Comparative studies of acarine limb regeneration, apolysis, and ecdysis

J. Insect Phys-iol.,1972,Vol. 18, pp. 2319to 2336. PergambnPress. Printed in Great Britain

COMPARATIVE STUDIES OF ACARINE LIMB REGENERATION, APOLYSIS, AND ECDYSIS C. L. ROCKETT*

and J. P. WOODRING

Department of Zoology, Louisiana State University, Baton Rouge, Louisiana (Received 26 May 1972) Abstract-A comparative study of acarine limb regenerative capacity, apolysis, and ecdysis was based on nine species representing the five major suborders of Atari. An autotomy plane was lacking in all. The ixodid completely regenerated limbs (regardless of site of amputation) after one apolysis. The argasid required two apolyses to completely regenerate amputated limbs. The uropodid regenerated a maximum of two distorted segments beyond the site of amputation. In the gamasid, the site of amputation generally formed the terminal end of the limb in the next instar. Regardless of the number of apolyses or site of amputation, the astigmatid and cryptostigmatids possessed limbs in subsequent instars which displayed segmental reduction and/or distortion proximal to the site of amputation. Over 90 per cent of the prostigmatids died following amputation of any part of a leg. Following amputation the haemolymph coagulated quickly and a good clot formed in the ixodid, gamasid, uropodid, and astigmatid. The clot was diffused in the cryptostigmatids. Coagulation was very slow in the argasid, but a clot eventually formed. Coagulation did not occur in the prostigmatid, and a clot was never observed. The limbs of the gamasid, argasid, uropodid, prostigmatid, and both cryptostigmatids apolysed in place. The pharate form of the gamasid, uropodid, and argasid was mobile. The pharate form of the prostigmatid and both cryptostigmatids is immobile, but withdrew the newly formed limbs from the old leg hulls prior to ecdysis. The ixodid and astigmatid had an extended inactive stage in each instar during which the limb regressed to the coxal region and the pharate limb grew into the ventral exuvial space. At ecdysis the cuticle split anteriorly in the gamasid, argasid, ixodid, and uropodid, while the cuticle split posteriorly in the astigmatid and both superior cryptostigmatids. The cuticle split in the prostigmatid around the middle of the mite. INTRODUCTION THE

REGENERATIVE

of lost appendages.

capacity of arthropods is generally limited to the replacement Limb regeneration has been reported in Myriapoda, Crustacea,

Insecta, and Arachnida; however, most studies on arthropod regeneration have centred on the larger crustaceans and insects. Regeneration studies on non-acarine * Present address: Department of Biology, Bowling Green State University, Bowling Green, Ohio 43403. 2319

2320

C. L. ROCKETTANDJ. P. WOODRING

arachnids have been done by WOOD (1926), BONNET (1930), VACHON(1957), and ROSIN and SHULOV (1963). WOODRING (1969) made preliminary observations on apolysis and limb regeneration in the mite, Caloglyphus boharti, and found that limb regeneration is very limited. The completion of a half-segment represented the maximum regeneration in this species. He suspected that the limb bud must contain a sufficient number of cells from each former leg segment to form a normal limb. An important element of arthropod limb regeneration is that limb regeneration takes place between apolyses and that the regenerated appendage does not become functional until the following apoiysis. Any discussion on regeneration will of necessity also involve a study of mechanisms of apoIysis fmoulting), which refers to the periodic and complex process of loosening the old cuticulum accompanied by the formation of a new cuticulum. Ecdysis refers only to the actual process of casting the exuviae. The pharate form refers to the fully formed next instar which is still within the old exuviae. Almost all Atari hatch from an egg to a hexapod larva. Following the larval instar there are a variable number of octopod nymphal instars. Ixodid ticks have one and some argasid ticks have up to eight nymphal instars. Most other acarine groups have either two or three nymphal instars. In some mites there is a very brief or no period of quiescence prior to ecdysis. In this case, the pharate form in the latter period of any instar is active and is still covered by the old exuviae. In other mites there is a long period of inactivity (immobility) associated with apolysis; the pharate form remains inactive within the old exuviae until ecdysis. For those mites we refer to the active and inactive stage within each instar. The purpose of this study was to compare limb regeneration and mechanisms of apolysis in the five major suborders of Atari.

MATERIALS

AND METHODS

Nine species, representing the five major groups (Table 1) of Acari, were collected and cultured in the laboratory for regeneration studies (Table 1). All limb amputations were done with a knife-like chip of razor blade inserted in a biological pin holder. Prior to receiving amputations, the mites were temporarily anaesthetized with carbon dioxide (dry ice) and subsequently placed on a ‘cutting’ substrate of histological paraffin. Sites of amputation and in vivo moulting processes of the smaller species were checked by microscopic examination of temporary I-I,0 mounts, but with the larger species, such as the ticks, the site of amputation was verified by examination under a stereoscopic microscope at 200 x . Excluding the Prostigmata, stock cultures of all species of acarines utilized were maintained in plastic vials with stainless steel wirecloth tops and plaster of Parischarcoal (9 : 1) covered bottoms. Excluding the argasid ticks, culture vials were maintained in desiccators with a relative humidity of 95 per cent and room temperaechinopus, Scheloribntes vzudus, and ture of approximately 25°C. Rbixogl~hus f)amaew sp. were cultured respectively on diets of wheat germ, ground mushroom,

STUDIESOF ACARINELIMB REGENERATION, APOLYSIS,AND ECDYSIS TABLE l-ACARINE

2321

REPRESENTATIVES SELECTED FOR REGENERATION EXPERIMENTATION

Suborder and group MESOSTIGMATA (GAMASINA) (UROPODINA) METASTIGMATA

PROSTIGMATA (PROMATA) ASTIGMATA (ACARIDIA) CRYPTOSTIGMATA (PYCNONOTICAE) (PORONOTICAE)

Species

Family

Macrochelidae Uropodidae

&l. muscaedomesticae (Stop.) F. ugituns (Banks)

Ixodidae Argasidae

A. americanurn (L.) A. radiates Raillet

Tetranychidae Pterygosomidae

T. neocaledonicus Andre P. podapolipophagus (Cunliffe)

Acaridae

R. echinopus (Fomouze & Robin)

Damaeidae Oribatulidae

Damaeus sp.

S. nudus Woodring

and Brewer’s yeast. Mucrocheles muscaedomesticae was cultured in containers containing cow manure where free living nematodes and some manure served as a food source. The other mesostigmatid mite (Fuscuropoda agitans) was cultured on either a diet of Brewer’s yeast or wheatgerm Ticks are blood sucking ectoparasites and require a blood-meal prior to apolysis. All stages of Amblyomma americanum were fed on guinea pigs (Cavia porcellus) by placing ticks in bottomless cage-vials which had been previously taped to the shaved back of a guinea pig. Upon completion of feeding, the engorged ticks were collected and stored in culture vials. All stages of the soft tick, Argas radiatus, were fed on young chickens (Gallus domesticus) with the tick-containing capsule taped to their axillary region. Upon completion of feeding, the engorged argasid ticks were placed in vials and subsequently stored in desiccators (90% r.h.) at 32°C until moulting occurred. The soft ticks were subjected to a photoperiod of 8 hr of light and 16 hr of darkness after the procedure of MEDLEY and AHRENS (1970). Tetranychus neocaledonicus were cultured in the laboratory on lima bean leaves, which were rimmed with Tanglefoot to prevent escape of the mites. An individual leaf was placed on a pad of wet cotton in a Petri dish and placed under a fluorescent bulb at 25°C. Pimeliaphilus podapolipophagus is a parasite of cockroaches and was simply cultured in glass cockroach-rearing cages containing the German cockroach, Blatella germanica. RESULTS

OF AMPUTATIONS AND

AND OBSERVATIONS ECDYSIS

OF APOLYSIS

General In all groups (suborders) of acarines tested, clean cut amputations on proximal, medial, and distal limb segments on one or more legs with subsequent recovery

C. L. ROCKETTAND J. P. WOODRING

2322

after apolysis were accomplished. The time of amputation on the acarine specimens was generally varied to include both young and old individuals of a particular instar. There was no evidence of an autotomy plane in any of the species examined. Aspects of apolysis and ecdysis in the nine study species were observed and compared. Mesostigmata Mamocheles muscaedomesticae. Twenty-two specimens survived one to three leg amputations per mite for a total of 35 amputated limbs (Table 2). All immature instars appeared equally tolerant to single or multiple limb amputation, and less than 15 per cent died prior to apolysis. If more than four legs were amputated at one time, the specimen lost mobility and subsequently starved. Multiple amputations vs. single amputations on a single specimen did not affect the regenerative TABLE 2-M.

muscaedomesticae

SURVIVING LEGAMPUTATION

Remaining leg * segments after amputation

No. of animals+ and instar of amputation

19 2 2% 2Q 2&

1 dn 3 dn 1 pn 1 pn 2 dn 1 dn 1 pn 3 dn 11 3 dn 1 dn 5 dn 1 dn 1 dn 1 pn 3 dn 3 dn 1 dn 2 dn

2%

3 3 3* 3+ 3% 4 4; 4% 5 5 56 5Q 6-c

Resultant segments in the adult 1 2 29 2Q 2* 2% 3 3 39 3Q I+ d 4 4* 4% 5+ld 5 54 5% 6 + claw

* A leg of M. mwcaedomesticae is composed of six segments ; a coxa, trochanter, femur, genu, tibia, and tarsus. Two segments remaining means only the coxa and trochanter remain after amputation. -1_M. muscaedomesticae has three immature instars; larva (I), protonymph (pn), and deutonymph (dn). c, means only the claw was cut off; d, indicates a distorted segment (see text).

STUDIES

OF ACARINE

LIMB

REGENERATION,

APOLYSIS,

AND

ECDYSIS

2323

capacity. The time of amputation during a particular instar appeared to play a role in the tolerance of the mite to amputations. Specimens amputated in the latter phases of an instar did not produce much cuticle over the amputated stump, and many of these died within a few hours after ecdysis. None of the amputated specimens of M. muscaedomesticae displayed any regenerative capacity (Table 2), and the site of amputation was most often the definitive end of the appendage after apolysis. In only one specimen was a small growth present distal to the point of amputation in the next developmental stage. This may have been due to small amounts of soft tissue being left beyond the site of amputation at the time of operation, which could have become covered and protected by coagulated haemolymph. In two of the amputated specimens, some reduction was evident after ecdysis. If only the tarsal claws were amputated, these appeared after the next ecdysis. This should not be construed as evidence of regenerative capacity because the tarsal claws are cuticular structures and are normally replaced at every apolysis. The haemolymph of M. muscaedomesticae coagulated rapidly at the point of amputation, and this produced a well-defined and rigid clot. The clot was generally localized only on the distal part of the terminal segment and only in two specimens was a clot observed to fill up an entire segment. Those segments that were completely filled with coagulated haemolymph appeared small and distorted after apolysis. A few hours prior to ecdysis, the hysterosoma of the immature stages of M. muscaedomesticae became distended, but the mites still retained mobility. Ecdysis was a relatively fast process, and the animals were inactive for only a few minutes. The mechanism of apolysis appeared to be similar to that in other arachnid groups and in certain insects. The leg epidermis separated from the cuticle, and a new cuticle with a claw was secreted within the old leg cuticle, the pharate mite being active during this process. Ecdysis began with the mite rocking back and forth for a few minutes and simultaneously expanding and contracting its body. The cuticle split along the anterior edge of the dorsum and with continued body contractions, the old cuticle slid in a posterior direction and became bunched up behind the fourth coxae. In the meantime, the new limbs were withdrawn from the old cuticular hulls, and the old exuviae was finally kicked off by the legs. Total time required for ecdysis was approximately 45 min. Fuscuropoda agitans. Nineteen specimens of F. agitans survived various single limb amputations (Table 3). F. agitans did not tolerate limb amputations as well as M. muscaedomesticae; approximately 30 per cent of the amputated specimens died prior to apolysis. The results of amputations were extremely variable with 10 of the 19 amputated specimens definitely displaying a limited amount of regeneration. Only one specimen displayed a regenerated segment which was normal in size and shape with a slightly abnormal setal pattern. In all other amputated specimens displaying regenerated portions of a limb, the regenerated segments were all distorted in shape, smaller in size, and displayed abnormal setal patterns. Six of the amputated specimens also displayed obvious reductions or loss of segments

C. L. ROCKETTANDJ. P. WOODRING

2324

proximal to the site of the cut. Coagulation of haemolymph in F. agitans was rapid, and bleeding ceased almost immediately after the cut was made. Generally, a firm, localized clot in the distal part of the remaining terminal segment resulted. TABLE 3-F. Remaining leg* segments after amputation 2 26 3 4 4 4 4 49 4+ 5 5 5f 5Q 5g

ugitans SURVIVING LEGAMPUTATION No. of animals-l_ and instar of amputation 11 11 21 21 21 1 pn ldn 21 11 21 1 pn 11 11 1 pn

Resultant segments in the next instar l+ld 2+ld 3+ld 3+ld 4+2d 4+2d 4+2d 4+ld 4+2d 5+ld S+ld 5$ 5+ld 3+ld

* A leg of F. ugituns is composed of six segments: a coxa, trochanter, femur, germ, tibia, and tarsus. Three segments remaining means only the coxa, trochanter, and femur remain after amputation. t F. ugituns has three immature instars: larva (I), protonymph (pn), and deutonymph (dn). d, Indicates a distorted segment.

The apolysis in F. agitans was similar to that in M. muscaedomesticae. The leg epidermis separated from the old cuticle, and the new cuticle and claw was secreted within the old leg cuticle. This separation of limb epidermis from the cuticle and secretion of new cuticle did not impede mobility. The apolysing mite was not as active as the nonapolysing mites, but walked if slightly prodded. Ecdysis in F. agitans started with the anterior and lateral splitting of the cuticle. The emerging mite pulled the legs partially free and pushed up the top of the exuviae. Ecdysis was completed generally in less than 1 hr. Metastigmata Approximately 40 specimens of A. americanurn Amblyomma amekcanum. received either single or multiple amputations. Amputations were performed on larval and nymphal instars both before and after engorgement and on both proximal and distal segments of all four pairs of legs at one time or another. Typical of hard ticks, A. americanurn has only one nymphal instar in its life cycle. There was 100

STUDIESOF ACARINE LIMBREGENERATION, APOLYSIS,ANDECDYSIS

2325

per cent survival of all single amputated A. americanurn. Approximately 20 specimens had two or three limbs amputated, and over 75 per cent survived. The maximum number of limbs amputated on any one specimen that survived and apolysed was four. This tolerance for amputations applied only to active specimens before and after engorgement. Amputation of a limb on an apolysing quiescent stage resulted in the death of the specimen. Every specimen (100 per cent) which survived amputation and apolysed regenerated appendages which were normal in size, form, and setal patterns. Unlike many of the insects that require several successive apolyses to obtain a completely normal appendage, A. amevicanum only required one apolysis. Regardless of the point of amputation on larvae or nymphs, a perfect limb appeared in the next instar. The haemolymph of A. americantim coagulated almost immediately at the point of amputation, where a firm clot quickly developed. Immediately after the cut was made, the tick could walk on the leg and no bleeding occurred. Upon completion of engorgement, which usually required 5 days, the tick larvae remained active for another 4 to 5 days. The larvae then became quiescent and remained in that condition for approximately 13 to 15 days. BALASHOV(1963) states that the entry of host blood into the tick’s gut serves as a stimulus to initiate the apolytic process. The life cycle time of A. americanum did not appear to be appreciably affected by amputations. Non-amputated larvae and amputated larvae respectively required an average of 23 and 24 days from the start of feeding until ecdysis. A sequential series of apolysing larvae were fixed in Brazil’s solution and sectioned at 8 to 10 p using a ribboning methacrylate method (WOODRING, 1970). On the first day of larval apolysis, the limb epidermis detached from the old cuticle. From the second to fifth day of apolysis there was a gradual regression of the soft limb tissue from the tarsus back to the level of the genu. The regressing tissue lost most signs of segmentation. Starting on about the sixth day of apolysis, when the regressing tip of the larval leg had reached the level of the femur, the main mass of regressing limb tissue had formed an oval swelling (or bud) in the coxal region of the pharate form and commenced to elongate in a ventro-lateral direction into the exuvial space. On about the seventh day of apolysis, the elongating nymphal limb had already begun to show signs of segmentation; the still shrivelling larval leg had regressed to the coxa. From the eighth to about the twelfth day of apolysis, the larval stub disappeared and the now segmented nymphal leg elongated to its full length. The nymphal legs came to lie folded upon themselves in the ventrolateral exuvial space, and remained in that position until ecdysis. Ecdysis began with the tearing of cuticle around the area of the scutum. From the scutum, this split continued around the lateral sides of the body until it reached a point immediately behind the fourth pair of legs. The tick split the cuticle by rhythmically expanding and contracting its body. The expansion movements were very strong while the cuticle was in the process of splitting and subsided as the posterior dorsal portion of the exuviae slid in a posterior direction. This required

2326

C. L. ROCKETT AND J. F. WOODRING

approximately 30 min. At this time, body movements were restricted to the unfolding and stretching of legs, which required about 20 min. Next, the emerging tick removed the mouthparts from under the old scutal plate, stretched the legs to their full length, and walked out of the exuviae. Total time required for ecdysis was approximately 50 min. Argas radiatus. The regenerative capacity of A. radiatus was observed in ten specimens, including both the larval and the nymphal instars. A. radiatus appeared to be only moderately tolerant to limb amputations. Approximately 50 per cent of the amputated larvae and 25 per cent of the amputated nymphs died; the larvae usually died within 1 hr after the amputation. was performed. The sites of amputations of A. radiatus were varied, with amputations being performed on both proximal and distal leg segments. Haemolymph coagulation was very slow in A. radiatus, and it often required 10 to 15 min for a limb to stop bleeding after amputation. Following haemolymph coagulation, a firm clot appeared at the site of amputation. If not prevented from walking prior to clot formation, the specimen bled profusely from the stump. Therefore, the majority of amputated specimens were prevented from walking until coagulation of haemolymph occurred. Every specimen which survived amputation and apolysed regenerated limbs that were somewhat smaller than a normal leg; however, the shape of each segment and the chaetotaxy was perfectly normal. Regardless of the site of amputation, all of the amputated specimens displayed regenerated limbs after a single apolysis which were 70 to 80 per cent of the length of a normal leg. For example, of two protonymphs receiving amputations on the right second leg, one received an amputation between the coxa-trochanter joint and the other had only the right tarsus removed. Both specimens displayed regenerated appendages in the next instar 70 to 80 per cent of the length of a normal leg. Double amputation on single specimens did not affect the average size of the regenerating limbs. Two amputated protonymphs were allowed to undergo two successive apolyses. In both cases the regenerated limb had attained the normal size following the second apolysis. This is similar to the situation encountered in many insects and non-acarine arachnids. The amputated appendage does not attain normal size until a number of successive apolyses has occurred. Unlike A. americanum, A. radiatus did not withdraw the legs prior to actual ecdysis nor did it have a quiescent stage for apolysis. Apolysis in A. radiatus was similar to that described already for M. muscaedomesticae. The leg epidermis separated from the cuticle, and a new cuticle complete with claw and setae was secreted within the old cuticular covering. The pharate form was mobile. Ecdysis started with the cuticle splitting at the anterior end of the tick, and the split progressed in a posterior direction along the lateral sides of the exuviae to the fourth pair of legs. The tick then pushed against the dorsum of the exuviae, which resulted in the dorsal half of the exuviae sticking up at a 70” angle. Ecdysis was completed by the tick pulling its legs out of its exuvial hulls and completely emerging from the exuviae. Time of ecdysis was less than 1 hr.

STUDIES

OF ACARINE

LIMB

RRGENBRATION,

APOLYSKS,

AND

ECDYSIS

2327

Prostigmata Tetranychus neocabdonicus. Approximately 75 specimens, both larval and nymphal stages, of T. neocaledonicus received amputations. Only six deutonymphs and one protonymph of the 75 amputated specimens survived single amputations and apolysed. Three of the seven surviving specimens died within a few hours after ecdysis. The amputated limb in these three specimens became stuck to the leaf substrates by what appeared to be haemolymph oozing from the leg stump. The hysterosoma soon collapsed, and the specimens apparently died of exsanguination or desiccation. Two other surviving specimens were carefully observed during the final phases of ecdysis, when both appeared to have difliculty in extricating the previously amputated limb from the cuticufar hull. In one specimen, the previously amputated limb was stuck to the exuviae and was the last limb to be withdrawn from the exuvial limb hulls. The other specimen never did successfully emerge from its exuviae; and 24 hr after the emergence of the other legs from the exuviae, it was still jerking on the amputated leg in an attempt to free itself. The haemolymph of T. neocaledonicus did not coagulate, and a recognizable clot had not formed 1 hr after amputation nor 24 hr after amputation. The majority of specimens receiving amputations were active and usually survived until the day following amputation; however, all but seven subsequently died before the start of apolysis. Control non-amputated specimens maintained under identical culture conditions lived and completed development. How did the seven specimens of T. neocaledonicus survive through the first apolysis ? Probably some physical obstruction (substrate debris or the like) prevented blood or water loss from the leg stumps. The leg stumps of the seven specimens surviving through apolysis were examined, and none appeared to have any cuticle on the distal portion of the amputated limb. It seems that during apolysis of T. neocaledonicus no new cuticle is formed over a cut end of an amputated leg. The lack of cuticle over the point of amputation could explain the difficulty of withdrawal of the amputated limb from its exuviae and the subsequent bleeding described above. No specimen of T. neocaledonicus displayed any regeneration. The few apolysing specimens displayed a reduction of about two leg segments. The terminal portion of the freshly amputated limb was usually uneven (ragged), and never developed a good clot or cuticular cap. T. neocaledonicas has a larval and two nymphal, instars, each with a 2 to 3 day inactive stage during which apolysis occurred. In the last 6 to 8 hr of apolysis, the cuticle appeared dry and semi-white in colour, and the newly formed cuticle of the legs of the next instar was deposited within the old leg hulls. Just before the start of ecdysis the fully formed legs were withdrawn from the old leg hulls and came to rest in the ventral exuvial space. The actual splitting of the exuviae occurred around the girth of the mite between the second and third pair of legs. The split extended completely around the mite and resulted in an anterior and posterior exuvial portion. The mite generally backed out of the anterior portion of the exuviae and then walked out of the posterior part. Ecdysis required as little as five minutes.

C. L.

2328

Pimeliaphiluspodapolipophagus. Approximately 15 but all prior to Within 1 hysterosomas of mites collapsed death soon they had of desiccation exsanguinization.

specimens received following amputations, It appeared

Astigmata echinopus. amputations (Table apolysis and of these

Ten specimens of echinopus survived Only 2 12 amputated died prior received amputations two legs.

4-R.

leg * segments after amputation 2%

* leg of femur, germ, trochanter

echinopus

LEG AMPUTATION

of animalsl_ instar of

in the adult

11 2 tn ltn

l+ld Id

5 5 54 5Q

1 1 1

l+ld 2+ld

5+

11

2+ld

echinopus is of six and tarsus. segments remaining after amputation. R. echinopus three feeding instars: larva tritonymph (tn). non-feeding, phoretic d, Indicates distorted segment.

a coxa, only the

and

protonymph (pn), was not

regeneration was by R. Except for protonymph, all specimens surviving and apolysis varied of the segments resulting segmental and/or distortion. amputations were than the segments and generally stumpin form. an amputated to undergo successive apolyses larva to did not in further or development the amputated In one specimen, amputation the third tarsus resulted the subsequential stage lacking third and left legs. accessory effect be attributed an unnoticed however, the feel that is unlikely following all the exact of amputation presence of clot was by placing mite in water-filled depression and examining with a field microscope. (1970) also similar accessory in performing on the related astigmatid Caloglyphus boharti.

STUDIES

OF ACARINE

LIMB

REGENERATION,

APOLYSIS,

AND

ECDYSIS

2329

Haemolymph coagulation in R. ecJGzopus was rapid, and a distinct clot was formed within a few hours after amputation. The clot position did not appear to govern the form of the leg in the next instar. The clot usually appeared in the segment proximal to the site of amputation, but in most cases the same leg in the next instar showed two or more fewer segments. It is of interest to note that in the single specimen which did not show this reduction of segments, the clot occurred only on the distal end of the amputated segment. In working with Caloglyphus boharti, WOODRING (1969) noted that the position of the clot that formed after leg amputation governed to a large extent the form of the leg in the next instar. It is suspected that the placement of R. echinopus in water mounts to check for the position of clots might have affected the clot position and caused the clot to progress further up the leg. Each instar of R. echinopus had an active and inactive stage, the latter consistently of 1 day duration. Apolysis appeared similar to that observed in C. boharti by WOODRING (1969). At the beginning of the inactive stage the soft leg tissue rapidly dedifferentiated and regressed into and formed a coxal limb bud, which then elongated and differentiated into a new limb extending into the ventral exuvial space. Only in the Astigmata and the ixodid ticks (Metastigmata) was this very unusual process observed. Ecdysis began with the lateral splitting of the cuticle at the posterior end of the body; then the split ran anteriorly to the level of the fourth pair of legs. The mite protruded its hysterosoma from the split, straightened its legs, and backed out of the exuviae. Cryptostigmata Scheloribates nudus. Twenty specimens survived one or two leg amputations per mite for a total of 25 amputated limbs (Table 5). About 20 per cent of the specimens operated upon died prior to apolysis, which indicated only a moderate tolerance to leg amputation. No regeneration was displayed by S. nudus. Segment reduction, distortion, or both occurred in all cases. The amount of segmental reduction was varied and appeared to be independent of the site of amputation. For example, amputations of only tarsi in four different tritonymphs resulted in reductions to the tibia, genu, femur, or coxa respectively in the next instar. The least amount of reduction was one segment, and the maximum four. In one case the corresponding adult leg was completely missing, including the complex acetabulum formed by the coxa in adult oribatids. The majority of distorted segments occurring in the next instar of the amputated specimens was slightly smaller than a normal segment, and were often flask shaped with a long terminal seta at its distal end (Fig. 1). Specimens receiving double amputations did not display any greater segmental reduction or distortion than many specimens receiving single amputations. Haemolymph coagulation in 5’. nudus was rapid, as indicated by the lack of bleeding following amputations. The position of the clot was difficult to determine because the cuticle was light brown in colour and near the same colour as that of

C. L. ROCKETTANDJ. P. WOODRING

2330

TABLE 5-S. Remaining leg* segments after amputation 1 2 2 2* 2+ 2% 3 3 3 3 3Q 4 :* 5 5 5 5 5 5%

nudus SURVIVING LEG AMPUTATION No. of animals-/and instar of amputation 1 pn 1 pn 1 tn 11 4 tn 1 dn 11 1 pn 1 dn 2 tn 1 tn ltn 2tn 1 pn 1 dn 1 tn 1 tn 2tn 1 tn 1 tn

Resultant segments in the adult mite 1 2 l+ld 0 1 1 1 1 1 2+ld 1 1 2+ld 1 2+ld 1 l+ld 2+ld 4+ld 3+ld

* A leg of S. nudus is composed of six segments: a coxa, trochanter, femur, genu, tibia, and tarsus. Two segments remaining means only coxa and trochanter remain after amputation. + S. nudushas four immature stages : larva (l), protonymph (pn), deutonymph (dn), and tritonymph (tn). d, Means a distorted segment (see text).

the clot. In all specimens with discernible clots, reduction of the limb in the next instar always extended further proximally than the position of the clot. Each immature instar of S. nudus had an active and inactive stage, the latter lasting 2 to 3 days. Amputation of a specimen in the active stage did not lengthen the quiescent period. Apolysis occurred in the inactive stage, during which the leg epidermis separated from the old cuticle and a new cuticle was secreted with the old leg hull. The fully formed limbs were withdrawn from the old leg hull and were extended into the ventral exuvial space 6 to 12 hr before ecdysis, depending on the instar. Ecdysis began with the old cuticle splitting laterally on the posteriormost end of the body and continuing anteriorly to the level of the fourth pair of legs. The hysterosoma then partially protruded from the split and remained in that position for 2 to 4 hr until the legs and mouthparts became sufficiently sclerotized. When the legs had hardened, they were used to push the individual from the old cuticle.

STUDIES OF ACABINE LIMB REGENERATION,APOLYSIS, AND ECDYSIS

FIGURE

Amputation Normal

limb

lxodid Typical

Argasid

Uropodid

Appearance

Gamasina

of Limb

in the

2331

I

Astigmata

Cryptostigmafa

Next

after

lnstar

Prostigmata

Amputation

FIG. 1. Generalized results of limb amputation in the Atari showing the maximum regeneration in each group (or the minimum of segmental reduction) after a single apolysis. Regeneration occurred in ixodids, argasids, and uropodids, though regeneration in the latter was very limited. No regeneration occurred in gamasina, astigmata, cryptostigmata, or prostigmata; instead, a variable degree of segmental reduction and/or distortion occurred. The few prostigmata that survived amputation and apolysed tore the new limb cuticle from the exuviae. See text for further details.

Dapnaeus sp. Seven specimens of an undetermined Damaeus species survived limb amputations (Table 6). The tolerance of this species to limb amputations was low; approximately 40 per cent of amputated specimens died prior to apolysis. TABLE 6-Damaeus Remaining leg* segments after amputation

i

:r 4

4& 5 5

sp. SURVIVING LEG AMPUTATION

No. of animals~ and instar of amputation 11 1 pn IQ-I 1 dn 1 pn 2tn

Resultant segments in the next instar I+ 1 2 1 1+ 1 3+ 1 1

* A leg of Damaeus sp. is composed of six segments : a coxa, trochanter, femur, genu, tibia, and tarsus. Two segments remaimng means only the coxa and trochanter remain after amputation. I- Damaeus sp. has four immature stages: larva (l), protonymph (pn), deutonymph (dn), and tritonymph (tn). d, Means a distorted segment (see text).

2332

C. L. ROCKETT ANDJ. P. WOODRING

As with S. nudus, Damaeus sp. did not display any regeneration of limbs, and segmental reduction and distortion were common. All of the distorted segments were less than half of their original size, possessed normal cuticle, and lacked extra or abnormal setae. Haemolymph coagulation was rapid, and as with S. nudus, the position of the clot did not appear to govern the appearance of the limb in the next instar. A number of specimens displayed well-defined clots, but the limbs in the next instar had regressed past the clot site. Apolysis in Damaeus sp. was similar to that described for S. nudus. Prior to the beginning of ecdysis, the new limbs were withdrawn from the old limb hull and extended into the ventral exuvial space. One of the reasons this species was studied was to see if the limbs in an oribatid characterized by very long and thin legs would also be withdrawn prior to ecdysis. That the pharate form during the inactive stage can withdraw legs three times the body length from the old cuticular hull while still encased in the old exuviae is remarkable. Only the fact that the cuticle of the new limb was not yet sclerotized made this act of contortion possible.

SUMMARY

AND

DISCUSSION

Within the Atari there was a tremendous variance of regenerative capacity, which ranged from the ability to regenerate perfect and complete appendages in a single apolysis to the absence of a regenerative capacity with concurrent segmental reduction or even death following the loss of an appendage (Fig. 1); however, species within the same subordinal group (Table 1) displayed pronounced similarities in regenerative capacities. In many arthropods, the presence of an autotomy plane is indicative of a certain amount of regenerative capacity. None of the acarine species involved in this study have an autotomy plane. Pulling on a limb of any of the test acarine species never resulted in breakage at a specific point; the breakage was always random. The only report of an autotomy plane in the Atari was by VITZTHUM (1943) for one of the primitive mites, Opilioacarus. The only group which was able to regenerate completely normal appendages Up to four legs were amputated at the coxa-trochanter on was the Metastigmata. various nymphs, and all survived amputations and regenerated complete appendages in one apolysis. To our knowledge, no insect, spider, or any other arthropod has been reported to display a regenerative capacity which equals that of the hard tick, A. americanurn. In the majority of arthropods which display regeneration of limbs, complete removal of a limb usually necessitates several successive apolyses to get complete regeneration of a normal sized leg. Such was the regeneration of legs in the soft tick, A. radiatus. In the next instar the regenerated appendage was normal in form but was always about 7.5 per cent of its normal length. For some unexplained reason, the site of amputation did not appreciably affect the size of the regenerate. A. radiatus required at least two apolyses to regenerate a full-sized normal appendage.

STUDIES

OF ACARINE

LIMB

REGENERATION,

APOLYSIS,

AND

ECDYSIS

2333

The Mesostigmata was the only other group of Atari to display any regenerative capacity at all. M. muscaedomesticae did not display any regenerative capacity, and the site of amputation was usually the definitive end of the new appendage after ecdysis. F. agitans displayed a limited amount of regeneration, where in some specimens a distorted segment appeared beyond the site of amputation after apolysis. The maximum amount of regeneration displayed was two distorted segments. The majority of amputated specimens of F. agitans displayed distortion on either the amputated segment or segment immediately proximal to the amputated segment. A great amount of segmental reduction followed amputation in the next instar of R. echinopus, and allowing a specimen to undergo several successive apolyses did not appear to enhance the regenerative capacity. WOODRING(1969) noted that the astigmatid mite, C. boharti, displayed some very limited regeneration in a very small percentage of amputees, wherein with intersegmental amputations of a segment the terminal half of the segment was regenerated whole and normal. He further noted that a high percentage (33 per cent) of adult legs displayed one, two or even three segments fewer than remained on the amputated nymphal leg stump. In no instance was an entire segment regenerated. From WOODRING’S (1969) observations on C. boharti and our observations on R. echinopus, it appears that limb regeneration in the astigmatid family Acaridae is essentially non-existent. Neither cryptostigmatid mites, S. nudes or Damaeus sp., tolerated leg amputations very well. Both displayed very similar regenerative capacities, characterized by segmental reduction, distortion, or both. Segmental reduction was often extensive and sometimes resulted in the complete absence of a partially amputated leg following apolysis. The prostigmatid species appeared to be the least tolerant to amputations. Less than 10 per cent of the amputated specimens of T. neocaledonicus survived amputations, and these all displayed some segmental reduction. The site of amputation on the leg was varied in all of the species studied. The site of amputation did not appreciably affect the amount of regeneration exhibited. Excluding amputations on quiescent stages in the process of apolysing, the time of amputations in the instar also did not appear to affect the amount of regeneration displayed, Amputation on quiescent stages was fatal, which was probably due to the general obstruction of the mechanism of apolysis. The clotting and wound healing capabilities of the amputated specimens within the five suborders were varied. Coagulation of haemolymph seemed in general to involve a thickening of the haemolymph through an unknown process, and a solid clot would form. In all suborders except the Prostigmata, new cuticle was secreted over the wound at apolysis. In the ixodids, mesostigmatids, astigmatids, and cryptostigmatids, the haemolymph quickly coagulated and a distinct clot was formed. In the Ixodidae and Mesostigmata the resultant clot following amputation was usually localized at the point of amputation. R. echinopus and Damaeus sp. commonly displayed recognizable clots on one or two segments proximal to the site of amputation. S. nudus did not usually form localized clots,

2334

C. L. ROCKETT AND J. P. WOODRING

but rather formed diffused clots in one or more proximal segments. In the argasid coagulation required 10 to 1.5min, and a firm clot formed only after 1 to 2 hr. The blood of Prostigmata did not seem to coagulate, and it did not appear to ever form a clot. Likewise, new cuticle was never formed to cover the leg stump at apolysis. The mode of apolysis and ecdysis varied among the Atari (Fig. 2), but was consistent in any given subordinal group. An inactive stage preceding ecdysis was FIGURE

2

FIG. 2. Schematic diagram of apolysis and of ecdysis in the Atari. Only legs I and IV and the chelicerae are shown; legs II and III and the pedipalps were omitted in order to avoid unnecessary clutter. Starting with the active feeding stage (which could be in any instar), note by following the three arrows that there are three basic mechanisms of apolysis among Atari. The Argasida, Uropodida, and Gamasina (left) remain mobile throughout apolysis (active pharate form), ecdyse by splitting the cuticle at the anterior end (ecdysis type l), and withdraw the legs at ecdysis. The Prostigmata and Cryptostigmata (centre) are immobile throughout apolysis, withdraw the legs prior to ecdysis and ecdyse either by splitting the cuticle around the girth of the mite (ecdysis type 2) or by splitting the cuticle at the posterior end (ecdysis type 3). The Astigmata and Ixodida (right) are immobile throughout apolysis, the limbs regress toward the coxa and grow anew from the coxal region into the ventral exuvial space, and ecdysis is either of type 1 (Ixodida) or of type 3 @stigmata). For further details see text.

lacking in all instars of M. muscaedomesticae, F. agitans, and A. radiatus. In these three species, the leg epidermis separated from the old cuticle and a new cuticle complete with setae and claws was secreted within the old cuticular covering.

STUDIFS

OF ACARINE LIMB REGENERATION,

APOLYSIS,

AND ECDYSIS

2335

During apolysis the pharate form was active and retained its mobility up to the moment of -ecdysis. R. echinopus and A. americanurn both had a relatively long inactive stage in each instar prior to ecdysis. Apolysis began with both species becoming swollen and immobile, and the leg tissue progressively dedifferentiated and regressed into a mound of tissue in the coxal region. Elongation and differentiation of the ‘coxal bud’ in a ventro-medial direction resulted in the limb of the next instar growing into the ventral exuvial space. The regressing leg tissue in ticks did not appear to dedifferentiate as extensively as the regressing leg tissue of the astigmatids. S. nudus, Damaeus sp., and T. neocaledonicus also had an inactive stage in the latter part of each instar during which apolysis occurred. In these three species the epidermis separated from the cuticle and secretion of new cuticle, complete with setae and claws, occurred within the old leg hull. Long before the start of ecdysis, the limbs were withdrawn from the old leg cuticle and subsequently came to lie within the ventral exuvial space. The mode of cuticular splitting, signalling the start of ecdysis, varied among the subordinal groups (Fig. 2). In the Mesostigmata and Metastigmata, ecdysis began with the old cuticle splitting on the anterior end of the animal, and the split progressed in a posterior direction along the lateral sides of the exuviae. In the Prostigmata, the splitting of the exuviae occurred around the girth of the mite between the second and third pair of legs. The Astigmata and superior Cryptostigmata developed the initial split at the posterior end of the body, and the split progressed along the lateral sides to the level of the fourth coxae. There was no correlation of regenerative capacity with the type of apolysis. In both the astigmatid and the ixodid, limb regression occurred during apolysis but regeneration of limb occurred only in the latter. The type of apolysis was similar in the argasid and gamasid, but only in the argasid did regeneration occur. In Goss’ (1969) summary 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, presence, and growth of nerves, and with the hormone (ecdysone) titres in the haemolymph. We have shown that neither the site of amputation, the number of legs amputated, nor the time of amputation relative to the next ecdysis has any influence on the extent of regeneration in the Atari. Essentially nothing is known, of acarine hormones. Many authors have demonstrated that in various non-arthropods, the regeneration of lost body parts depends on the presence of a nerve supply. The process of regeneration in insects has been studied many times by many authors; however, the r81e of nerves on regeneration is still open to question. NUESCH (1968) states that among insects the beginning of regeneration seems to be independent of the nervous system. BODENSTEIN(1955) noted that in the roach, Periplaneta americana, the stumps of the ventral nerve cord grew so rapidly that it was impossible to obtain regenerated legs without nerves. The majority of acarine specimens subjected to amputation displayed movement in the remaining stump following amputation. This would appear to be indicative of a nerve supply still being present in at least

C. L. ROCKETTAND J. P. WOODRING

2336

the proximal regions of the stump. Those not displaying movement in the amputated limb might be assumed denervated, but no appreciable difference of regenerative capacity was noted in the mobile or non-mobile limb stumps. In dealing with acarine regeneration, the probability exists that the presence or absence of post-larval mitoses might, in itself determine the amount of regenerative capacity displayed by various acarine groups. In working with C. boharti, WOODRING (1969) noted that it was likely that these mites have a constant cell number after the larval instar because no mitoses were observed during elongation of the new leg. Boudreaux (1971, personal communication) observed which

post-larval

post-larval

mitoses

mitoses

in sections

of spider mites.

have been observed

stated that he has never The

are the ticks.

only acarines in BALASHOV (1963)

the moulting process of the tick, Hyalomma asiaticum, and noted that extensive mitosis occurred during the first period of apolysis. We sectioned A. americanurn larvae and found extensive mitoses in the epidermis and limb tissue

studied

starting. on the second day of feeding

and continuing

with less extensive mitoses

over the next 5 days. Further work is in progress to determine the extent and occurrence of post-larval mitoses in all of the major groups of acarines. REFERENCES BALASHOV Y. S. (1963) Anatomical-histological peculiarities of moulting of the tick Hyalomma asiaticum (Acarina: Ixodidea). Zool. Zhur. 42, 345-357. BODENSTEIND. (195.5) Contributions to the problem of regeneration in insects. r. exp. Zoo& 129,

209-224.

BONNET P. (1930) La mue,

l’autotomie et la regenerations chez les araignees, avec une etude des dolomides d’Europe. Bull. sot. Hist.nat. Toulouse 59, 237-700. GOSSR. J. (1969) Principles of Regeneration. Academic Press, New York. MEDLEY J. and AHRENSE. (1970) Life history and bionomics of two American species of fowl ticks of the subgenus Persicargas. Ann. ent. Sot. Am. 63, 1591-1594. NUESCHH. (1968) The role of the nervous system in insect morphogenesis and regeneration.

A. Rev. Ent. 13, 27-44. ROSIN R. and SHULOVA. (1963) Studies on the scorpion Nebo hierochonticus. Proc. ~001. Sot. Lond. 140, 547-575. VACHON M. (1957) La regeneration appendiculaire chez les scorpions (Arachnides). C. R. Acad. Sci., Paris 244, 2556-2559. VITZTHUM G. (1943) Acarina. In Bronn’s Klassen und Ovdnungen des Tierreichs, Band 5, Abteilung 4, Buch 5, pp. l-101 1. Akademische Verlagsgesellschaft Leipzig. WOOD F. D. (1926) Autotomy in Arachnida. 3. Morph. 42, 143-195. WOODRING J. P. (1969) Preliminary observations on moulting and limb regeneration in the mite Caloglyphus boharti. J. Insect Physiol. 15, 1719-1728. WOODRINGJ. P. (1970) Comparative morphology, homologies, and functions of the male system in oribatid mites (Arachnida: Atari). _7. Morph. 132, 425-451.