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Microorganisms associated with the two-spotted spider mite Tetranychus urticae
JOURNAL
OF
INVERTJCBJl.ITJ:
PATHOLOGY
Microorganisms
17,
48-52 (1971)
Associated Mite
with
the
Tetranychus
Two-Spotted
Spider
urticae’
H. D. SOLOGIC* AND J. G. RODRIGUEZ? Depariment
of Entomology,
Universily Received
oj Kentucky, April
Lezinyton,
Kenlucky
.JO606
9, 1970
Microorganisms were isolated from samplings of I’etranychus urticae from a greenhouse bean culture. Twenty-five isolations were made over the period of 1 year; 4 organisms were found consistently, with a 5th isolated only occasionally. All bacteria occurring internally were also found externally. More than 30 generations of germfree mites were reared on bean seedlings in the environment of an isolator with no apparent ill effects. Six common antibiotic materials were incorporated in a synthetic diet and fed to developing mite adults; none of the materials produced harmful effects when fed at 1000 ppm. Overall conclusions reached were that the microorganisms found in association with 1’. ztrlicae are not essent.ial to its well-being.
pathogenic on acarines are also limited; some attention has been given to tetranychid In our continuing studies in the nutritional physiology of the two-spotted spider mites in particular. Steinhaus (1959) ; Smith mite, Tetranychus urticae, it was deemed et al. (1959); Smith and Cressman (1962); prudent to investigate the nature of micro- and Gilmore and Munger (1963) have organisms that may be found in association studied various aspects of virus diseasesof with the phytophagous mite. It is of para- the citrus red mite. Carver and Canerday mount importance to know, for example, if (1968) have reported on studies with a any microorganism may be contributing to fungus pathogenic to Tetranychus spp. the nutritional requirements of this species. Apparently there is no record of pathogenic The nature and significance of microorga- bacteria attacking mites. At least 1 study of the microorganisms nisms associated with acarines in general associated with tetranychid mites has been have received little attention. It is known reported (Bailey and Canerday, 1968), and that various ticks, which are all blood this was conducted concurrently with our feeders, have microorganisms or rickettsiastudy. They collected 3 species of spider like intracellular symbionts (Steinhaus 1946, mites from cotton in several areas of Ala1949). Unlike ticks, certain other bloodbama. They removed any external microsucking mites have mycetomes made up of organisms from the mites by washing the symbiont-containing mycetocytes. latter with a series of detergent rinses. Any The literature and data on microorganisms microorganisms that) survived were con1 The investigation was supported in part by sidered to be of internal origin. INTRODUCTION
National Science Foundation Grants GB-4293 and GB-20339. This paper (70-7-38) is published with the permission of the IIirect,or of the Kentucky Agricultural Experiment Station. 2 Graduate Assistant and Professor of Ent,orespectively. The authors gratefully molow, acknowledge the assistance of L)r. It. H. Weaver, Professor of Microbiology, University of Kentucky, in the development of this study.
METHODS
AND PROCEDURE
To st’udy the microorganisms associated internally with the mite it was first necessary to develop a suitable isolation technique. Several procedures were compared for efhciency; the one selected consisted of 48
MLCROORGANISMH
ASSOCIATED
rxt tbrnal &erilization of the mites followed by macrrntion. _\[it#rs were rinsed directly from foliage into xkrile vials with a ,‘iooo solution of mercuric chloride. The vials were sealed and agitated for about 2 min, aft,er which the holutioll was decanted off, the mites were rinsed 8 tdmes with sterile water and finally rinsed wklr t,hioglycolate broth. This rinse was incubat.ed and observed as a control on tht, external st,erilization procedure. Approximately 300 of t,he externally st,erilizfbd, rinsed mites were macerated in a hanging drop slide with a glass rod and suspended in 2 ml of thioglycolate broth; this suspension was used as t’he inoculum for a second tube containing about, 10 ml of t,hioglycohte brotll. A third tube was inoculated wit,h 3 ml of broth subject,ed to the maceration procedure, without mites, as a st.eri1it.v cont,rol on the procedures and materials. All t,ubes were incubated at, 27°C. Tlltt sterility control was observed at intervals for 2 wk. If no growth was observed after 2-I hr, streak plates were made from the cult ur(B tubes onto nut’rient agar, brainheart infusion agar, and Sabouraud agar. Isolates ivrre selected from these plates for further study on the basis of differences in coloni:d morphology and gram reactions, and wt’rc rrstrcalied to ensure a pure cult,ure (if growth developed in the control tube srlbsrcluellt to the 24hr observation, isol:tt f’s from these plates were discarded). Stock cultures of the isolates were maint:tint4 OII nutrient agar and Kligler’s Iron :~g:ir. T\\cnty-five successful isolations were made from :I single greenhousepopulatBionof 7’. ,W~~NW reared on bean plants over a period of 1 gwr. I\licroorg:misms occurring externally were isolnt~cdby rinsing the mites taken from the greenllouse cult,ure with 2 ml thioglycolate broth, and t.his rinse was used as the inocuIum for tubes of the samebrot,h. No nt#tempt w:is mndc to identify all organisms ioslated, :k ttrth purposf~ fat* these isolat,ions was
WITH
XITE
4!i
simply to determine whether t,he organisms isolat,ed internally were peculiar to the gut or also occurred ext,ernally. Isolation techniques used were identicsl to those for internal examination. Colonieb on isolation plates resembling those found from internal isolations were selected YJIC I compared, with the int,ernal isolatrs t’ol, reaction with standard biochemical identifi cation tests. Gut smears were made by piercing thc~ body wall and cecum of the mite with a glass microcapillary tub?. The capillary tube and it,s cont,ents were crushed on a slide, smearrtl, air-dried, and gram stained for examination I. To ascertain whet,lter ?‘. IL&W could livcb successfully without microorganisms, mitt’s were reared aseptically from surface-strri lized eggs on sterile bean plants and froni surface-sterilized deutonymphs OJ~ :I chtmi tally defined diet incorpor:lt.ing various anti biotics. In the first procedure, bean seeds wart’ gas-sterilized with methyl bromide, t 11(‘11 germinated in sterile ago pour pkes. Tht* sterile seedlings were then transferred to small dishes of sterile sand in an aseptic glove box for continued growth. Tn-o-day-oltl mite eggs were surface-sterilized by holding in 5 5%formaldehyde for .3--Bmin, rinsed x times with st,erile distilled water, and transferred to sterile plant tissue in petri disllr,-. After hatching, the sterile mites were tlltlll t,ransferred to the sterile plants in t tttx ~IOVP box. Sterility controls with thioglycol:~t t’ broth accompanied tach stage of t 11(xoptar:lCon: tllr: production of bean seedlings, t 11~1 sterilization of tht> tyqp, mid the tr:msffkr of the mites. In the secortd procedure, mitts :Lborlt. three-quarters of thta n-ay into tIlta deutonymphal quiescent, st,agewere sterilized \vitlr 5 9’0formalin, rinsed, and iransferrrd to tile feeding chamber u’llcre they emerged ;W adults. The method of rearing mites :uld the chemically defined diet as describ4 by Walling rt al. (1968) wery Iwed. \‘:kriolw
50
SOLOGIC AND RODRIGUEZ
antibiotics were incorporated into the diet at only occasionally. TKO gram-negative rods concentrations ranging from 1000 to 10,000 of the family Enterobncteriacene, 1 gramppm, and t,he percent mortalit,y was re- positive spore-forming bacillus, and 1 gramcorded after a period of 10 days (Fig. 1). positive non-spore-forming rod were obSteriUy tests were made from time to time tained with consistent frequency. A gram positive staphylococcus was observed 5 which proved to be negative. times. RESULTS All bacteria occurring internally were also found externally. It is of interest to note Ohat The number and general type of organisms although fungi were consistently found found in this study are shown in Table 1. externally none were ever recovered from From 25 isolations made, 4 organisms were internal isolations. Isolations were made over found consist’ently, with a 5th being isolated a period of a year with no change in the microflora being observed. Examination of TABLE 1 gut smears corroborated the data obt’ained MICROORGANISMS ISOLATED FROM EXTERNALLY by isolation procedure. STERILIZED Tetranychus urticae, OVER A PERIOD OF A ~-YEAR SAMPLING PROGRAM IN WHICH It was found possible to rear this mite in 25 SAMPLES WERE STUDIED the aseptic environment of a glove box using germfree procedure with no apparent detriFrequency mental effect#sfor more than 30 generations. Organism of The antibiotic materials tested, with t*he occurrence exception of Aureomycin at exceedingly high dosage of 10,000 ppm, appeared to have no Gm(-) Enterobacteriaceae sp. 1 25 Gm(-) Enterobacteriaceae effect on the mite. Mortalit8y was recorded sp. 2 21 Gm(+) Spore-forming bacillus 21 after a lo-day period of feeding on a chemiGm(+) Non-spore-forming rod 20 cally defined diet (Fig. 1). Variation occurred Gm(+) Staphylococcus 5 in percent mortality in the separat’eexperi-
A B G D E F
-
VANCOMYCIN OXYTETRACYCLINE BACITRACIN STREPTOMYCIN AUBEOMYCIN PENICILLIN
1 2 3 c
HYDROCHLORIDE
-
1000 PPM 5000 PPM 10,000 PPM CONTROL
1 20
A FIG.
material.
1. Percent
mortality
0 of Tetranychus
C urticae
D after
10 days
E feeding
F on diets
containing
antibiotic
MICROORGANISMS
ASSOCIATED
rnents, but there was no significant difference bet\vtw treatment and controls in the individual test)s. Aureomycin at over 6000 ppm is toxic to t,he mite, and its effect is not reIatrtl to tlw sterilization of the mite gut. Tests rnatlt~ for stwility of procedure mere neg:lti\-tb.
l
WITH
MITE
5 I
52
SOLOGIC
Pathology,” York. E. in European 435-437.
STEINHAUS,
757 A. red
pp.
McGraw-Hill,
AND
New
1959. Possible virus disease mite. J. Insect Pathol. 1,
RODRIGUEZ
M. V., WHITE, D. C. AND RODRIGUEZ, J. G. 1968. Characterization, distribution, catabolism, and synthesis of the fatty acids of the two-spotted spider mite, Tetranychus urticae. J. Insect Physiol. 14, 1445-1458.
WALLING,
OF
INVERTJCBJl.ITJ:
PATHOLOGY
Microorganisms
17,
48-52 (1971)
Associated Mite
with
the
Tetranychus
Two-Spotted
Spider
urticae’
H. D. SOLOGIC* AND J. G. RODRIGUEZ? Depariment
of Entomology,
Universily Received
oj Kentucky, April
Lezinyton,
Kenlucky
.JO606
9, 1970
Microorganisms were isolated from samplings of I’etranychus urticae from a greenhouse bean culture. Twenty-five isolations were made over the period of 1 year; 4 organisms were found consistently, with a 5th isolated only occasionally. All bacteria occurring internally were also found externally. More than 30 generations of germfree mites were reared on bean seedlings in the environment of an isolator with no apparent ill effects. Six common antibiotic materials were incorporated in a synthetic diet and fed to developing mite adults; none of the materials produced harmful effects when fed at 1000 ppm. Overall conclusions reached were that the microorganisms found in association with 1’. ztrlicae are not essent.ial to its well-being.
pathogenic on acarines are also limited; some attention has been given to tetranychid In our continuing studies in the nutritional physiology of the two-spotted spider mites in particular. Steinhaus (1959) ; Smith mite, Tetranychus urticae, it was deemed et al. (1959); Smith and Cressman (1962); prudent to investigate the nature of micro- and Gilmore and Munger (1963) have organisms that may be found in association studied various aspects of virus diseasesof with the phytophagous mite. It is of para- the citrus red mite. Carver and Canerday mount importance to know, for example, if (1968) have reported on studies with a any microorganism may be contributing to fungus pathogenic to Tetranychus spp. the nutritional requirements of this species. Apparently there is no record of pathogenic The nature and significance of microorga- bacteria attacking mites. At least 1 study of the microorganisms nisms associated with acarines in general associated with tetranychid mites has been have received little attention. It is known reported (Bailey and Canerday, 1968), and that various ticks, which are all blood this was conducted concurrently with our feeders, have microorganisms or rickettsiastudy. They collected 3 species of spider like intracellular symbionts (Steinhaus 1946, mites from cotton in several areas of Ala1949). Unlike ticks, certain other bloodbama. They removed any external microsucking mites have mycetomes made up of organisms from the mites by washing the symbiont-containing mycetocytes. latter with a series of detergent rinses. Any The literature and data on microorganisms microorganisms that) survived were con1 The investigation was supported in part by sidered to be of internal origin. INTRODUCTION
National Science Foundation Grants GB-4293 and GB-20339. This paper (70-7-38) is published with the permission of the IIirect,or of the Kentucky Agricultural Experiment Station. 2 Graduate Assistant and Professor of Ent,orespectively. The authors gratefully molow, acknowledge the assistance of L)r. It. H. Weaver, Professor of Microbiology, University of Kentucky, in the development of this study.
METHODS
AND PROCEDURE
To st’udy the microorganisms associated internally with the mite it was first necessary to develop a suitable isolation technique. Several procedures were compared for efhciency; the one selected consisted of 48
MLCROORGANISMH
ASSOCIATED
rxt tbrnal &erilization of the mites followed by macrrntion. _\[it#rs were rinsed directly from foliage into xkrile vials with a ,‘iooo solution of mercuric chloride. The vials were sealed and agitated for about 2 min, aft,er which the holutioll was decanted off, the mites were rinsed 8 tdmes with sterile water and finally rinsed wklr t,hioglycolate broth. This rinse was incubat.ed and observed as a control on tht, external st,erilization procedure. Approximately 300 of t,he externally st,erilizfbd, rinsed mites were macerated in a hanging drop slide with a glass rod and suspended in 2 ml of thioglycolate broth; this suspension was used as t’he inoculum for a second tube containing about, 10 ml of t,hioglycohte brotll. A third tube was inoculated wit,h 3 ml of broth subject,ed to the maceration procedure, without mites, as a st.eri1it.v cont,rol on the procedures and materials. All t,ubes were incubated at, 27°C. Tlltt sterility control was observed at intervals for 2 wk. If no growth was observed after 2-I hr, streak plates were made from the cult ur(B tubes onto nut’rient agar, brainheart infusion agar, and Sabouraud agar. Isolates ivrre selected from these plates for further study on the basis of differences in coloni:d morphology and gram reactions, and wt’rc rrstrcalied to ensure a pure cult,ure (if growth developed in the control tube srlbsrcluellt to the 24hr observation, isol:tt f’s from these plates were discarded). Stock cultures of the isolates were maint:tint4 OII nutrient agar and Kligler’s Iron :~g:ir. T\\cnty-five successful isolations were made from :I single greenhousepopulatBionof 7’. ,W~~NW reared on bean plants over a period of 1 gwr. I\licroorg:misms occurring externally were isolnt~cdby rinsing the mites taken from the greenllouse cult,ure with 2 ml thioglycolate broth, and t.his rinse was used as the inocuIum for tubes of the samebrot,h. No nt#tempt w:is mndc to identify all organisms ioslated, :k ttrth purposf~ fat* these isolat,ions was
WITH
XITE
4!i
simply to determine whether t,he organisms isolat,ed internally were peculiar to the gut or also occurred ext,ernally. Isolation techniques used were identicsl to those for internal examination. Colonieb on isolation plates resembling those found from internal isolations were selected YJIC I compared, with the int,ernal isolatrs t’ol, reaction with standard biochemical identifi cation tests. Gut smears were made by piercing thc~ body wall and cecum of the mite with a glass microcapillary tub?. The capillary tube and it,s cont,ents were crushed on a slide, smearrtl, air-dried, and gram stained for examination I. To ascertain whet,lter ?‘. IL&W could livcb successfully without microorganisms, mitt’s were reared aseptically from surface-strri lized eggs on sterile bean plants and froni surface-sterilized deutonymphs OJ~ :I chtmi tally defined diet incorpor:lt.ing various anti biotics. In the first procedure, bean seeds wart’ gas-sterilized with methyl bromide, t 11(‘11 germinated in sterile ago pour pkes. Tht* sterile seedlings were then transferred to small dishes of sterile sand in an aseptic glove box for continued growth. Tn-o-day-oltl mite eggs were surface-sterilized by holding in 5 5%formaldehyde for .3--Bmin, rinsed x times with st,erile distilled water, and transferred to sterile plant tissue in petri disllr,-. After hatching, the sterile mites were tlltlll t,ransferred to the sterile plants in t tttx ~IOVP box. Sterility controls with thioglycol:~t t’ broth accompanied tach stage of t 11(xoptar:lCon: tllr: production of bean seedlings, t 11~1 sterilization of tht> tyqp, mid the tr:msffkr of the mites. In the secortd procedure, mitts :Lborlt. three-quarters of thta n-ay into tIlta deutonymphal quiescent, st,agewere sterilized \vitlr 5 9’0formalin, rinsed, and iransferrrd to tile feeding chamber u’llcre they emerged ;W adults. The method of rearing mites :uld the chemically defined diet as describ4 by Walling rt al. (1968) wery Iwed. \‘:kriolw
50
SOLOGIC AND RODRIGUEZ
antibiotics were incorporated into the diet at only occasionally. TKO gram-negative rods concentrations ranging from 1000 to 10,000 of the family Enterobncteriacene, 1 gramppm, and t,he percent mortalit,y was re- positive spore-forming bacillus, and 1 gramcorded after a period of 10 days (Fig. 1). positive non-spore-forming rod were obSteriUy tests were made from time to time tained with consistent frequency. A gram positive staphylococcus was observed 5 which proved to be negative. times. RESULTS All bacteria occurring internally were also found externally. It is of interest to note Ohat The number and general type of organisms although fungi were consistently found found in this study are shown in Table 1. externally none were ever recovered from From 25 isolations made, 4 organisms were internal isolations. Isolations were made over found consist’ently, with a 5th being isolated a period of a year with no change in the microflora being observed. Examination of TABLE 1 gut smears corroborated the data obt’ained MICROORGANISMS ISOLATED FROM EXTERNALLY by isolation procedure. STERILIZED Tetranychus urticae, OVER A PERIOD OF A ~-YEAR SAMPLING PROGRAM IN WHICH It was found possible to rear this mite in 25 SAMPLES WERE STUDIED the aseptic environment of a glove box using germfree procedure with no apparent detriFrequency mental effect#sfor more than 30 generations. Organism of The antibiotic materials tested, with t*he occurrence exception of Aureomycin at exceedingly high dosage of 10,000 ppm, appeared to have no Gm(-) Enterobacteriaceae sp. 1 25 Gm(-) Enterobacteriaceae effect on the mite. Mortalit8y was recorded sp. 2 21 Gm(+) Spore-forming bacillus 21 after a lo-day period of feeding on a chemiGm(+) Non-spore-forming rod 20 cally defined diet (Fig. 1). Variation occurred Gm(+) Staphylococcus 5 in percent mortality in the separat’eexperi-
A B G D E F
-
VANCOMYCIN OXYTETRACYCLINE BACITRACIN STREPTOMYCIN AUBEOMYCIN PENICILLIN
1 2 3 c
HYDROCHLORIDE
-
1000 PPM 5000 PPM 10,000 PPM CONTROL
1 20
A FIG.
material.
1. Percent
mortality
0 of Tetranychus
C urticae
D after
10 days
E feeding
F on diets
containing
antibiotic
MICROORGANISMS
ASSOCIATED
rnents, but there was no significant difference bet\vtw treatment and controls in the individual test)s. Aureomycin at over 6000 ppm is toxic to t,he mite, and its effect is not reIatrtl to tlw sterilization of the mite gut. Tests rnatlt~ for stwility of procedure mere neg:lti\-tb.
l
WITH
MITE
5 I
52
SOLOGIC
Pathology,” York. E. in European 435-437.
STEINHAUS,
757 A. red
pp.
McGraw-Hill,
AND
New
1959. Possible virus disease mite. J. Insect Pathol. 1,
RODRIGUEZ
M. V., WHITE, D. C. AND RODRIGUEZ, J. G. 1968. Characterization, distribution, catabolism, and synthesis of the fatty acids of the two-spotted spider mite, Tetranychus urticae. J. Insect Physiol. 14, 1445-1458.
WALLING,