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Initiation and orientation of the symbiote migration in the human body louse Pediculus humanus L.
J. Invrcr Physiol.. Vol. 28. No. 5. pp. 417-422, 1982 Printed in Grrut Britain
0022-1910/82/050417-06$03.00/O 0 1982 Perqumon
Press Lrtl
INITIATION AND ORIENTATION OF THE SYMBIOTE MIGRATION IN THE HUMAN BODY LOUSE PEDICULUS HUMANUS L. M. W. EBERLE and D. L. MCLEAN Department
of Entomology, (Rrceiced 27
University
May
of California.
Davis, CA 95616. U.S.A
1981; rerised6 Norember
1981)
Abstract-- Bacterial symbiotes in the human body louse Pedichs humanus migrate from the mycetome to the lateral oviducts during the adult moult. Experimental results are presented suggesting that the symbiote migration is initiated by a humoral factor associated with the adult moult: that the factor is present in both sexes but the male mycetome is unable to respond to it; and that the symbiotes arc attracted by the female reproductive tract. Ke,r Word 1nde.u. Pediculus humanus, symboite, mycetome, migration
INTRODUCTION THE:HUMANbody louse Pediculus humanus L. depends upon bacterial symbiotes for the B vitamins nicotinic acid. pantothenic acid and beta-biotin (PUCHTA 1955, B~JC‘HNER 1965). These symbiotes are transmitted across the ovary to all of the female louse’s progeny. However, before transovarial transmission can occur, the symbiotes must migrate from a mycetome located on the ventral surface of the midgut to the female reproductive tract. RIES (1931) reported that this transfer occurs between the apolysis and the ecdysis of the adult moult. The symbiotes exit from the mycetome. move posteriorly on the ventral surface of the midgut directly to the lateral oviducts and invade the lateral oviduct cells (Figs. la, 2, 3). The migration completely empties the mycetome of symbiotes. From the lateral oviduct cells, the symbiotes move through the ovarian pedicels into the developing eggs (Fig. lb). The male louse, unlike the female, permanently retains its symbiotes in the mycetome. We have attempted to answer three questions pertaining to this symbiote migration that have not been asked previously: (a) how is the migration initiated in the female; (b) why is the migration absent from male lice; and (c) how is the migration oriented toward the female reproductive tract? To answer question (a). we tested three hypotheses: (1) the symbiote migration is initiated by a brain hormone: (2) the migration is initiated by a hormone from the female reproductive tract; and (3) the migration is initiated by a factor associated with the adult mouit. These hypotheses were tested by, respectively, decapitating lice three days before the symbiote migration; removing the female reproductive tract 9 days before the symbiote migration; and preventing or inducing the adult moult with hormonal treatments to determine if the symbiote migration would be correspondingly prevented or induced. To answer question (b). we tested two hypotheses: ( I ) the symbiote migration does not occur in male lice because males lack the stimulus that initiates the 417
migration; and (2) the initiating stimulus for the migration is present in both sexes. but the male mycetome is unable to respond to it. The first hypothesis was tested by transplanting female mycetomes into male lice 3 days before the symbiote migration. If the female mycetomes fail to function normally in the male lice, the hypothesis is confirmed. The second hypothesis was tested by transplanting male mycetomes into female lice 3 days before the migration; this hypothesis is confirmed if the male mycetomes fail to respond to the initiating stimulus present in the female lice. For the question of how the symbiote migration is oriented toward the female reproductive tract (question c), we hypothesized that the migrating symbiotes are attracted by the female reproductive tract. This hypothesis was tested by transferring the female reproductive tract from its usual position posterior to the mycetome to a position anterior to the mycetome 3 days before the symbiote migration. We observed whether the migrating symbiotes changed the direction of their migration in response to the change in the location of their target. MATERIALS
AND
METHODS
Organisms The Culpepper strain of Pediculus humanus was used in our experiments; this strain is adapted for feeding on rabbits and thus removes the need for a human host. The Culpepper strain was obtained from Insect Control and Research, Inc. of Baltimore, Maryland. Following Cole’s recommendations (Cole 1966). we maintained the colony at 2883OC and approx. 607; r.h. The lice resided on corduroy patches stacked in petri dishes. Cloth was a suitable substrate because human body lice normally live and oviposit in clothes. The lice were fed daily by placing the corduroy patches on the shaved abdomen of a rabbit immobilized in a stanchion. Within 30min, the lice migrated to the skin of the rabbit. engorged and
418
M. W. EBERLEand D. L. MCLEAN
returned to the corduroy patches. The lice exhibited little tendency to wander into the fur of the rabbit, and any strays were removed from the rabbit after feeding. The corduroy patches were changed on a daily basis while the lice were ovipositing, and the eggs that were oviposited within each 24 hr period were raised as a separate cohort. The major events of the lifecycle were predicted according to the following timetable, which takes the date of oviposition as day 1: hatching, day 10; first nymphal moult, day 14; second nymphal moult. day 17; adult moult with symbiote migration, day 20. Surgical
procedures
These procedures included decapitation, transplantation of mycetomes, removal of female reproductive tracts and transfer of female reproductive tracts to a position anterior to the mycetome. In all procedures, the lice were first immobilized by pressing them against the adhesive surface of a piece of black electrical tape attached by transparent double-coated tape to the bottom of a small petri dish. They were immersed in saline composed of 9.0 g NaCl, 0.2 g KCI. 0.2 g MgCl. 0.2 g CaCl,, 0.2 g NaHCO, and 8.0 g glucase dissolved in 1000 ml of distilled water. The saline
Fig. l(a). Diagram Fig. I(b). Diagram
of symbiotes of symbiotes
was decanted after surgery to allow the surgical wound to close through the coagulation of haemolymph. Lice were decapitated by ligaturing between the head and thorax with a human hair, then removing the head with a No. 15 scalpel blade. To prevent starvation, headless lice were fed daily with titrated human blood injected rectally. The apparatus for injecting the blood consisted of a fine capillary tube attached by a flexible hose to a syringe pump (Sage Instruments). The portion of the line not filled with blood was filled with the saline previously described. The tip of the capillary tube was inserted into the rectum of the louse, and a volume of blood equivalent to a normal blood meal was injected. The blood flowed freely from the rectum into the midgut and appeared to be digested normally. Mycetomes were transplanted by puncturing the ventral abdominal cuticle of the mycetome donor to one side of the mycetome and drawing the midgut and the attached mycetome out of the puncture with foreceps (Dumont No. 5). The mycetome was detached by trimming away the midgut with a No. 15 scalpel blade. To minimize osmotic damage, the mycetome was immediately reimplanted into a recipient louse by puncturing the abdominal cuticle
migrating from the mycetome to the lateral human body louse. After RIES (1931). from lateral
oviduct cells infecting RIES (1931).
a develo;;ng
oviducts
in the female
egg in the ovary.
After
Fig. 2. Scanning
electronmicrograph
of symbiotes
exiting
from the female mycetome.
Fig. 3. Scanning
electronmicrograph
of symbiotes
invading
a lateral
oviduct
-
_-- --
__- --
-
-
-_
-_
-.
-_“. -..
-..
-.,
-I
-..
Fig. 4. Transmission electronmicrograph showing a band of striated muscle (arrow) one of the chambers that contain symbiotes (s) in the mycetome.
-”
-_
-
encircling
-
-
-
-
-
-
-
-
_-
..-- ,-
.-
-,- -
-
-
--
-
Fig. 5. Transmission electronmicrograph showing bands of striated muscle (arrows) a symboite-containing chamber. Muscles are cut in cross-section.
-~-.li--‘-~‘._.“_ -,...,,-”
encirchg
Initiation and orientation of symbiote migration and inserting the mycetome through the puncture with the aid of a slender probe constructed from a capillary tube drawn out over a flame. Female reproductive tracts were removed by puncturing the ventral abdominal cuticle over the median oviduct. grasping the median oviduct with forceps and drawing the median oviduct, the lateral oviducts and the ovaries out of the louse. If the intent was to transfer the reproductive tract to a position anterior to the mycetome, a second puncture was made anterior to the mycetome and the reproductive tract was inserted through it and positioned against the midgut surface at approximately the same distance from the mycetome as it normally lies.
These consisted of preventing the adult moult by topically applying a juvenile hormone analogue and inducing the adult moult by injecting 20-hydroxyecdysone. The juvenile hormone analogue (1 l-methoxy-3,7,1 l-trimethyl-2,4-dodecadienate; Altosid 5E, Zoecon Co.) was applied to corduroy patches at a concentration of 1000 pg/cm*. This concentration was obtained by diluting Altosid SE (0.6g of active ingredient per ml) 1:lO with acetone, then combining 3.27 ml of this dilution with 2.50 ml of vegetable oil (added as a spreading agent) and 4.23 ml of acetone. One ml of this mixture was applied to each 5 cm’ patch. Treated patches were always used singly, never stacked. Lice were placed on the treated patches 2 days after the moult to the second instar and remained on them permanently. While on the treated patches, they moulted through three further nymphal instars and died attempting to moult to a fourth. The treatment prevented the adult moult by allowing only nymphal moults. The 20-hydroxy-ecdysone (Calbiochem) was dissolved in 95’)~”ethanol and diluted with the saline described previously to a concentration of 6pg/pl in 4”” ethanol. Two ~1 were injected into the haemocoel of each louse with the apparatus used for rectal-blood feeding. The adult moult occurred 2 days after the injection. Normal lice could not be used with ecdysteroid injections because there was too little time between their second nymphal moult and their adult moult to permit the experimental induction of an adult moult. In their place. lice were used that were unable to moult unless injected with 20-hydroxy-ecdysone. They were obtained by placing second-instar nymphs on Altosid-treated corduroy patches, prepared as described above, 2 days after the moult to the second instar. They were removed immediately after moulting to the third instar, and their reproductive organs were then removed to prevent fatal vaginal prolapse in the fourth instar. Rather than the usual adults, the fourth instars were nymphs that lacked external genitalia and retained the symbiotes in the mycetome. However, because the Altosid treatment was very brief. they retained the adult characteristic of not moulting. They remained fourth-instar nymphs permanently. unless injected with 20-hydroxy-ecdysone whereupon they moulted to adults.
At the conclusion
ary to determine whether symbiote migration had occurred. This was done by dissecting each louse, transferring the mycetome and the reproductive tract to separate drops of saline on the same microscope slide, crushing these organs with gentle pressure on the cover slips, and examining them for symbiotes with a phase-contrast microscope. If the migration had occurred, the symbiotes were found only in the reproductive tract. If the migration had not occurred, the symbiotes were found only in the mycetome.
RESULTS Question itiated?
Hormonal treatments
of each experiment,
it was necess-
421
(a): How
is the symbiote
migration
in-
Experiment 1. Ten female nymphs were decapitated 3 days before the symbiote migration to test the hypothesis that migration is initiated by a brain hormone. Contrary to the hypothesis, migration occurred in all 10 headless lice at the usual time following the apolysis of the adult moult. Experiment 2. Reproductive tracts were removed from 20 female nymphs 9 days before the symbiote migration to test the hypothesis that the migration is initiated by a hormone from the female reproductive tract. Contrary to the hypothesis, symbiotes left the mycetome during the adult moult in all 20 females. We were unable to determine where the symbiotes migrated in the absence of lateral oviducts, their usual target. Experiment 3. Twenty female nymphs were prevented from moulting to adults with topical application of Altosid to test the hypothesis that a factor associated with the adult moult indicates symbiote migration. All 20 failed to initiate the migration though they lived sufficiently long to moult through five nymphal instars. Thus, migration was prevented by preventing the adult moult. In a second test of the hypothesis, 10 female fourth-instar nymphs were injected with 20-hydroxy-ecdysone to induce their adult moult. All 20 initiated the symbiote migration during the moult. As a control, 10 female fourthinstar nymphs were injected with only the ethanolsaline used to dilute the ecdysteroid: none moulted or initiated the migration. Thus, symbiote migration was initiated by initiating the adult moult. These results confirm the hypothesis. Question (b): Why is the migration absent ,from male lice? Experiment 1. Ten female mycetomes were transplanted into male lice three days before the symbiote migration to test the hypothesis that males lack the stimulus that initiates the migration. Contrary to this hypothesis, all 10 female mycetomes released their symbiotes during the adult moult of their male hosts. This result indicates that the initiating stimulus is not transmitted neuronally since the transplanted mycetomes could not have retained intact innervation. Experiment 2. Ten male mycetomes were transplanted into female lice three days before the symbiote migration to test the hypothesis that male mycetomes are unable to respond to the initiating stimulus. All 10 male mycetomes failed to release their sym-
422
M. W. EBERLEand D. L. MCLEAN
biotes, though the female mycetomes of their hosts released theirs. This result confirms that male mycetomes are unable to respond to the initiating stimulus. Question (c): How is the migration oriented toward the reproductive tract?
female
Ten female reproductive tracts were transferred to a position anterior to the mycetome three days before the symbiote migration to test the hypothesis that the symbiotes are attracted by the female reproductive tract. Migrating symbiotes located and invaded all 10. This result has two explanations: either the symbiotes changed the direction of their migration, or they dispersed into the haemolymph and circulated randomly throughout the louse until they reached the lateral oviducts by chance. We believe that the latter explanation is unlikely because RIES (1931) reported that migrating symbiotes do not disperse in the haemolymph but rather move directly from the mycetome to the lateral oviducts, and because it is difficult to envision how hundreds of symbiotes could arrive at the lateral oviducts simultaneously after randomly circulating throughout the louse. Assuming that the symbiotes did reverse the direction of their migration, the results suggest that the symbiotes are attracted by the female reproductive tract.
DISCUSSION How is the symbiote migration initiated? The results indicate that the initiating stimulus is a factor associated with the adult moult. This factor probably does not originate in the brain or the female reproductive tract. It is not transmitted neuronally and therefore probably is humoral. To these results should be added an observation that we made while examining excised mycetomes with a phase-contrast microscope: the mycetomes are capable of muscular contraction. If the mycetome is stripped of its syncytial covering, these contractions are clearly visible and resemble the peristaltic contractions of the midgut. We examined mycetomes with a transmission electron microscope and observed that the symbiote-containing chambers were encircled with numerous bands of striated muscle (Figs. 4, 5). These muscles may play a role in the initiation of the symbiote migration by expressing the symbiotes from the mycetome. The role of the initiating factor may be to increase the frequency and the amplitude of the mycetome’s contractions. Unfortunately, the results provide little information about the identity of the initiating factor. The factor may be a hormone from a site other than the brain or the female reproductive tract; it may be a change in haemolymph chemistry associated with the adult
moult, such as a change in pH; or it may be a combination of factors, such as the low level of juvenile hormone and the high level of 20-hydroxy-ecdysone that accompany the adult moult in many insects. Why is the symbiote migration absent from male lice? The results indicate that male mycetomes are unable to respond to the initiating factor, which is present in both sexes. RIES (1931) reported a difference between the structures of male and female mycetomes that may account for the inability of male mycetomes to respond. Unlike the female mycetome, the male mycetome does not reorganize its internal structure in preparation for the symbiote migration. The female mycetome fuses the l&l6 internal chambers into a central cavity in which the symbiotes are loosely gathered. This reorganization releases the symbiotes from their confinement in the chambers and positions them directly over the site of their future expulsion from the mycetome. Without this preparation, it is difficult to envision how the symbiotes could escape from the mycetome. In contrast, the male mycetome retains the l&16 internal chambers. How is the symbiote migration oriented toward the female reproductive tract’? The results, as we interpret them, suggest that the symbiotes are attracted by the female reproductive tract. The mechanism for this attraction is unknown. Possibly the female reproductive tract releases a symbiote attractant which forms a gradient in the haemolymph that the symbiotes follow to the source. The mechanism for attracting the symbiotes probably is separate from the mechanism that initiates the migration, since the former appears to involve the female reproductive tract while the latter appears to be independent of it. Thus, the regulation of the symbiote migrate may involve two mechanisms, one to initiate the migration and one to orient it. Aclinow/edgements--We are grateful to Dr. EUGENE GERBERG and Mr. M. COLE for providing us with the Culpepper strain of Pediculus humanus. Professors BRUCE HAMMOCK, HENRY HAGADORN, CHARLES JUDSON. ANDY MCCLELLAND, HARRY KAYA and Drs. RICHARD SHUKLE and STEVEN AULT provided valuable suggestions and criticisms. Dr. LYNN S. KIMSEY executed the drawing for Fig, 1.
REFERENCES BUCHNER P. (1965) Endosynbiosis of Animals with PIanr Microoryanisms. Revised English version. Interscience Publishers, New York. COLE M. (1966) Body lice. In Insect Colonization and Mass Producbon (Ed. by SMITH C. N.), pp. 15-24. Academic Press, New York. PUCHTA 0. (1955) Experimentelle Untersuchungen iiber di Bedeutung der Symbiose der Kleiderlaus Pediculus restimenti Burm. Z. ParasitKde. 17, l-40. RYESE. (1931) Die Symbiose der Lause und Federlinge. Z. Morph. Okol. Tiere 20, 233-367
0022-1910/82/050417-06$03.00/O 0 1982 Perqumon
Press Lrtl
INITIATION AND ORIENTATION OF THE SYMBIOTE MIGRATION IN THE HUMAN BODY LOUSE PEDICULUS HUMANUS L. M. W. EBERLE and D. L. MCLEAN Department
of Entomology, (Rrceiced 27
University
May
of California.
Davis, CA 95616. U.S.A
1981; rerised6 Norember
1981)
Abstract-- Bacterial symbiotes in the human body louse Pedichs humanus migrate from the mycetome to the lateral oviducts during the adult moult. Experimental results are presented suggesting that the symbiote migration is initiated by a humoral factor associated with the adult moult: that the factor is present in both sexes but the male mycetome is unable to respond to it; and that the symbiotes arc attracted by the female reproductive tract. Ke,r Word 1nde.u. Pediculus humanus, symboite, mycetome, migration
INTRODUCTION THE:HUMANbody louse Pediculus humanus L. depends upon bacterial symbiotes for the B vitamins nicotinic acid. pantothenic acid and beta-biotin (PUCHTA 1955, B~JC‘HNER 1965). These symbiotes are transmitted across the ovary to all of the female louse’s progeny. However, before transovarial transmission can occur, the symbiotes must migrate from a mycetome located on the ventral surface of the midgut to the female reproductive tract. RIES (1931) reported that this transfer occurs between the apolysis and the ecdysis of the adult moult. The symbiotes exit from the mycetome. move posteriorly on the ventral surface of the midgut directly to the lateral oviducts and invade the lateral oviduct cells (Figs. la, 2, 3). The migration completely empties the mycetome of symbiotes. From the lateral oviduct cells, the symbiotes move through the ovarian pedicels into the developing eggs (Fig. lb). The male louse, unlike the female, permanently retains its symbiotes in the mycetome. We have attempted to answer three questions pertaining to this symbiote migration that have not been asked previously: (a) how is the migration initiated in the female; (b) why is the migration absent from male lice; and (c) how is the migration oriented toward the female reproductive tract? To answer question (a). we tested three hypotheses: (1) the symbiote migration is initiated by a brain hormone: (2) the migration is initiated by a hormone from the female reproductive tract; and (3) the migration is initiated by a factor associated with the adult mouit. These hypotheses were tested by, respectively, decapitating lice three days before the symbiote migration; removing the female reproductive tract 9 days before the symbiote migration; and preventing or inducing the adult moult with hormonal treatments to determine if the symbiote migration would be correspondingly prevented or induced. To answer question (b). we tested two hypotheses: ( I ) the symbiote migration does not occur in male lice because males lack the stimulus that initiates the 417
migration; and (2) the initiating stimulus for the migration is present in both sexes. but the male mycetome is unable to respond to it. The first hypothesis was tested by transplanting female mycetomes into male lice 3 days before the symbiote migration. If the female mycetomes fail to function normally in the male lice, the hypothesis is confirmed. The second hypothesis was tested by transplanting male mycetomes into female lice 3 days before the migration; this hypothesis is confirmed if the male mycetomes fail to respond to the initiating stimulus present in the female lice. For the question of how the symbiote migration is oriented toward the female reproductive tract (question c), we hypothesized that the migrating symbiotes are attracted by the female reproductive tract. This hypothesis was tested by transferring the female reproductive tract from its usual position posterior to the mycetome to a position anterior to the mycetome 3 days before the symbiote migration. We observed whether the migrating symbiotes changed the direction of their migration in response to the change in the location of their target. MATERIALS
AND
METHODS
Organisms The Culpepper strain of Pediculus humanus was used in our experiments; this strain is adapted for feeding on rabbits and thus removes the need for a human host. The Culpepper strain was obtained from Insect Control and Research, Inc. of Baltimore, Maryland. Following Cole’s recommendations (Cole 1966). we maintained the colony at 2883OC and approx. 607; r.h. The lice resided on corduroy patches stacked in petri dishes. Cloth was a suitable substrate because human body lice normally live and oviposit in clothes. The lice were fed daily by placing the corduroy patches on the shaved abdomen of a rabbit immobilized in a stanchion. Within 30min, the lice migrated to the skin of the rabbit. engorged and
418
M. W. EBERLEand D. L. MCLEAN
returned to the corduroy patches. The lice exhibited little tendency to wander into the fur of the rabbit, and any strays were removed from the rabbit after feeding. The corduroy patches were changed on a daily basis while the lice were ovipositing, and the eggs that were oviposited within each 24 hr period were raised as a separate cohort. The major events of the lifecycle were predicted according to the following timetable, which takes the date of oviposition as day 1: hatching, day 10; first nymphal moult, day 14; second nymphal moult. day 17; adult moult with symbiote migration, day 20. Surgical
procedures
These procedures included decapitation, transplantation of mycetomes, removal of female reproductive tracts and transfer of female reproductive tracts to a position anterior to the mycetome. In all procedures, the lice were first immobilized by pressing them against the adhesive surface of a piece of black electrical tape attached by transparent double-coated tape to the bottom of a small petri dish. They were immersed in saline composed of 9.0 g NaCl, 0.2 g KCI. 0.2 g MgCl. 0.2 g CaCl,, 0.2 g NaHCO, and 8.0 g glucase dissolved in 1000 ml of distilled water. The saline
Fig. l(a). Diagram Fig. I(b). Diagram
of symbiotes of symbiotes
was decanted after surgery to allow the surgical wound to close through the coagulation of haemolymph. Lice were decapitated by ligaturing between the head and thorax with a human hair, then removing the head with a No. 15 scalpel blade. To prevent starvation, headless lice were fed daily with titrated human blood injected rectally. The apparatus for injecting the blood consisted of a fine capillary tube attached by a flexible hose to a syringe pump (Sage Instruments). The portion of the line not filled with blood was filled with the saline previously described. The tip of the capillary tube was inserted into the rectum of the louse, and a volume of blood equivalent to a normal blood meal was injected. The blood flowed freely from the rectum into the midgut and appeared to be digested normally. Mycetomes were transplanted by puncturing the ventral abdominal cuticle of the mycetome donor to one side of the mycetome and drawing the midgut and the attached mycetome out of the puncture with foreceps (Dumont No. 5). The mycetome was detached by trimming away the midgut with a No. 15 scalpel blade. To minimize osmotic damage, the mycetome was immediately reimplanted into a recipient louse by puncturing the abdominal cuticle
migrating from the mycetome to the lateral human body louse. After RIES (1931). from lateral
oviduct cells infecting RIES (1931).
a develo;;ng
oviducts
in the female
egg in the ovary.
After
Fig. 2. Scanning
electronmicrograph
of symbiotes
exiting
from the female mycetome.
Fig. 3. Scanning
electronmicrograph
of symbiotes
invading
a lateral
oviduct
-
_-- --
__- --
-
-
-_
-_
-.
-_“. -..
-..
-.,
-I
-..
Fig. 4. Transmission electronmicrograph showing a band of striated muscle (arrow) one of the chambers that contain symbiotes (s) in the mycetome.
-”
-_
-
encircling
-
-
-
-
-
-
-
-
_-
..-- ,-
.-
-,- -
-
-
--
-
Fig. 5. Transmission electronmicrograph showing bands of striated muscle (arrows) a symboite-containing chamber. Muscles are cut in cross-section.
-~-.li--‘-~‘._.“_ -,...,,-”
encirchg
Initiation and orientation of symbiote migration and inserting the mycetome through the puncture with the aid of a slender probe constructed from a capillary tube drawn out over a flame. Female reproductive tracts were removed by puncturing the ventral abdominal cuticle over the median oviduct. grasping the median oviduct with forceps and drawing the median oviduct, the lateral oviducts and the ovaries out of the louse. If the intent was to transfer the reproductive tract to a position anterior to the mycetome, a second puncture was made anterior to the mycetome and the reproductive tract was inserted through it and positioned against the midgut surface at approximately the same distance from the mycetome as it normally lies.
These consisted of preventing the adult moult by topically applying a juvenile hormone analogue and inducing the adult moult by injecting 20-hydroxyecdysone. The juvenile hormone analogue (1 l-methoxy-3,7,1 l-trimethyl-2,4-dodecadienate; Altosid 5E, Zoecon Co.) was applied to corduroy patches at a concentration of 1000 pg/cm*. This concentration was obtained by diluting Altosid SE (0.6g of active ingredient per ml) 1:lO with acetone, then combining 3.27 ml of this dilution with 2.50 ml of vegetable oil (added as a spreading agent) and 4.23 ml of acetone. One ml of this mixture was applied to each 5 cm’ patch. Treated patches were always used singly, never stacked. Lice were placed on the treated patches 2 days after the moult to the second instar and remained on them permanently. While on the treated patches, they moulted through three further nymphal instars and died attempting to moult to a fourth. The treatment prevented the adult moult by allowing only nymphal moults. The 20-hydroxy-ecdysone (Calbiochem) was dissolved in 95’)~”ethanol and diluted with the saline described previously to a concentration of 6pg/pl in 4”” ethanol. Two ~1 were injected into the haemocoel of each louse with the apparatus used for rectal-blood feeding. The adult moult occurred 2 days after the injection. Normal lice could not be used with ecdysteroid injections because there was too little time between their second nymphal moult and their adult moult to permit the experimental induction of an adult moult. In their place. lice were used that were unable to moult unless injected with 20-hydroxy-ecdysone. They were obtained by placing second-instar nymphs on Altosid-treated corduroy patches, prepared as described above, 2 days after the moult to the second instar. They were removed immediately after moulting to the third instar, and their reproductive organs were then removed to prevent fatal vaginal prolapse in the fourth instar. Rather than the usual adults, the fourth instars were nymphs that lacked external genitalia and retained the symbiotes in the mycetome. However, because the Altosid treatment was very brief. they retained the adult characteristic of not moulting. They remained fourth-instar nymphs permanently. unless injected with 20-hydroxy-ecdysone whereupon they moulted to adults.
At the conclusion
ary to determine whether symbiote migration had occurred. This was done by dissecting each louse, transferring the mycetome and the reproductive tract to separate drops of saline on the same microscope slide, crushing these organs with gentle pressure on the cover slips, and examining them for symbiotes with a phase-contrast microscope. If the migration had occurred, the symbiotes were found only in the reproductive tract. If the migration had not occurred, the symbiotes were found only in the mycetome.
RESULTS Question itiated?
Hormonal treatments
of each experiment,
it was necess-
421
(a): How
is the symbiote
migration
in-
Experiment 1. Ten female nymphs were decapitated 3 days before the symbiote migration to test the hypothesis that migration is initiated by a brain hormone. Contrary to the hypothesis, migration occurred in all 10 headless lice at the usual time following the apolysis of the adult moult. Experiment 2. Reproductive tracts were removed from 20 female nymphs 9 days before the symbiote migration to test the hypothesis that the migration is initiated by a hormone from the female reproductive tract. Contrary to the hypothesis, symbiotes left the mycetome during the adult moult in all 20 females. We were unable to determine where the symbiotes migrated in the absence of lateral oviducts, their usual target. Experiment 3. Twenty female nymphs were prevented from moulting to adults with topical application of Altosid to test the hypothesis that a factor associated with the adult moult indicates symbiote migration. All 20 failed to initiate the migration though they lived sufficiently long to moult through five nymphal instars. Thus, migration was prevented by preventing the adult moult. In a second test of the hypothesis, 10 female fourth-instar nymphs were injected with 20-hydroxy-ecdysone to induce their adult moult. All 20 initiated the symbiote migration during the moult. As a control, 10 female fourthinstar nymphs were injected with only the ethanolsaline used to dilute the ecdysteroid: none moulted or initiated the migration. Thus, symbiote migration was initiated by initiating the adult moult. These results confirm the hypothesis. Question (b): Why is the migration absent ,from male lice? Experiment 1. Ten female mycetomes were transplanted into male lice three days before the symbiote migration to test the hypothesis that males lack the stimulus that initiates the migration. Contrary to this hypothesis, all 10 female mycetomes released their symbiotes during the adult moult of their male hosts. This result indicates that the initiating stimulus is not transmitted neuronally since the transplanted mycetomes could not have retained intact innervation. Experiment 2. Ten male mycetomes were transplanted into female lice three days before the symbiote migration to test the hypothesis that male mycetomes are unable to respond to the initiating stimulus. All 10 male mycetomes failed to release their sym-
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biotes, though the female mycetomes of their hosts released theirs. This result confirms that male mycetomes are unable to respond to the initiating stimulus. Question (c): How is the migration oriented toward the reproductive tract?
female
Ten female reproductive tracts were transferred to a position anterior to the mycetome three days before the symbiote migration to test the hypothesis that the symbiotes are attracted by the female reproductive tract. Migrating symbiotes located and invaded all 10. This result has two explanations: either the symbiotes changed the direction of their migration, or they dispersed into the haemolymph and circulated randomly throughout the louse until they reached the lateral oviducts by chance. We believe that the latter explanation is unlikely because RIES (1931) reported that migrating symbiotes do not disperse in the haemolymph but rather move directly from the mycetome to the lateral oviducts, and because it is difficult to envision how hundreds of symbiotes could arrive at the lateral oviducts simultaneously after randomly circulating throughout the louse. Assuming that the symbiotes did reverse the direction of their migration, the results suggest that the symbiotes are attracted by the female reproductive tract.
DISCUSSION How is the symbiote migration initiated? The results indicate that the initiating stimulus is a factor associated with the adult moult. This factor probably does not originate in the brain or the female reproductive tract. It is not transmitted neuronally and therefore probably is humoral. To these results should be added an observation that we made while examining excised mycetomes with a phase-contrast microscope: the mycetomes are capable of muscular contraction. If the mycetome is stripped of its syncytial covering, these contractions are clearly visible and resemble the peristaltic contractions of the midgut. We examined mycetomes with a transmission electron microscope and observed that the symbiote-containing chambers were encircled with numerous bands of striated muscle (Figs. 4, 5). These muscles may play a role in the initiation of the symbiote migration by expressing the symbiotes from the mycetome. The role of the initiating factor may be to increase the frequency and the amplitude of the mycetome’s contractions. Unfortunately, the results provide little information about the identity of the initiating factor. The factor may be a hormone from a site other than the brain or the female reproductive tract; it may be a change in haemolymph chemistry associated with the adult
moult, such as a change in pH; or it may be a combination of factors, such as the low level of juvenile hormone and the high level of 20-hydroxy-ecdysone that accompany the adult moult in many insects. Why is the symbiote migration absent from male lice? The results indicate that male mycetomes are unable to respond to the initiating factor, which is present in both sexes. RIES (1931) reported a difference between the structures of male and female mycetomes that may account for the inability of male mycetomes to respond. Unlike the female mycetome, the male mycetome does not reorganize its internal structure in preparation for the symbiote migration. The female mycetome fuses the l&l6 internal chambers into a central cavity in which the symbiotes are loosely gathered. This reorganization releases the symbiotes from their confinement in the chambers and positions them directly over the site of their future expulsion from the mycetome. Without this preparation, it is difficult to envision how the symbiotes could escape from the mycetome. In contrast, the male mycetome retains the l&16 internal chambers. How is the symbiote migration oriented toward the female reproductive tract’? The results, as we interpret them, suggest that the symbiotes are attracted by the female reproductive tract. The mechanism for this attraction is unknown. Possibly the female reproductive tract releases a symbiote attractant which forms a gradient in the haemolymph that the symbiotes follow to the source. The mechanism for attracting the symbiotes probably is separate from the mechanism that initiates the migration, since the former appears to involve the female reproductive tract while the latter appears to be independent of it. Thus, the regulation of the symbiote migrate may involve two mechanisms, one to initiate the migration and one to orient it. Aclinow/edgements--We are grateful to Dr. EUGENE GERBERG and Mr. M. COLE for providing us with the Culpepper strain of Pediculus humanus. Professors BRUCE HAMMOCK, HENRY HAGADORN, CHARLES JUDSON. ANDY MCCLELLAND, HARRY KAYA and Drs. RICHARD SHUKLE and STEVEN AULT provided valuable suggestions and criticisms. Dr. LYNN S. KIMSEY executed the drawing for Fig, 1.
REFERENCES BUCHNER P. (1965) Endosynbiosis of Animals with PIanr Microoryanisms. Revised English version. Interscience Publishers, New York. COLE M. (1966) Body lice. In Insect Colonization and Mass Producbon (Ed. by SMITH C. N.), pp. 15-24. Academic Press, New York. PUCHTA 0. (1955) Experimentelle Untersuchungen iiber di Bedeutung der Symbiose der Kleiderlaus Pediculus restimenti Burm. Z. ParasitKde. 17, l-40. RYESE. (1931) Die Symbiose der Lause und Federlinge. Z. Morph. Okol. Tiere 20, 233-367