J. Insect Physiol., 1969, Vol. 15, pp. 1719 to 1728. Pergamon Press.

Printed in Great Britain

PRELIMINARY OBSERTiATIONS ON MOULTING AND

LIMB REGENERATION IN THE MITE CALOGLYPHUS
BOHARTI”

J. P. WOQDRING

Department of Zoology, Louisiana State University,

Baton Rouge, La. 70803

(Received 1.5 April 1969; revised 9 May 1969)

Abstract-Each immature instar of Caloglyphus boharti consists of an active

and inactive stage. At the beginning of the inactive stage the soft leg tissue
rapidly dedifferentiates and regresses into and forms a coxal limb bud, which
elongates and differentiates into a new limb extending into the ventral exuvial
space. Amoebocytes are conspicuously active during limb regression, though
their r61e is unclear, and they were not involved with limb elongation. Although
the appearance of the limb in the resultant adult depends on the position of the
wound clot, leg regeneration of amputated nymphal legs was clearly very limited.
This indicates that the limb bud must contain a sufhcient number of cells from
each former leg segment in order to form a normal limb.

INTRODUCTION

REGENERATIVE powers have become reduced perhaps as a result of increased

structural complexity in arthropods, and only seldom is more than limb regeneration
possible. Limb regeneration occurs in Crustacea, Insecta, Arachnida, and
Myriapoda, and is most often associated with autotomy. In Goss (1969) sum-
mary of regeneration in animals. it is pointed out that the extent of leg regeneration
in arthropods may be correlated with the point of amputation of the limb, the time
of amputation relative to the next ecdysis, presence of an autotomy plane, pre-
sence and growth of nerves, and with the hormone (ecdysone) titres in the haemo-
lymph.
A most conspicuous feature of arthropod limb regeneration is that tissue re-
generation takes place between apolyses, but that the regenerates do not become
functional until after the next ecdysis. Since moulting plays such a decisive role in
arthropod limb regeneration, a brief description of acarine apolysis is in order.
Three apolysis mechanisms occur, which I will term types A, B, and C. Apolysis
in Mesostigmata and Metastigmata (type A) is similar to that in other arachnid
orders and to that in some insects. The leg epidermis separates from the cuticle,
and a new cuticle is secreted within the old leg hull. At ecdysis, the body cuticle
splits and the animal partially emerges from the exuviae. The last step involves
pulling the new limbs from the old cuticular covering. Apolysis is comparatively
* Supported by NSF grant GB-7982.

1719
1720 J. P. WOODRING

fast, and the mite is inactive for only a brief time. The non-parasitic Prostigmata
(spider mites and others) and the Cryptostigmata (oribatids) form the new limb
also within the old leg cuticle, but they withdraw the new legs from the old leg
hull many hours (or even days) before actual ecdysis. This is type B. In neither
type A nor type B acarine moulting is there appreciable reorganization or regression
of the soft tissues of the limbs.
Other Prostigmata (chiggers and water mites) and the Astigmata (which
includes Caloglyphw boharti CROSS, 1969) illustrate type C apolysis. The old leg
tissue dedifferentiates and regresses back into a limb bud in the coxal region, and
the new limb grows into the exuvial space from the bud. One variation of type C
apolysis, which was described by JONES (1950, 1954) involves essentially a double
moult between active stages (chiggers and water mites).
The typical postembryonic development of mites includes a hexapod larva
followed by two or three octopod nymphal instars. Mites with types B and C
apolysis, but not with type A, have each instar conspicuously divided into an active
and inactive stage. The onset of the inactive stage is the beginning of apolysis.
At 22°C the duration of the active stage tritonymph of Cdoglyphus boharti was
60 to 72 hr, and the duration of the inactive stage was 20 to 26 hr.
Aside from the work of JONES (1950, 1954), HUGHES (1964), KANTJNGOand
NAEGELE(1964), and KANUNGO(1969), very little is known about acarine moulting
processes. KANUNGO and NAEGELE (1964) described the activity and migration
of the coelomocytes-amoebocytes of C. berlesei: Essentially nothing is known of
limb regeneration in Atari.

MATERIALS AND METHODS

Stock cultures of C. boharti were maintained in screw-cap jars (4 x 5 cm)
with 6 to 8 mm of plaster of Paris-charcoal (9 : 1) serving as a substrate. Proto-
nymphs and tritonymphs were isolated, and upon entering the inactive stage were
placed ventral side up in water on a depression slide. A cover-slip covered about
one-eighth of the depression, and the specimens were wedged into the angle
formed by the edge of the depression and the underside of the cover-slip. By
periodically adding water to replace that lost through evaporation, the regression
of old leg tissue and the formation of the new legs was observed through the
transparent cuticle in viva with a bright-field microscope (200 to 400 x ).
At selected intervals, inactive tritonymphs were fizzed in either 10% formalin
or alcoholic Bouin’s, embedded in Paraplast, sectioned at 6 IL, and stained with
Delafield’s haematoxylin and with eosin Y.
Active stage proto- and tritonymphs of C. boharti for amputation experiments
were immobilized with ether vapours (10 to 1.5 min) and placed in a drop of water
in the cavity of a shallow depression slide. Individual specimens were slid with a
needle out of the water up on to the dry side of the depression. As the water
that had adhered to the mite evaporated, the specimen became lightly stuck to the
glass surface. Before complete evaporation of adhering water, a leg was extended
so that the mite became lightly stuck with a selected leg flat on the glass surface.
 MOULTING
ANDLIMBREGENERATION
IN A MITE 1722

Using sharpened insect pins, the extended leg was amputated at selected points.
The specimens were transferred back to the water and the exact point of amputation
continned. About 90 per cent survived and eventually moulted to adults, which
were then mounted in Foyer’s chloral hydrate mounting medium and examined for
extent of leg regeneration.

THE MOULTING PROCESS

During the latter part of the active stage of each instar, the opisthosoma
became distended, the animal stopped feeding, and eventually all movements
ceased. When the mite no longer moved in response to touch, the inactive stage
had begun. The epidermis immediately started to separate from the cuticle, and
amoebocytes appeared in the exuvial space. A half-hour after the start of the
inactive stage all muscle connexions to the cuticle were broken.
In the inactive tritonymph leg, regression commenced about 30 mm after the
onset of the inactive period. Only 45 to 60 min were required for the tip of the
regressing leg tissue to retreat from the tarsus to the level of the trochanter. Cer-
tain demarcation lines of the soft leg tissue were seen in z&o to retreat as the leg
tip retreated, indicating a regression or shrinking of the limb rather than a dis-
solving or lysis of the tip. Sections of these regressing limbs showed the tissues
to be less distinct than the same tissue in the limbs of active stage nymphs. Cell
boundaries became indistinct, epidermal cells became cuboidal, nerve cells became
indistinguishable from epidermal cells, and muscle cells lost their cross-striations
and typical fibrous appearance.
Regressing limb tissue in sections gave the impression of dedifferentiation,
wherein the most proximal dedifferentiated cells merged into the coxal region to
form the beginnings of the coxal bud. The remaining regressing tissue sequentially
merged into this bud, and clearly contributed to its enlargement. Regression was
almost completed 1.5 to 2 hr after the start of the inactive stage, and the new
limb began immediately to elongate from the coxal bud. The coxal or new limb
bud first became recognizable in viva when the retreating old limb tissue reached
the level of the trochanter. A limb bud did not develop if that particular leg was
amputated during the preceding active stage or preceding instars.
Amoebocytes were very motile on and around the regressing leg tissue, but
phagocytosis was not conspicuous and the r6le of these cells in regression remains
unclear. The amoebocytes were shown by KANUNGO and NAEGELE(1964) to be
modified coelomocytes that migrate through the epidermis at the onset of the
inactive stage (apolysis), and these authors concurred with JONES(1950) that these
cells are probably involved with or responsible for the dissolution of soft limb
tissue. A dissolving or partial lysis of leg tissue of C. boharti involving the amoebo-
cytes was neither proved nor disproved.
A few amoebocytes remained in each empty leg segment as the soft tissue
regresses, and upon completion of regression they accumulated on the old epimere
cuticle. The amoebocytes gradually became less motile, and did not appear to
1722 J. P. WOODRING

play a role in elongation or differentiation of the new limb. Though some of the
amoebocytes probably re-enter the haemocoel, many are lost at ecdysis.
Serial sections of the bud before elongation started showed it to be composed
mainly of bundles of recognizable (but unattached) muscle cells, the whole being
covered with a single layer of cuboidal epidermal cells. Certain cells with filamen-

leg bud I

FIG. 1. Schematic ventral view of left coxal region (1.5 hr) showing the end of the
regression phase and the beginning of bud elongation.

tous cytoplasmic extentions were assumed to be presumptive sensory neurons.

The bud became twice as long as thick within 30 to 45 min, and three ‘proto-
segments’ were recognizable (Fig. 2). The proximal two protosegments were the
trochanter and femur. During the next hour these three protosegments became
more delineated, and the terminal one budded off another segment, which im-
mediately started to bud off yet another. The last three segments in order of
appearance were of course the genu, tibia, and tarsus. The elongating limb tissue
became less dense with increased length, and bundles of muscles were seen to
separate and remain in the various segments as they were formed. What were
thought to be elongating sensory neurones were also observed. Mitosis was
never seen in any cells of the early bud or of the elongating new limb.
Though each segment was oval in shape and lacked cuticle or setae, all leg
segments were recognizable 3 to 3.5 hr after the start of the inactive stage. Within
the next hour each segment independently elongated, each became more cylindrical
in shape, and the intersegmental boundaries became more distinct. Finally, the
pretarsus quickly budded off (10 min) and began to dedifferentiate. The growth
and elongation took place in a ventro-medial direction, so that the new limb came
to lie in the ventral exuvial space. At the end of this hour (4.5 to 5 hr from the
start) a thin cuticle became visible, the first large sensory setae were discernible,
and the new limb had achieved about three-quarters of its destined total length.
Simultaneously and internally, the epidermal cells became more flattened and the
 MOULTING AND LIMB REGENERATION IN A MITE 1723

muscles became more fibrous in appearance. It was assumed that cuticular-

muscle connexions were made at this time.
Full elongation of all segments with the appearance of all setae was apparent
8 hr after the beginning of the inactive stage. Cuticular sclerotization was apparent
in z&o after 12 to 16 hr, the first spastic leg twitches occurred between 18 to 20 hr,
and actual release from the exuviae occurred after 22 to 26 hr. Table 1 summarizes
these events.

(2.0 hrs)

protosegment I

protosegment III (trochanter)

. / (3.0 hrs)
r coxa

FIG. 2. Schematic lateral views of limb bud 1 elongation of inactive stage of

C. bohmti tritonymph. The average extent of elongation is given in hours after
the onset of the inactive stage. The 2- and 2*5-l-n elongations are shown as
idealized sections, while the 3- and 3*5-hr elongations are shown in external view.

LIMB AMPUTATIONS
Successful leg amputations were performed on the active stages of 10 larvae,
14 protonymphs, and 45 tritonymphs of C. boharti. All larval legs were cut at
approximately the trochanter-femur joint, and from 8 of these larvae the coxa or
trochanter was terminal in that leg of the resultant adult. This indicates no re-
generation, even though the animal had apolysed three times. The other 2 resultant
adults showed two abnormal (and very small) segments beyond the trochanter,
which indicates either a limited regeneration or that some soft tissue of some distal
segment remained with the larval leg stump after amputation.
The results of protonymphal and tritonymphal leg amputations are summarized
in Fig. 3. Though various legs at various points were amputated in the active
stages of the two nymphal instars, the results are summarized as though only two
types of operation were performed: intrasegmental (23 times) or intersegmental
(36). It made no difference which leg was involved, no difference if it was a proto-
nymphal or tritonymphal leg amputated, and no essential difference whether the
1724 J. P. WOODRING

amputation was mid-tibia or mid-femur. A mid-tibia amputation, for example,

was as apt to result in an abnormal adult tibia as a mid-femur amputation to result
in an abnormal adult femur.

‘TABLE ~-SUMMARY OF EVENTS DURING THE INACTIVE STAGE OF TRITONYMPH C. bohurti

AT 22°C

Cumulative time
W Event(s)

0 All movement of the active stage ceases, and the inactive stage starts
O-O.5 Epidermis loosens from the cuticle and amoebocytes appear in the
exuvial space
0.5 Soft tissue of limbs starts to regress
0*.5-1.5 Soft leg tissue regresses from tarsus to trocbsnter, and amoebocytes
very active
1.3-2.0 Limb buds appear as regression is completed
2.0-2.6 Buds elongate and differentiate into three protosegments and
smoebocytes become less active
2.6-3.5 All segments are formed, and the pretsrsus buds off
34-45 Each segment independently elongates and becomes more distinct
44-5.0 Cuticle secretion starts, and first large sensory setae sprout
5.0-6.0 New limb three-quarters of total destined length, and muscles
differentiated and attached
8.0-9.0 Leg fully elongated and all setae present
12-16 Cuticle thickens and some sclerotization evident
18-20 First limb movement noted
22-26 Ecdysis

See text for further details.

The position of the clot that formed after leg amputation, relative to the point
of amputation, governed to a large extent the form of the leg in the next instar.
The clot often formed at the wound surface, but in diminishing frequency it
formed at the joints of more proximal segments (Fig. 3). The clot appeared to be
composed primarily of leg tissue cells with some coelomoeytes and did not seem
to involve plasma components. Clots at joints one or more segments proximal
to the point of amputation were composed of a mixture of a variable number of
cells of all the distal (to the clot) segments remaining on the stump.
Intersegmental amputations were consistently cleanly cut, but the intraseg-
mental amputations (at the joints) sometimes resulted in the joint separating before
the internal tissue was severed. This meant that some soft tissue from segments
distal to the joint cut would remain, or some soft tissue from more proximal
segments were pulled out and discarded with the cut-off segments. The clot in
the latter situation would then form much more proximally than otherwise. This
would account for the high percentage (33 per cent) of adult legs displaying one,
two, or even three segments fewer than remained on the amputated nymphal leg
stump.
 MOULTING AND LIMB REGENERATION IN A MITE 1725

A study of Fig. 3 will show that limb regeneration in C. bolzarti is very limited
In several instances of intersegmental amputations the terminal segment was
regenerated whole and normal. This completion of a half-segment represents the
maximum regeneration in this species. In no case was a whole, normal segment

FIG. 3. Summarized results of limb amputation of active stage proto- and

tritonymphs of C. bohurti. Twenty-three intrasegmental amputations (upper
half) and 36 intersegmental amputations (bottom half) were performed. The
stippled areas show the possible positions of the wound clot. The numbers
given in each coxa represent how often this result was obtained from the total
of 59 nymphal amputations. The arrows start at the point of amputation (dotted
line) on the nymphal leg, lead through the clot position, and end at the various
possible forms of the corresponding adult leg.

regenerated beyond the segment or leg joint at or on which the cut was made.
Abnormal parts of segments beyond the point of amputation occurred 8 times
out of 36 intersegmental amputations, and it is assumed this resulted from some
trace of the soft tissue of a more distal segment remaining after amputation.
With both intersegmental and intrasegmental amputations some resultant
adult limbs had none to two segments less than remained on the amputated
nymphal leg stump, and sometimes in addition had one to three abnormal to
highly abnormal segments. For example, if the tarsus was cut from leg 1 of the
95
1726 J. P. WOODRING

active tritonymph, the adult leg 1 could appear with a normal trochanter, an ab-
normal femur, a very abnormal genu, and no tibia. The assumption is made in this.
example that the clot formed at the trochanter-femur joint and that the clot con-
tained many femur cells, few germ cells, and no tibia1 cells.
Legs other than those operated upon were affected in eleven instances (from
a total of 59 amputations) to the extent shown in Table 2. Some of these accessory
effects may be attributed to inadvertant injury or even unnoticed amputations of
limbs other than that limb being deliberately amputated, but it seems likely some
of these accessory effects are not accidental. In the family Acaridae only the genus
Lardogl’yphus is characterized by possessing forked claws, and the occurrence of
forked claws on all unamputated legs of a C. boharti can only be explained by
assuming a mutation or a widespread effect of the amputation of a single leg.

TABLE ~-LEGS AFFECTEDOTHER THAN THE LEG DELIBERATELYAMPUTATEDOF C. boharti

NYMPHAL STAGES

Instar of Amputated Other leg(s) Appearance of affected, non-amputated

operation leg affected leg in the adult

Protonymph L(IV) L(II1) Tibia abnormal, tarsus lacking

Protonymph L(II1) L(Iv) Tibia abnormal, tarsus lacking
Tritonymph R(II1) L(III) Claw missing
Tritonymph L(II1) L(IV Claw missing
Tritonymph R(II1) R(IV Tibia and tarsus abnormal, claw present
Tritonymph R(II1) R(IV) Tarsus abnormal, claw lacking
Tritonymph L(II1) L(IV) Tarsus abnormal, claw lacking
Tritonymph R(II1) L(II1) Tarsus abnormal, claw lacking
Tritonymph L(Iv) L(II1) Genu and tibia abnormal, tarsus lacking
Tritonymph R(II1) R(IV Trochanter to tarsus abnormal, claw
lacking
Tritonymph R(I) All Forked claws on all other legs

L: left leg; R: right leg.

There was no evidence of an autotomy plane in C. boharti limbs. Pulling on a

nymphal leg always resulted in an intersegmental break, but which joint separated
was unpredictable.
In crowded stock cultures of this species approximately 1 per cent of the adults
normally have one or more missing terminal segments on any one leg. Nymphal
instars temporarily buried in food (yeast) added to the culture would be apt to
lose a leg to indiscriminantly feeding fellow mites.

CONCLUSIONS AND DISCUSSION

At the start of the inactive stage (apolysis) of the various instars of C. boharti
the epidermis separated from the cuticle and the soft leg tissues regressed rapidly
into a simultaneously developing coxal limb bud. Though regression appeared to
involve primarily cell dedifferentiation and retreat, a lytic process may also occur
during regression. Whether Iysis is extensive or limited to certain tissues or even
 MOLJLTING AND LIMB REGENERATION IN A MITE 1727

to certain cells could not be proven. It is attractive to interpret amoebocyte

activity as being lytic and/or phagocytic, but they could instead function to cause
(possibly through secretions) the dissociation of the soft leg tissue. The mechanism
of regression then remains unclear, and the role of the amoebocytes uncertain.
The amoebocytes are not involved in the elongation or segmental differentiation
of the new leg.
The elongation and differentiation of the new limb during the inactive trito-
nymphal stage of C. boharti did not appear different from the embryonic develop-
ment of the legs nor different from the development of the fourth pair of legs
before or during the resting stage larvae of most mites. The rapid elongation and
development of the new limb involved a simultaneous movement and differentiation
of epidermal, muscle, and nervous tissue, resulting within 1 hr in the appearance
of all leg segments. It is not impossible that these mites have a constant cell
number, as no mitoses were observed during elongation of the new leg.
A whole, normal segment was never regenerated beyond the segment or joint
at which the amputation was made. Completion of a half-segment after one moult
represented the maximum extent of regeneration of limb tissue. Abnormal adult
leg segments resulted from either soft tissue from more distal segments remaining
on the stump or from the variation in formation and position of the clot formed
after amputation. The further proximal the clot formed the more mixed was the
segmental origin of the cells composing the clot. Missing segments in addition to
those removed through amputation were partially due to pulling of soft tissue
from stump segments during amputation.
The limited regenerative capacities of limbs indicate that the developing limb
must contain a sufficient number of cells from each former leg segment in order to
form a new leg with a normal number of normal segments. This could mean that
the dedifferentiated limb cells, which are at least morphologically altered, retain
their segmental identity during regression, and, further, that these cells regress
into the coxal bud in an orderly sequence.
Since the coxal bud size corresponded to the number of nymphal leg segments
amputated (the more segments removed the smaller the bud), it may be argued
that as many new segments are sequentially formed as cells are available in the bud
and that segmental identity of the cells is unnecessary. A constant cell number for
this species would explain why no new cells are produced if cells (or cells from
specific segments) were missing in the coxal bud, regardless of possible segmental
identity of coxal bud cells.
The abnormal segments of various sixes, doubled setae, and occasional forked
segment reflected the mixing of various segmental cells in the wound clot, which
would indicate retention of segmental identity. If, for example, the clot contained
cells from the femur, genu, and tibia, then the number of cells should be sufficient
to form a normal femur in the resultant adult. Instead, a smaller and abnormal
femur, genu, and tibia were formed. If the clot only contained femur cells, as
when genu and tibia1 soft tissue was pulled from the nymphal leg stump during
amputation, then only a femur was formed.
1728 J. P. WOODRING

If indeed post-embryonic mitoses occur, then the lack of limb regeneration

would most logically be attributed to a lack of directives from segmentally specific
cells that never got into the coxal bud. Lack of regeneration would be difficult to
explain if the coxal bud cells could multiply and new leg segments are formed ac-
cording to the number of cells available.
NUESCH (1968) states that though the beginning of regeneration may be in-
dependent of the nervous system in insects, the growth of the new limb needs
the influence of innervation. The growth and differentiation of muscles seem
particularly dependent upon motor neurones. Since in C. bohurti all leg tissue
(including nervous) regresses at each apolysis, an influence of the nervous system
upon lack of leg regeneration would in this species have to reflect the loss and
failure of replacement of nervous tissue at amputation. The normal elongation
and differentiation of the non-amputated limb could still be mediated through
nerves.
HUGHES (1964) states that during nymphal apolysis neurosecretory granules
disappear from certain cells in the supra-oesophageal ganglion of Acarus sire, and
he assumes these secretions to be involved in the ecdysial process and, perhaps,
with differentiation of ensuing tissue. No gland analogous to the crustacean
Y-organ or the insect prothoracic gland has yet been identified in mites, so specu-
lation of the role of hormones on acarine moulting or leg regeneration would be
premature.
If tissue lysis were the chief process of limb regression instead of tissue de-
differentiation and retreat, then one would have to say that cell fragments or RNA
of the correct amount and cellular origin directs the development of the new leg.
Missing or altered segments in legs other than those amputated can best be ex-
plained if tissue lysis were providing cell fragments to the elongating new limb
via the exuvial fluid. This would mean the limb buds are de nova, somewhat like
the normal appearance of the fourth pair of legs in the protonymphs, and that
their growth was directed by specific determinants of the old limb.

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HUGHSS T. E. (1964) Neurosecretion, ecdysis, and hypopus formation in the Acaridei.
Acarologia VI, 338-342.
JONES B. M. (1950). Acarine growth: A new ecdysial mechanism. Nature, Lond. 166,
908-909.
JONESB. M. (1954) On the role of the integument in acarine development and its bearing on
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