtained by the split hemp-seed technique or by the whole hemp-seed extract procedure. Whole hemp seeds were split in half, placed on agar, and inoculated with 1 ml of washed 5-day-old culture of the fungus. After several days, when the hemp seed was overgrown with fungus, the halves were placed into CaCl, (10-3M) for 12 hr and then transferred to pans containing larvae. The whole hemp-seed extract was an easier and much more productive method of obtaining zoospores. Whole hemp seed (5 g) was ground in 100 ml 0.05 M phosphate buffer, pH 7.0. The suspension was stirred for several hours until mcst of the fibrous material of the seed was suspended. The suspensionwas filtered through cheesecloth and the extract was incorporated into agar at the level of 1 mg protein per ml. The plate was inoculated with 1 ml of a j-dayold culture and allowed to grow for 5 days. The plates were placed in a dessicator (CaCI,) under low vacuum. The cultures were kept in the dessicator for 5 days, removed, flooded with 1O-3M CaCl,, and allowed to stand overnight. The agnr was rut
Growth ar2d preparation. Larvae of Culex pipiem quinyuefasciatus were used in this study. The mosquitoes were obtained from U.S. Public Health Service Disease Control Laboratory in Savannah, Georgia and maintained by Dr. S. Romney of this department. Czlle.2larvae were grown in quasisynchronous culture at 28OC in pans containing about 2.5 liters of water with a population density of about 500 larvae per pan; nutrient source was a pellet of Ken-L-Ration dog meal. Uninfected larvae, designated as control populations, were collected at specific time intervals, washed thoroughly with cold distilled water, counted, suspended in pH 7.0, 0.05 M phosphate buffer, and disrupted in a precooled glass homogenizer. (All subsequent operations were conducted at 2-5°C unless otherwise indicated.) The homogenate was centrifuged at 15009 for several minutes and the supernatant volume measured and retained for analysis. Infection studies. Zoospores were obtained as previously described and the detection of infection and monitoring of the rate was done by microscopical examination. The infected populations were sampled, washed, and counted as for control populations, but the infected larvae were disrupted in a Parr nitrogen bomb at 500 psi nitrogen pressure. The intact fungal cells and larval debris (Fig. 2C) were centrifuged at 15009 for several minutes to obtain a cell-free infected larval preparation. The cell-free extracts were assayed for ,&glucosidase and glucose-6-phosphate dehydrogenase, because these are marker enzymes for the fungus. If the fungus had been disrupted to any great extent these activities would be demonstrable (Table 2). Control experiments with uninfected and infected lnrvac indicated clcnrly that, no
BIOCHEMISTRT
OF
3IOSQUITO
TABLE EFFECTS
OF
Control
Infected a Abbreviations: (2-acetamido-2-deoxy-6-D
TECHNIQUES
I~ISKUPTION
Age
Protein fLg/larva
Ilays 7
H 4.8
10
17.8
17.1
4.x
4.5
70
11.0
14.6
(Glucose-B-Phosphate glucosidase); H (glass
Enzyme
(trivial
cu-galactosidase &galactosidase a-glucosidase fl-glucosidase N-acetyl-p-1%glucosaminidase Glucose-B-phosphate dehydrogenase Trehalase L-asparaginase L-sspartic acid transcarbamylase “Glutamate-oxalacetate transaminase “Glutamate-pyruvate aminase
name)
OF ENZYMKS Culex Lagenidium (PYG)a -
+ -
tr +++ -
+ + +
+++ +++ +++
tr + + + -
+
+
tr
+++
tr
-
Lhnv.4~:~ GlcNAc mu/larva
I-I 0
0
H 1.7
C 1.3
‘2 1
2.2
2.1
8 *5
8.1
0
0
0
0
1.7
1.3
2.6
2.1
1.4
2.1
7.4
8.X
II 0
c 0
‘2 2
c
(B-Dglucosidase); cavitation).
GlcNAc
2
IN LagenidiwrL giganteum pipiens quinque.fasciatus Culex
OF Cu/ex
p-Glc mIJ/larva
dehydrogenase); o-Glc homogenized) ; C (Nitrogen
TABLE DISTRIBUTION
E'RI:P.UL\TIONS
G-g-Pas, mU/larvn
7
G-6-Pase
I
ON CELL-FILI:E
c 4 .i
39.5
IKFECTION
Enzyme
AND IN UNINFECTED
(trivial
name) .____~ transcarbamylase
L-ornithine L-glutaminase o-diphenol oxidase Lipase (non-specific) Acid phosphatase Alkaline phosphatase Protease Isocitrate dehydrogenase Lactic Acid dehydrogenase Urease Uricase
Lagenidium +++ ++ ++ +++ ++ + +++ ++
Culex + tr +++ ++ -t+ ? -
trans-
a PYG grown; there are changes - (not present); + (low); + +
in transaminase (average); +++
significant difference in protein or enzyme occurred when either the homogenization or the cavitation technique was employed (Table 1). Thus, in the interest of ease, control larvae were always homogenized, whereas infected larvae were disrupted by cavit.ation. This latter process ensured us that no material from the fungus leaked into the preparation (Fig. 2C). Pnlameters
measured
Insect enzym,es. o-diphenol: oxygen oxidoretluctase (E.C. 1.10.3.1, trivial name
when grown in NH*+ salt (very active); tr (trace).
medium.
o-diphenol oxidase) was assayed at 30% by t’he method of Fling et al. (1963) using L-DOPA as the substrate. One unit of enzyme produces a change in absorbance of 1.35/min. This enzyme was selected because it is a typical insect enzyme and because of its importance in the deferrye mechanism of the larvae. Aspartate: 2-oxyglutarate aminotransfcrase (E.C. 2.6.1.1, aspartate aminotransferase or glutamate-osalacetate transaminase) was assayed with Statzyme GOT (Worthington Biochemical Corporation). One enzyme unit is equal to a decrease in
296
DOMNAS,
GIEBEL
absorbance of O.OOl/min at 30%. This enzyme was selected for study because it would indicate whether or not the amino acids were being depleted. Orthophosphoric monocstcr phosphohydrolase (E.C. 3..1.3.1, alkaline phosphatase) was assayed with p-nitrophenylphosphate in 0.1 M Trizma buffer, pH 9.0. The reaction was stopped with 0.5 M KaOH and the absorbance of p-nitrophenolatc ion read at 420 nm. One enzyme unit is equal to 1 pmole of p-nitrophenolate produced in 30 min at 30°C. The study of the behavior of this enzyme would indicate whether the host was using up or mobilizing its phosphate reserves. (E.C. a7 a’-Glucoside-1-glucohydrolase 3.3.1.28, trehalase) was assayed with glucose oxidase (Worthington Biochemical Corporation) (McInnis and Domnas, 1973). One enzyme unit is equal to 1 pmole of glucose produced in 30 min at 30%. This enzyme was studied because of its importance to larval carbohydrate metabolism. Chitobiose acetamidodeoxyglucohydrolase (E.C. 3.2.1.29, chitobiaee) was assayed with p-nitrophenol p-D-glucoseaminidc. The reaction was stopped with 0 5 di NaOH and the absorbance of p-nitrophenolate read at 420 nm. One unit enzyme is equal to 1 pmole of y-nitrophenolate produced in 30 min at 30%. This enzyme was investigated because of its role in chitin metabolism. A connection between infection through the exoskeleton and chitobinsc activity was thought likely. All measurements were mado with a Hitachi Perkin-Elmer Model 139 spect,rophotometer equipped with a constant tcmperature cell attachment set for 30GC. The pH of optimum activity and cold :tability for the enzymes described above were determined, and as a general rule, all enzyme assays were performed as soon as possible after obtaining the cell-free preparation. Other prrrametem. (1) Protein was measured by the Lowry et al. (1851) procedure using crystalline bovine albumin (Sigma) R’: the standard.
AND
MCINNIS
(2) Total free amino acids were determined by two methods and both procedures gave similar results. The cell-free extract from the larvae was treated with an equal volume of cold 95% alcohol and allowed to stand for 7 days at 5°C. At the end of this time, precipitation was complete and aliquots of protein-free extract were analyzed by the procedure of Rosen (1957), using leucine as n standard. hlt.crnatively, a counted number of uninfected larvae were homogenized in a given volume of 95% cold ethanol and allowed to stand for 7 days in the cold. After centrifugation, infected larval aliquots were tested as before by the Rosen (1957) procedure. The last procedure was not employed for infected larvae. (3) Total sugar was assayed by the phenol-sulfuric acid method of Dubois et al. (1956) on alcohol extracts prepared by the first amino acid procedure described above. The study of the behavior of the amino acids and sugars was of importance as a general criterion of response to the pathogen by the host. Thin layer chromatography of glucose rind trehalose. Larval alcohol extracts were chromatographed one dimensionally on ;:ilica gel G plates with butanol:ethanol: acetone :water (5 :4 :3 : 21 or formic acid : butanone : t-butanol :water (3 : 6 :8 :3) solvent systems (Procaro, 1963). The sugars were visualized by an anisaldehyde :sulfuric acid spray (Lewis and Smith, 1969). Where possible, the accumulated data arc expressed on a per larva basis and each parameter measured was repeated on five different infected populat,ions. The data shown herein represent typical values ohtnincd from the experiments. RESULTS
Microscopical
Observations
Stage I infection (Figs. 1A and B). The earliest indication of infection was the presence of a “bore hole” (Fig. IB, arrow) produced by the hyphal initial penetrating
BIOCHEMISTRY
FIG. 1. (A) Stage II infection
Stage (x20)
OF
RIOSQUITO
I infection, cephalic region ; (D) Stage III infection
(x20); (x12).
‘97
INFECTION
(B)
“borehole”
mow
(x52);
CC)
through the integument, and is often de- some 24-32 hr after initiation of infection tectablc by melanin deposition (Fig. 3). with spores (Fig. 1B). The melanin reaction has been frequently Singe II infection (Fig. 1C). An extenobserved in similar pathologies (Madelin, sive ramification of hyphae in the cephalic 1963; David, 1968; Sanassi and Oliver, region and some hyphae have entered the 1971). The initial lesions nearly always approthorax (arrows). The larvae were still peared in the posterior cephalic segment vigorous and alive in this stage.
298
IIOMNAS,
FIG. 2. (‘4) Sporangium sporangia (arrow) ; (C) gium on hemp seed half
GIEBEL
AND
with spores (Nomarski cavitated larval preparation (x34).
&aye III infectiolz (Fig. 1D). Hyphal segmentation in the thorax and anterior abdominal segments generally was evident. Each hyphal segment represents a potential
.\ICINISlS
intprfcrrnce, x213) ; (B) d~wl Inrv:~ with with intact fungi (X213) ; (I>) spornn-
sporangium vae were still unresponsive Stage T1’
(Willoughby, 1989). The laralive, hut were lethargic ancl to meclianival stimuli. irzfection (Figs. 2X and B‘I.
BIOCHEMISTRY
FIG. 3. Melanin
clcposition
OF
after
JIOSQUITO
pcnetrntion
Larval death and sporulation of the fungus. The larvae generally die soon after the cephalic, thoracic, and upper abdominal segments are filled with the fungus. Eight to 20 hr fo1lowin.g death, some of the hyphal segments function as sporangia (Fig. 2A) and release zoospores. The spores that are released can infect healthy larvae. BIOCHEMICAL
STUDIES
The results of an enzyme survey of both host and parasite are listed in Table 2. It was found that four enzymes, trehalase, L-aspartic acid transcarbamylase, glutamate-oxalacetate transaminase, and lipase (nonspecific) demonstrate comparable activity in both the host and the parasite. r,-ornithine transcarbamylase was present in
299
INFECTION
of hyphnl
initial
(x2.50).
both, but was of very low activity in the mosquito larva. Trehalase and lipase were found to be stable after storage at -1O’C for 7 days. Other enzymes, such as o-diphenol oxidase, were found to be unstable, with loss of activity after 7-9 days. Although Trebatoski and Haynes (1969) reported the presence of both acid and alkaline phosphat’es in Culex pipiens, only the alka,line phosphatase was found in our species. Comparison of the developmental data for total protein (Fig. 4)) free amino acids (Fig. 5), and total sugars (Fig. 6) for infccted versus uninfected populations indicates a similar generalized pattern for all t’hree parameters ; a period of increase or stasis followed by a sharp decline of the
300
DOMNAS,
GIEREL
parameters in the infected populations. The rates of increase of these parameters in the infected populations were demonstrably less than the rates for uninfected populations.
2ool-----1 I .H
-
3 120._;u _ .g s E
P
/ I /
so_ 40-
, ,a”
oc** 0
’
’ 40
’
’ SO
’
’ I20
’
’ 160
’
’ 200
’
Hours FIG.
4. Development
in healthy populations.
(O-----O)
of total protein per larvae and infected (e-----e)
FIG. 5. Effect of Lagenidium free amino acids in C&x ~~yn:;l populations; l -----a
infection on total larvae (O-----O, infected popula-
FIG. 6. Effect of Lagenidiunn total free sugars in Culex populations normal; @-----a, infected larvae).
mycosis (O-----O,
on
AND
MCINNIS
Total protein (Fig. 4) increased from 40 pg/larva at 110 hr to 170 pg/larvae at 180 hr. In contrast, total protein in the infected larvae demonstrated an increase durin.g the first 50 hr of infection, at a rate less than that for t,he infected populations, followed by a decline during the remainder of the infection period. Total free amino acid concentration in uninfected populations showed a net increase from about 30 X 1O-3 @lole/larva (as leucine) at 110 hr to 90 X lo-” pmolc/ larva at 170 hr, followed by a drop at 190 hr. In the infected populations, however, only a slight increase or a stasis period occurred during the first 50 hr of infection, followed by a sharp decrease during the next 10 hr, with concentration remaining constant during the remainder of the infection test period. The total sugar of control population larvae increased from 2 pg/larva at 110 hr to 9 pg/larva at 155 hr, followed by a decline to less than 4 pg at 170 hr. In infected populations, an increase in sugar concentration was observed during the first 15 hr, followed by a 15hr stasis period, after which another increase in concentration occurred which was followed by a sharp decline from 7 p&/larva at 150 hr to less than 1 pg/larvae at 160 hr (Fig. 3). The enzymes chitobiase, trehalase, alkaglutamate-oxalacctate line phosphatase, transaminasc, and o-diphenol oxidase (Figs. 7-11) demonstrated similar development patterns in uninfected populations: an increase in activity with an increase in larval age. Glutamate-oxalacetate transaminase demonstrated a similar pattern, but a decrease was observed in the later stages of development, at about 180 hr (Fig. 10). In infected populations, chitobiase, trehalase, and alkaline phosphatase exhibit,ed an increase in activity during the first. 15 hr of infection, with a decline occurring after this period (Figs. 7-9). The enzymes o-diphenol oxidase and glutamateoxalacetate transaminase, however, demonstrated different patterns in infected popu-
BIOCHEMISTRY
50-
0
2400
/
,= b 30 x fn C
J’ /
J’ /
<20z Ii
/ d m---o
.
IO iA,0
140
160
160
200
HOUCS
Fro. 7. Time-course development in normally developing Culex larvae and infected C&x larvae (O-----e).
MOSQUITO
301
IXFECTIOIi
zymes, but the increase lasted through the first 50 hr of infection at a rate equal to that of the uninfected population. At the end of this period, the enzyme activity in t,he infect,4 populations dropped sharply, whereas the activity in the uninfected populations increased (Fig. 10). The enzyme o-diphenol oxidase (Fig. 11) exhibited an immediate decline in activity observed 30 hr after the initiation of infection. The behavior of t,his enzyme was of interest since it tended to disappear on infection. When t,hese data were plotted as specific activity (Fig. 12)) it was noted that there was a considerable stimulation of the enzyme in the infected relative t.o the control followed by a rapid decline of enzyme. Specific activity plots of the data simply show a general decrease of the enzyme in infected
-
/
ti
: 0
OF
of chitobiasc (O-----O)
‘““I’
_;,
1
h Fro. S. Effect of trchalase development and (O-----O) (O-----O).
Lagenicliwn in norma! infected
infection on C&x larvae CUkX larvae
,o 3 E
OL 100
’
I
I
I
120
I
140
I
,
I
160
,
180
_ 200
Hours
FIG. 10. iransaminase and infected
Responses of glutamate-oxalacetate in normai Culex larvae (0 -----0) larvae (a-----@).
Q / :
-20-o FIG.
normal fected
9. Behavior Culex larvae by Lagenidium
:' P Hours
of alkaline phosphatnse (O-----O) and larvae spores (O-----a).
/' in in-
lations. The transaminase increased in the infected larvae as did the other three en-
p-0' ,+A I , 120
/' I
,
140
I
I_
160
180
I 200
HOUB FIG.
normal infected
11. Behavior of o-diphenol Culex larvae (O-----O) with Lagenidiun~ (a-----
oxidase in and larvae 0).
302
DOMNAS,
I
1
,
,
,
I
,
GIEBEL
,
0.6 -
FIG. 12. Specific activities of o-diphenol in normal and infected Culez larvae.
oxidase
when compared to normal larvae. As explained by Cant.ino (1961), it is best to use a natural biological unit, like the larva, since it shows the amount of enzyme in the organism. DISCUSSION
One of the major problems which confronted us was a method of separating the fungus from the infected larva in order to assay various parameters. In our studies on the fungus, we observed that mature cultures could be subjected to pressure of 2000 psi in the Parr Nitrogen Disruption Bomb without cell breakage occurring. Uninfected larvae of Mea, however, could be disrupted at pressure between 500-1000 psi. Thus, when infected larvae (late Stage II, Stage III, and Stage IV) were subjected to pressures of 500 psi in the Parr disruption bomb, the larvae were completely disrupted but the fungal subthalli remained intact (Fig. 2C). Stage I and early Stage II infectons, however, could not be separated because the mycelial matter was very fragile and ruptured under 500 psi pressure, but it is very unlikely that significant amounts of parasitic protein are mixed with host material. The cell-free larval extract was assayed for the fungal enzymes p-glucosidase and glucose-Bphosphate tlrhyclro-
ANDMCINNIS
gcnase (Lang and Stephan, 1967) and no significant. activity was found (Table 1). It would appear from these controls that little fungnl material was present in the larval extract. Another problem of concern to us was the synchrony of infection. Were the larvae being infected in a short time period, or were the infections occurring over a broad time span? Larvae at the same stage of infecti;n were harvested and their protein concentration compared with that of another infected population whose larvae were randomly sampled. The protein profiles of two experiments were found to be quite clot, and it was concluded that the infection was occurring in a short time period. Infection of the larvae occurs via spores which pen&ate through the integument (Fig. 3). Similar observations have been reported with other fungi (Benz, 1963), but the mechanism of penetration is not known. Some investigators have speculated on the role of chitinase (Benz, 1963), but the presence of this enzyme has not been demonstrated in a11cxscs. A chitinase was not detccted in I,. giganteum, and growth of the fungus on chitin and glucosamine was poor to zero. Our data, however, does not specifically exclude the possibility that the spore may have a chitinase system, since we have not yet, been able to investigate its physiology and biochemistry. Penetration of the integument could conceivably occur via proteolytic and lipolytic activity, with the hyphal initial pushing through weaker parts of the integument or amorphous regions of the chitin layer via mechanical action ; a nonspecific lipase and a proteases have been detect’ed in L. giganteum, with both enzyme systems having relatively high activity (Table 2). Once initial infection had occurred. Ilowever, rapid growth of the fungal parasite ensued, with larval death 60-72 hr later. Protein synthesis in infected larvae slowed down almost from the first day of infection, continued to decelerate, and wcntunlly ccnscd. The data shown here
BIOCHEWSTRY
OF
MOSQUITO
were obtained from still living organisms, yet, “dead” larvae possessed measurable protein and residual enzymic activities. ,4t death, trehalase and glutamate-oxaloacet,ate transminase were still detectable; the free amino acids were very low, and chromatograms of extracts from dead larvae showed that trehalose was absent and glucose was very low. The loss of necessary amino acids would of necessity stop protein synthesis even though the protein synthesizing systems were still present. The results, in effect, seemed to indicate that the organisms were dying of starvation, a conelusion also reached in the case of the Japanese beetle milky disease. (Bulla and St.. *Julian, 1972). One parameter presented aspects anomalous to the overall general pathological picture. The enzyme, o-diphenol oxidase, disappeared in extracts of infected larvae in about 40 hr. The behavior of this enzyme was interesting because of its association with clit,icle sclerotization (Hackman, 1971). Since t’he concentration of enzyme/larva dropped after penetration of the integument, i.e., Stage I, it is unlikely that the enzyme was being inactivated because of a reaction initiated by spore penetration. It would appear more likely that inactivation could be a supression of a defense mechanism. The disappearance of o-diphenol oxidase could indicate an inhibiCon at the biosynthetic or even at the subunit assembly stage (Jolley and Mason, 1965). Further work should help to clarify this point, particularly with regard to tyrosine levels and other aromatic amino acids. REFERENCES BENZ,
G. 1963. “Insect Pathology” (E. A. Steinhnus, ed.) Vol. 1, pp. 229-338. Academic Press, New York. BULLA, L. A., AND ST. JULIAN. G. 1972. Biorhemistry of milk disease: Radiorespirometry of ppruvate. acetate, succinate, and glutamate oxidation by healthy and diseased Japanese beetle larvae. J. Znvertebr. Pathol., 19, 12&124. CANTINO, E. C. 1961. The relationship between
INFECTION
303
biochemical and morphological differentiation in nonfilamentous aquatic fungi. Symp. Sot Geta. Microbial., 11, 243-271. DAVID, W. A. L. 1968. “Insects and Physiology” (J. W. L. Beament and J. E. Treheme, Eds.). 1st ed., 17-35. American Elsevier, New York. DUBOIS, M., GILLES, K. A., HAMILTON. J. K., REBERS, P. A. ASD SMITH, F. 1956. Colorimetric method for determination of sugars and related substan:*es. A&. Chem., 28, 350-356. FLING. M., HOROWITZ, r\T. H., AND HIGINEMAN, S. F. 1963. The isolation and properties of crystalline tyrosinase of Neurospora. J. Biol. Chem., 238, 2045-2053. GLEASON, F. H. 1968. Nutrional comparisons in the Leptomitales. Am. J. Botany, 55, 1003-1010. HACKMAN, R. H. 1971. The integument of the arthropoda. In “Chemical Zoology, VI. Arthropoda, Part B” (M. Florkin and B. T. Scheer, Eds.). Academic Press, New York. JOLLEY, R. L., AND MASON, H. S. 1965. The multiple forms of mushroom tyrosinase. J. Biol. Chem., 240, 1489-1490. LANG, E. A., AND STEPHAS, J. K. 1967. Nicotinamide-adenine dinucleotide phosphate enzymes in the mosquito during growth and aging. Biochem. J., 102, 331-337. LEWIS, B. A. AND SMITH, F. 1969. Sugars and derivatives. In “Thin Layer Chromatography”. (E. Stahl, ed.), pp. 507-837. Springer-Verlag, ?;cw York. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. AND RANDALL, R. J. 1951. Protein measurement with Folin phenol reagents. J. Biol. Chem., 193, 323-335. MADELIN, M. F. 1963. Insect Pathology, (E. A. Steinhaus, Ed.), Vol. 2, pp. 233-271. Academic Press, New York. MCINNIS, T. M., AKD DOMSAS, A. 1973. The properties of trehalase from the mosquito parasitizing mater-mold, Lngenidium sp. J. Invertebr. Pathol., 22, 313-320. PROCARO, C. A. 1963. The deteztion of trehalose in the larval hemolymph of Tororhynchitis tntilis Coker. MS. thesis, Department of of North Carolina, Zoology, University Chapel Hill, XC. ROSEN, H. 1957. A modified ninhydrin calorimetric analysis for amino acids. Arch. Biochem. Biophys., 67, 10-15. S.~NASSI, 8., AND OLIVER, J. H. 1971. Integument of the velvet mite, Dinothrombeum giganteum and histopathological changes caused by the fungus Aspergil1lL.s flavus. J. Invertebr. Pathol., 17, 354-365. TREBATOSKI, A. M., AND HAYNES, J. F. 1969. Comparison of enzymes of twelve specirs of
304
DOMNAS,
GIEBEL
mosquitos. Ann. Entomol. Sot. Amer., 62, 327-335. UMPHLETT, C. J. 1973. A note to identify a certain isolate of Lagenidium which kills mosquito larvae. Myeologia, 65, 970-972. UMPHLETT, C. J., AND HUANG, C. 1972. Experi-
AND
MCINNIS
mental infection of mosquito larvae by speties of the aquatic fungus Lagenidium. J. Invertebr. Pathol., 20, 321326. WILLOUGHBY, L. G. 1969. Pure culture studies on the aquatic phycomycete Lagenidium giganteum. Trau-. &it. Mvcol. Sot., 52, 393-410.