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The development of the Malpighian tubules ofCarausius morosus (Orthoptera)

By A. A. SAVAGE(From the Department of Zoology, University of Leeds; present address,

Wycliffe College, Stonehouse, Gloucestershire)

SummaryIn Carausius morosus Brunn (Phasmidae) the Malpighian tubules are morphologicallyand physiologically differentiated into 3 distinct types. The appendices are of unknownfunction, number about 31, and are inserted separately from one another over theposterior third of the mid-gut. The excretory and calciferous tubules are arranged ingroups at the extreme posterior end of the mid-gut. There are 20 to 37 groups eachconsisting of one excretory and 5 calciferous tubules opening into a small ampulla.

The tubules arise in 5, occasionally 6, generations; 2 in the embryo and one in thesecond, third, fourth, and sometimes in the fifth nymphal instars. The first embryonalgeneration is composed of the 24 to 37 appendices (pre-moult embryonal tubules), thesecond of the 20 to 27 excretory (post-moult embryonal) tubules. The number ofcalciferous (nymphal) tubules produced in each generation depends largely on thenumber of groups; there are usually 2 per group in the second and third instars andone in the fourth. Occasionally a single tubule is added to some groups in the fifthinstar. The appendices pass backwards and are attached to the anterior parts of thehind-gut; the excretories first pass forwards, then backwards and are attached to thehind-gut just anterior to the rectum, whilst the calciferous tubules are attached distallyto the fat-body.

Each tubule is composed of 5 rows of spirally arranged cells which appear as hexa-gons in surface view. The excretory tubules are differentiated into upper andlower segments and are attached to the gut by a series of short tracheae. The calci-ferous tubules have a distal region filled with a white crystalline substance and areserved by single spiral tracheae. The appendices are not differentiated into segmentsand have no tracheal supply.

The development of any one tubule comprises a period of initiation and elongationby cell-division, and a period of differentiation. The first period is restricted to thefirst few days of a stadium, i.e. is linked to cyclical development, whilst the secondcontinues through the remainder of the life of the insect.

The secondary tubules of any one group are added alternately on either side of thefirst formed tubule along the line of the posterior interstitial ring, and the arrangementof tubules is a result of curvature of this ring.

Tubule buds formed in a particular instar may complete their phase of cell-divisionin that instar or may remain dormant until the similar period in the succeeding instar.

The calciferous tubules undergo a rudimentary metamorphosis in the sixth (last)nymphal stage.

IntroductionT H E development of Malpighian tubules has already been studied in twospecies of Orthoptera, Blatta (Henson, 1944) and Schistocerca (Savage, 1956),and its relevance to general problems of insect development is set out in thesepapers. The present account is concerned with the same problems, and since

[Quart. J. micr. Scl., Vol. 103, pt. 4, pp. 417-37, 1962.]

418 Savage—Malpighian tubules of Carausius

the material has proved more favourable in some respects I have been able toamplify as well as confirm existing knowledge of the subject.

The stick insect has three types of tubule, each showing certain individualcharacteristics, though all are modifications of the basic developmental patternfound in the Orthoptera.

Materials and MethodsThe stick insects were bred in the laboratory through all stages of their life-

history. Carausius morosus is parthenogenetic and the population consistedentirely of females. The eggs were collected, placed on moist sand in pillboxes, and kept at about 20° C. After a period of 2 to 4 months the nymphshatched and were transferred to a rearing cage. The identification of the 7instars was based upon measurements of head-width and the degree ofdevelopment of the genitalia (Savage, 1957).

Examples of all stages were dissected and examined in 1% saline, or 30%alcohol, or after overnight staining in methylene blue in 1% saline. A numberof specimens were injected with a drop of methylene blue in 1% salineimmediately before they were killed for dissection. The dye is taken up by theMalpighian tubules, greatly facilitating dissection, and enabling the rapidremoval of tubules for examination in drops of insect haemolymph or salinewhilst they are still fresh. This method proved particularly useful for thedemonstration of cell-walls and nuclei. For microscopic preparations materialwas fixed in Carnoy's fluid, double embedded in celloidin and paraffin wax,cut into sections 8 to 10 /u. thick, and stained in Delafield's or Heidenhain'shaematoxylin with eosin or orange G as counter-stains. Whole mounts werestained in Delafield's haematoxylin.

The anatomy of the tubulesIn previous accounts of the anatomy of the alimentary canal of C. morosus

three types of tubular appendages, excluding the hepatic caecae, have beendescribed; the 'appendices of the mid-gut', the 'superior' tubules, and the'inferior' tubules (de Sinety, 1901; Ramsay, 1955). This analysis into threetypes depends upon differences of anatomy and function. All three types oftubules open into the mid-gut, the appendices separately and irregularly overits hinder parts, the superior and inferior tubules in groups round the mid-gut/hind-gut junction (fig. 1, A). The superior tubules are purely excretory infunction, correspond exactly to the Malpighian tubules of other Orthoptera,and may therefore be more appropriately called the excretory tubules. Theinferior tubules are filled with calcium salts in their distal parts and it has beensuggested (de Sinety, 1901) that they are concerned with the impregnation ofthe egg-shell with mineral salts. Morphologically, they are clearly modifiedMalpighian tubules but since their function is not excretory they will bereferred to as the calciferous tubules. The function of the appendices isunknown; their anatomy is not strictly comparable to Malpighian tubules ingeneral and their status as tubules will be considered later.

FIG. I. A, dorsal view of the hinder part of the gut in an adult. B, the arrangement of theexcretory and 5 nymphal tubules in a typical group together with a plan of their insertions,c, ditto, in a group with 4 nymphals. D, ditto, in a group with 4 nymphals. E, ditto, in a groupwith 6 nymphals. F, ditto, in a group with 2 excretories and 8 nymphals. G, second showingthe relative lengths of the nymphal tubules in a second instar nymph, H, ditto, in a fourthinstar nymph, a, appendix of the mid-gut; ca, calciferous (nymphal) tubule; ex, excretory(post-moult embryonal) tubule; hg, hind-gut; I, longitudinal muscle of hind-gut; mg, mid-gut;ot, opening of tubule group; sph, sphincter muscle; I to V, 1st to 5th nymphal (calciferous

tubules in order of initiation.


In the adult the excretory and calciferous tubules together number about140 and are arranged in 20 to 27 groups round the hind end of the mid-gut.Although the number of groups is variable, the morphological relationships ofthe tubules within each group are remarkably constant, only a few aberrationshaving been observed. Typically, each group consists of 6 tubules, an excre-tory tubule anteriorly with 5 calciferous tubules behind it. The 5 calciferoustubules are arranged one behind the excretory and 4 in 2 pairs behind thefirst (fig. 1, B). Each group of tubules opens into a shallow conical ampullaconsisting of mid-gut cells anteriorly and hind-gut cells posteriorly. It is notas definite a structure as in Schistocerca (Savage, 1956) and merely representsthe confluent bases of the tubules (similar to fig. 2, B). The ampullae opendirectly into the gut and are connected transversely by an annular groovewhich marks the mid-gut/hind-gut junction. The cells at the base of thisgroove are small and represent the posterior interstitial ring. The peritrophicmembrane extends backwards from the mid-gut over the openings of theampulla and since the hind-gut has deep longitudinal folds there is always anample channel for the flow of excretory materials (as in fig. 2, A). Thus thebasic features of the anatomy of this region are similar to the correspondingregion of Schistocerca (Savage, 1956), but rather simpler.

The excretory tubules pass anteriorly a short distance from their insertionson the mid-gut, turn sharply posteriorly and become attached at their distalends to the hind-gut just in front of the rectum (fig. 1, A). Each tubule is 25 to30 mm long and covered by a 'peritoneal coat' of thin squamous epitheliumand muscle-fibres. Throughout its length it is supplied by a series of shorttracheae from the tracheal system round the gut, each trachea dividing intoan ascending and descending branch on reaching the tubule. The tubule itselfconsists of two segments, an upper 075 to 2-0 mm long and 50 to 60 /x widewhich is clear and colourless, and a lower about 100 ju wide which is more opaqueand frequently contains crystals. Both segments possess a striated border. Thecellular construction of the two regions is similar, the only difference being thesmaller size of the cells of the upper segment, each consisting of 5 rows ofhexagonal binucleate cells arranged in a slight longitudinal spiral (fig. 5, c).

The calciferous tubules do not pass anteriorly but proceed directly back-wards from their points of insertion on the mid-gut, and become attacheddistally to the fat-body by a bunch of tracheae ending in a group of cells at thetip of the tubule (fig. 1, A). These cells were first described in Gryllidae bySirodot (1858) and when later described in Phasmidae were called the 'cells ofSirodot' (de Sinety, 1901). They are not present in the excretory tubules.Each calciferous tubule is about 25 mm long, covered by a 'peritoneal coat',and has a single spiral trachea running along its entire length. It is divisibleinto two segments an upper one 6 to 8 mm long, 200 to 300 fx wide, and a lowerone about 100 /u. wide. In contrast to the excretory tubules, the uppersegment is much the wider and is densely packed with white crystalline sub-stances whilst the lower is relatively clear and transparent. Both segmentshave a striated border and consist of 5 rows of spirally arranged hexagonal

FIG. 2. A, longitudinal section of a tubule group in a second instar nymph showing the originof the first nymphal. B, ditto, in third instar nymph, ex, excretory (post-moult embryonal)tubule; hg, hind-gut; mg, mid-gut; pir, posterior interstitial ring; pt, peritrophic membrane;

I, III, ist and 3rd nymphal tubules.


binucleate cells. Correlated with its larger size the cells of the upper segmentare larger than those of the lower and are frequently distorted by the pressureof the contents.

The third type of tubules of Carausius are the appendices of the mid-gut.They number 24 to 37 and are distributed irregularly over the hind third ofthe mid-gut. Each opens separately by a pyriform swelling, passes directlybackwards, and is attached distally to the hind-gut a few millimetres behindthe mid-gut/hind-gut junction (fig. 1, A). Each is a simple tube 8 to 10 mmlong, 50 fj- wide, and covered by a peritoneal coat but without any trachealattachments. The appendices are clear and colourless throughout and possessa striated border, but there is no suggestion of a division into segments. Theyare constructed of 5 rows of hexagonal binucleate cells except at the pyriformbase where the cells are irregular in shape and may contain up to 6 nuclei.

Thus the Malpighian tubules of Carausius are differentiated into 3 distinctforms. There can be no doubt of the homology of the excretory and calciferoustubules but the interpretation of the appendices is more doubtful. However,their cellular construction, 5 rows of binucleate cells, and the fact that they aremid-gut appendages point to their being true Malpighian tubules. As will beseen later, certain aspects of their development provide strong support forthis opinion.

The development of the tubulesThe 3 types of tubule differ not only in their anatomy and function but also

in their times of formation. Each type bears a definite invariable relationshipto the particular phase of the embryonic or post-embryonic period in which itarises. The embryonic development of Carausius lasts 2 to 4 months (at about200 C) and the eggs develop at varying rates. However, there is always a moultduring this period which enables embryogenesis to be divided into twoperiods, a 'pre-moult phase' and a 'post-moult phase'. Similarly, the post-embryonic development is divisible by moults into 7 instars, 6 nymphal, andthe imaginal (Savage, 1957). The appendices arise in the pre-moult embryonicphase, the excretory tubules in the post-moult embryonic phase, and thecalciferous tubules in the second to fourth (occasionally fifth) nymphal instars.No tubules are formed in the first, sixth, or imaginal instars although differen-tiation of the existing tubules continues.

Since the formation of each type of tubule is restricted to a certain period ofthe life-history they may be named according to their time of formation andcomparisons with other Orthoptera may be attempted on this basis. Theappendices are then called 'pre-moult embryonals', the excretory tubules 'post-moult embryonals', and the calciferous tubules 'nymphals'.

All 3 types of tubule, although differing in time of initiation and final posi-tion, originate from embryonic cells at the inner end of the proctodaeumwhich have been shown to be homologous with the anal half of the annulateblastopore (Henson, 1946). The appendices (pre-moult embryonals) ariseat the inner end of the proctodaeal invagination during the formation of the

Savage—Malpighian tubules of Carausius 423

mid-gut and as this is formed they are carried out on to its surface. Theexcretory (post-moult embryonal) and calciferous (nymphal) tubules arise inthe same embryonic zone when the mid-gut is complete and hence retain theiroriginal position.

The 3 types of tubule show the same basic form of cellular differentiation.There are 2 main phases; one of initiation and elongation by cell-division, andone of elongation and differentiation by cell enlargement. All the tubules

TABLE I

The numbers of Malpighian tubules in adults

Type of tubule

AppendicesCalciferous

Excretories

No. ofspecimensexamined

3°13

No. ofspecimensexamined

49

Range in no.of tubules

24-37108-129

Range in no.of tubules

20-27

Mean

3i117

Frequencydistribution

Number

2021222324252627

Frequency

213

28

16512

show 5 rows of cells in cross-section and it is only when they become functionalthat individual characteristics begin to appear.

The increase in the number of tubules. As already mentioned, the Malpighiantubules of Carausius arise in successive generations during the life-history.The appendices arise in the pre-moult phase of the embryo and, in allcharacteristics except size, their development is complete, so far as can beseen, by the time of the embryonic moult. The excretory tubules arise in thepost-moult phase and complete their development to a functional condition bythe time of hatching. The development of the nymphal tubules is restrictedto the second to fourth (occasionally fifth) instars inclusive. None of thetubules completes its development in the instar in which it originates but alldifferentiate together in the sixth instar. This mode of development is quitedifferent from that of Blatta (Henson, 1944) and Schistocerca (Savage, 1956)where the complete process of tubule development is repeated in each instar.In other words the strictly cyclical character of tubule development describedin other Orthoptera is not adhered to so rigidly in the nymphal tubules ofCarausius.

An analysis of tubule numbers is given in table 1. In any one individual thenumber of appendices is fixed in the embryo and shows no change thereafter.

2421.4 Gg


Between individuals the observed range of variation is 24 to 37, with a normalfrequency distribution.

The number of excretory tubules, each being the basis of a group, variesfrom 20 to 27. The frequency distribution here is not normal but bimodal,the majority of individuals possessing either 21 or 24 tubules.

The number of calciferous tubules is nearly always 5 per group and variationin the total number of tubules present is therefore dependent almost entirelyon variation in the number of excretory tubules. For this reason no separateanalysis of the nymphal tubules is given. Normally, of the 5 per group, 2arise in the second instar, 2 in the third, and one in the fourth.

The appendices of the mid-gut (pre-moult embryonals). During the early stagesof embryogenesis, before the formation of the tubules, the posterior partof the alimentary canal is represented by the proctodaeal invagination. Atthe blind inner end of this tube is a mass of cells which divide actively andform the hind part of the mid-gut. Soon after the beginning of this processactively dividing tubule buds appear in this same mass of cells; they immedi-ately begin to elongate posteriorly along the surface of the gut and soonbecome attached to the mesodermal covering of the proctodaeum. At thesame time their basal ends are carried forwards with the mid-gut as it extendsand on the completion of the pre-moult phase, when the mid-gut is a fullyformed tube, they have taken up their final positions as in the adult (fig. 3,.A, c, D).

As with all the tubules, the entire development of the appendices may bedivided into 2 phases; a period of initiation and elongation by cell-division,and a period of growth and differentiation by cell enlargement. Throughoutthe phase of cell-division the tubules are 17 to 19 fx wide, have a small lumenand a covering of mesodermal cells. They are composed of 5 rows of cells andmitoses are restricted to the longitudinal direction (fig. 4, B). The mitoticphase comes to an end before the embryonic moult and further elongation isbrought about by cell enlargement. At the time of hatching the appendicesare about 1 -5 mm long and 25 /x wide; throughout the remainder of the life ofthe insect they gradually increase in size though they remain smaller than theexcretory and calciferous tubules. They always consist of 5 rows of cells witha striated border but they are not differentiated into segments.

It will be noted from the preceding account that the appendices and mid-gut are derived from a mass of cells situated at the inner end of the proto-daeum. Henson (1946a) has shown that there is an embryonic zone of cells,,the posterior interstitial ring, at the inner end of the proctodaeum homologouswith the anal half of the annulate blastopore; and that tissue formed at itsanterior border is endodermal whilst tissue produced posteriorly is ectodermal(fig. 3, A). Hence the appendices and mid-gut must be interpreted as en-dodermal structures (fig. 3, c, D).

The post-moult embryonal and nymphal tubules arise in much the samemanner as the primaries and secondaries of Blatta (Henson, 1944) andSchistocerca (Savage, 1956) where it was shown that they are to be interpreted


FIG. 3. The theoretical interpretation of the proctodaeal invagination.A, the basic form of the proctodaeum. B, the origin of primary tubulesin Blatta, Schistocerca and Forficula. C, the origin of the pre-moultembryonals of Carausius. D, the origin of the post-moult embryonalsof Carausius and the more anterior position of the pre-moult em-bryonals. a, pre-moult embryonal (appendix) tubule; end, endoderm;ect, ectoderm; ex, post-moult embryonal (excretory) tubule; hg,hind-gut; nig, mid-gut; p, primary tubule; pir, posterior interstitialring; pr, 'proctodaeal' or met-enteric membrane. (Theoretical

diagrams based on camera lucida drawings.)


as endodermal derivatives. If this be admitted there is much similarity oforigin and anatomy between the appendices and Malpighian tubules proper(fig. 3, B, D). Similar observations have been made by Heymons (1897) onBacillus rossii (Phasmidae), but they were interpreted in a totally differentsense to support his theory of the ectodermal origin of the mid-gut. As a resultof this theory there have been many conflicting accounts of the origin of themid-gut (Hammerschmidt, 1910; Leuzinger, 1926; Thomas 1936).

In some of these accounts the excretory and calciferous tubules have beenreferred to as Malpighian tubules proper whilst the appendices have apparentlybeen regarded as different structures. It is here proposed that all 3 types oftubule should be regarded as true Malpighian tubules since their structureand origin are essentially similar. All are endodermal, they all arise in theposterior interstitial ring and have a similar type of development and cellularconstruction. Hence they may be regarded as homologous structures.

The excretory tubules [post-moult embryonals). At the time of the embryonicmoult the mid-gut is a fully formed tube with its line of demarcation from thehind-gut clearly defined immediately behind the met-enteric membrane (asin fig. 4, B). Soon after the moult 20 to 27 actively dividing tubule budsappear along this line of demarcation, which is thereby revealed as a zone ofembryonic cells at the mid-gut/hind-gut junction (fig. 4, A). The tubulestherefore arise in exactly the same manner as the first formed tubules of Blatta(Henson, 1944) and Schistocerca (Savage, 1956) and hence this region is to beinterpreted as the posterior interstitial ring. All the buds appear simulta-neously and are evenly distributed round the perimeter of the gut. The tubulerudiments continue their mitotic phase and gradually elongate, passing firstanteriorly and then turning sharply backwards to become attached to the hind-gut just anterior to the rectum. During this period the tubules are about 19 to23 /x wide, consist of 5 rows of cells covered by a layer of mesoderm (fig. 4,c), and mitoses are restricted to the longitudinal direction (fig. 4, A). At theend of this phase of cell-division the nuclei divide without a correspondingcell-division and the binucleate character of the cells is established (fig. 4, D).That this is a result of the final cell-division is shown by the fact that ata slightly earlier stage some of the cells still have only one nucleus (fig. 5, A).Cell-division ceases before hatching and all further increase in size is broughtabout by cell enlargement. Throughout the period of enlargement the tubulesmaintain the same basic type of construction of 5 rows of spirally arrangedbinucleate cells, but there is a change in cell shape. At the end of the mitoticphase the cells are more or less equal-sided hexagons (fig. 5, A) but theygradually change shape until in the adult they are relatively regular elongatedhexagons (as in fig. 5, c). It is clear that this change of shape is related to theprocess of elongation.

At hatching, each tubule is 2-5 to 3-2 mm long and divisible into an uppersegment 0-3 mm long and 22 /x wide and a lower segment about 30 JX wide.The tubule already appears to be fully functional, having a well-developedstriated border. During the remainder of nymphal development both segments

FIG. 4. A, the origin of a post-moult embryonal (excretory) tubule in a late embryo. B,longitudinal section of inner end of proctodaeum and the insertion of a pre-moult embryonal(appendix) tubule in an early embryo, c, transverse section of a post-moult embryonal tubulein a late embryo. D, ditto, nymphal tubule at the beginning of the phase of cell enlargement.E, ditto, lower segment of an adult nymphal tubule, a, appendix (pre-moult embryonaltubule); ex, excretory (post-moult embryonal) tubule; kg, hind-gut; m, mitosis; me, mesoderm;

mg, mid-gut; pe, peritoneal cell; pr, metenteric membrane; t, trachea.


c

FIG. 5. Surface views of tubules to show nuclei and cell shape, A, nymphal tubule at end ofmitotic phase, B, nymphal tubule of intermediate size, c, lower segment of an excretory

(post-moult embryonal) tubule in an adult.

gradually increase in size. In Schistocerca (Savage, 1956) there is no increasein the size of the proximal segment of the primary tubule after its initial differen-tiation at the time of upper. This difference between the two species may berelated to the difference in the function of the later formed tubules, since inSchistocerca the nymphal tubules are also excretory. In Carausius the processof tubule enlargement occurs in a 'stepped1 fashion during post-embryonic


development; the tubules gradually enlarge during each stadium but becomeslightly smaller after each moult. This is probably related to water loss atmoulting; a similar state of affairs exists in Schistocerca.

The fully differentiated excretory tubules of Carausius show the same gen-eral form as the Malpighian tubules of Blatta, Forficula (Henson, 1944,19466),Gryllus (Savage, unpublished), and Schistocerca. They are similar to theprimaries of Schistocerca in their mode of attachment to the rectum and in all5 species consist of a short clear upper segment and a long relatively opaquelower segment.

The calciferous tubules (nymphals). At the time of hatching there are 20 to 27post-moult embryonal tubules arranged equidistantly round the posterior endof the mid-gut. The 20 to 27 groups of tubules of the adult are produced bythe addition of 5 calciferous (nymphal) tubules, occasionally 4 or 6, to each ofthe embryonal tubules during the nymphal stages.

As in other Orthoptera, the nymphal tubules are initiated in the first fewdays after ecdysis, but Carausius is unusual in that this formation of tubules isrestricted to certain instars. Although there are 6 nymphal instars new tubulesappear in only the second, third, fourth, and occasionally fifth. The normalsequence is 2 new tubules in the second instar, 2 in the third, and one in thefourth. There are variations from this typical state of affairs in some specimenswhen 3 tubules are added in the second instar and the remaining 2 in thethird, or 2 may be added in the second instar, one in the third, and the remain-ing 2 in the fourth. A few groups were noted which possessed 4 or 6 tubulesinstead of the usual 5. In the cases where 6 nymphals were present it wasclear that the last one was initiated in the fifth instar whilst in other caseswhere 4 were present one of the tubules in earlier instars failed to develop.Thus whilst the time of initiation is usually constant it may be moved so thattubules develop in an earlier or later instar, except that no tubules arise in thefirst or sixth nymphal instars.

Although the number of tubule groups is variable, the morphologicalrelationships of the tubules within each group are constant. This results froma well-defined mode of development in which the succession of tubules frominstar to instar is remarkably uniform. The first nymphal tubule is addedimmediately behind the post-moult embryonal, the second behind the first butslightly to one side; the third level with the second but on the other side (fig.1, B). The fourth and fifth are added immediately posterior to the second andthird, the fourth behind the second and the fifth behind the third (fig. 1, B).The succession of tubules is easily recognizable since they vary in lengthaccording to their times of initiation (fig. 1, G, H). It is also noteworthy thattubules originating at the same time but in different groups in the same insectare of approximately the same length. Hence initiation and developmentmust proceed synchronously in all groups. Even when tubule measurementsin different specimens are compared their order of initiation is recognizableand shows the consistency of the processes concerned with their development(table 2).


The post-moult embryonal and nymphal tubules of Carausius are formedin the posterior interstitial ring and their positions of origin correspond exactlywith the primaries and secondaries of other Orthoptera previously described.The explanation of tubule succession already given for Schistocerca (Savage,1956) may be applied to Carausius in the following manner. Schistocerca has12 groups of tubules divisible into 2 distinct types: 6 primary groups placeddorso-laterally, laterally, and ventro-laterally; and 6 intercalary groups in theintervening positions. Each of the primary groups is formed round a primary

TABLE 2

The lengths of the nymphal tubules in each instar. The ranges in tubule length arein each case based on measurements of 40 tubules in a number of specimens

Instar

1st beginningend

2nd beginningend

3rd beginningend

4th beginningend

5th beginningend

6th beginningend

7th beginning

Length of nymphal tubules in mm

I

—1-4-2-01 -4-2-0

5-75-78-108-10

H-5-I311-5-1320-2220-22

II

———

0-5-1-00-5-1-01-5-2-01-5-2-0

5-65-6

9-0-10-59-0-10-516-1916-19

III

————

1-0-1-51-0-1-5

4-5-54-5-58-9-58-9-5

14-1714-17

IV

———

o-1-0750-1-0-75

2 - 4

2 - 45-7-55-7-5

12-1512-15

V

——————

1-0-1-51-0-1-5

4-54-5

10-1310-13

tubule whilst each intercalary group is formed round an embryonal secondarytubule. At the time of hatching there are 2 tubules in each primary group,one in each intercalary group, and during the nymphal stages further tubulesare added to each group. In the primary groups it was shown that the posteriorinterstitial ring (i.e. mid-gut/hind-gut junction) curved forwards around theprimary tubules and gave rise to nymphal or secondary tubules first to oneside and then to the other (fig. 6, H). In the intercalary groups it was shownthat the interstitial ring curved anteriorly, level with the embryonal secondarytubule and again nymphal tubules were produced alternately to either side(fig. 6, G). In Carausius the post-moult embryonals correspond in origin andposition with the embryonal secondaries of the intercalary groups of Schis-tocerca. It would seem that an essentially similar bending of the interstitialring may be presumed in Carausius (fig. 6, c).

It has already been shown that the last 4 nymphal tubules of Carausius areformed alternately to either side of the mid-line of the group and it is furthersuggested that the preceding tubule is similar. The condition of the interstitialring at the time of origin of the post-moult embryonal tubules is presumed tobe as in fig. 6, A. Later, the ring becomes curved into a hair-pin shape so that

Savage—Malpighian tubules of Carausius

H K MFIG. 6. The theoretical interpretation of the form of the posterior interstitial ring at the tubulegroups in Carausius, A to F; and Schistocerca, C, H. A, group with a single post-moult embryonalin a first instar nymph. B, ditto, with one additional nymphal in a second instar nymph, c,typical group in adult. D, E, two types of aberrant groups with 4 nymphal tubules. F, aberrantgroup with 6 nymphal tubules. G, 'intercalary' group. H,'primary'group, j to M, diagrams toshow the change in the form of the nymphal tubules of Carausius during their development(length only to scale), es, embryonal secondary tubule; ex, excretory (post-moult embryonal)tubule ;£, primary tubule; pir, posterior interstitial ring; I to VI, 1st to 6th nymphal secondary

tubules in order of initiation.


its inner margins are almost fused and new tubules are added along the 2 sidesof the curve. The first nymphal arises on the ring almost immediately behindthe post-moult embryonal (figs. 1, B; 2, A; 6, B). The second arises on theother limb of the curve and the remaining three alternately right and left alongthe interstitial ring (fig. 6, c). The insertion of the tubules (fig. 1, B) indicatesthat the inner margins of the ring may be closer together in the region of thepost-moult embryonal and first nymphal, but more widely separated in theregion of the second to fifth nymphals (fig. 6, c).

Further evidence in favour of this interpretation may be derived fromaberrations in the normal succession, from histological evidence and from itsgeneral applicability. The histological evidence, apart from the fact thattubules definitely originate in the interstitial ring (fig. 2, A, B), consists merelyin the observation that there is a tongue of ectoderm protruding between thebases of the last 2 tubules in the third instar, i.e. proving the existence ofa forward loop (fig. 6). This tongue is obliterated in the adult and theinterstitial ring appears to pass directly behind the tubule groups. Thisappearance is probably caused by the fusion of the 2 limbs of the forward loopof the interstitial ring.

Aberrations from the typical succession of tubules have been observed ina number of specimens. These aberrations are of few kinds and are readilyinterpreted in terms of the pattern of development described above. Certaingroups possess 4 or 6 nymphal tubules instead of the usual 5, arranged asshown in fig. 1, c, D, E. A comparison with the typical group (fig. 1, B) showsthat the aberrant pattern is the result of addition or subtraction of a singletubule, which can readily be explained in the same terms as the normal form.It will be seen from fig. 6, F that the interpretation of the succession of tubulesin a group with 6 nymphals is a mere extension of that put forward for thenormal type. Similarly, for the form with 4 nymphals (fig. 6, E) the last insuccession has failed to develop. In another aberration with 4 nymphals (fig.6, D) it is necessary to presume a slight difference in the form of the forwardloop of the interstitial ring; it is suggested that the close approximation of the2 limbs extends further backwards than in the normal group and brings thefirst 3 tubule rudiments into line behind each other (fig. 1, D). In rare casesgroups possessing 2 post-moult embryonals have been seen. Such groupsalways have an abnormally large number of nymphal tubules, as in the exampleshown which possesses 8 (fig. 1, F). The relationships of these latter cases tothe typical form remain uncertain, since it has not been possible to determinethe details of tubule arrangement and order of initiation. One specimen hasbeen observed in which a post-moult embryonal tubule was Y-shaped, asthough the primordium had split late in the cell-division phase. This suggeststhat the groups with 2 embryonals may be formed by splitting of the tubuleprimordium at a much earlier stage, i.e. the double group is the result of thesplitting of a single group and not the approximation of two originally separategroups.

The cellular development of a calciferous tubule, like that of the other types


of tubule, may be divided into 2 main periods; a phase of cell-division anda phase of cell enlargement. It first appears as a bud of actively dividing cellsin the posterior interstitial ring and gradually elongates by cell-division (fig. 2,A). Throughout this period it is 19 to 23 /x wide, always shows 5 rows of cellsin cross-section, has a wall one cell thick, and is covered by a thin layer ofmesodermal cells. The nuclei and cytoplasm stain darkly and there is a smalllumen but no striated border (fig. 4, c). The mitotic divisions are restrictedto the longitudinal direction (fig. 2, B) except for a slight spiral deviation andare related to the process of elongation since there is no increase in tubulewidth. The final nuclear divisions take place without a corresponding cell-division and thus establish the binucleate character of the cells, as in the post-moult embryonals (fig. 5, A). This rigid orientation of the cell-divisions bringsabout an increase in length of the tubules to about 1-4 mm but does notproduce any change in width, which remains 19 to 23 /x, nor does it disturb thearrangement in 5 rows of cells (figs. 6, j ; 7).

When the mitotic phase is complete, the striated border appears and cell en-largement sets in at the base of the tubules and gradually extends towards thetip. The tubule thus appears wider at the base and gradually tapers (fig. 6, K).This process continues in a similar manner until the tubule is 8 to 9 mm longwhen, for the first time, upper and lower regions, corresponding to theupper and lower segments of the fully differentiated tubule, become dis-tinguishable (fig. 6, L). At this stage the lower region is 6-o to 7-5 mm longby 50 /x wide and the upper region about z mm long and 30 fj, wide.

No further changes occur until the tubule has reached a length of 10 to 12mm when differentiation of the upper segment suddenly takes place. Thesmall cells of which it is composed rapidly increase in size so that they nowsurpass those of the lower segment. As a result the upper segment as awhole becomes much wider than the lower segment (figs. 6, M; 7). Theprocess of cell enlargement begins at its junction with the lower segmentand rapidly spreads towards the tip. The deposition of the white crystallinematerial, characteristic of the adult tubule, also begins in the proximal regionsof the segment and extends distally. From this stage onwards both segmentsgradually increase in length and diameter until the fully developed conditionis attained.

It has thus been shown that the individual tubules, like those of all Orthop-tera so far studied, pass successively through phases of initiation, cell-division,cell enlargement, and differentiation. The relations of these processes to thenymphal stages are rather different in Carausius from what they are in otherOrthoptera. In Blatta (Henson, 1944), Gryllus (Savage, unpublished), andSchistocerca new tubules arise after hatching and after each nymphal ecdysis.The mitotic phase is passed through in the first few days after ecdysis andfunctional differentiation is more or less complete by the time of the nextmoult.

In Carausius initiation and cell-division (mitotic phase) are likewise confinedto the first few days after ecdysis but cell enlargement thereafter proceeds


without reference to ecdysis and functional differentiation, as judged by theappearance of the upper segment, does not take place until the sixth instar.Thus although the development of nymphal tubules in Carausius is related tothe cyclical development as represented by instars, the form of the relationshipis different from that of other Orthoptera.

The typical sequence of events may be described as follows. No tubules

160 r

6 8 10 12Tubule length in mm

FIG. 7. Graph to show the increase in tubule width of the basal and distal regions in relationto tubule length in nymphal tubules.

arise in the first instar; 2 appear one after the other in the second instar. Thefirst of these rapidly completes the mitotic phase and thereafter continues itscell enlargement phase throughout the second, third, fourth, and fifth instars,finally becoming functionally differentiated in the sixth. The other secondinstar tubule usually does not complete its mitotic phase in this instar butremains dormant until immediately after the ecdysis to the third instar whena renewed period of cell-division occurs. This is completed within a few daysand development continues in a similar manner to that of its predecessor.Meanwhile z more tubules appear in this third instar, again successively, andthey usually behave in the same way as the second instar pair, one proceedingwith its development immediately and the other remaining dormant until thefourth instar. In the fourth instar one new tubule is initiated which maydevelop immediately or remain dormant until the fifth. No new tubules arise


in the fifth but a dormant fourth instar tubule will recommence cell-divisionand then develop in the usual way. All the tubules present continue to enlargeand by the end of the fifth instar the oldest tubules are 11 -5 to 13-0 mm long,the youngest only 4-0 to 5-0 mm (see table 2). During the sixth instardifferentiation of all the tubules takes place as they reach a length of 10 to 12mm; the older first, followed successively by the younger tubules. Furtherelongation occurs after differentiation throughout the sixth and imaginalinstars until the tubule is some 25 to 30 mm in length.

During the entire course of their development the nymphal tubules retainthe same basic composition of 5 rows of spirally arranged cells. Elongation isbrought about first by cell-division and later by increase of cell size andchange of cell shape. At the end of the mitotic phase the cells appear insurface view as more or less equal-sided hexagons without any particularorientation to the longitudinal axis of the tubule (fig. 5, A). During enlarge-ment they gradually become elongated hexagons with their long axes orientatedat a slight angle to the longitudinal axis of the tubule (fig. 5, B, C). Thisdeviation is related to the spiral course of the rows of cells. This arrangementis only disturbed in the upper segment of the fully differentiated nymphaltubule according to the degree of its distension. The cells may then range inform from the typical through intermediates to large irregular hexagons about200 JX across.

DiscussionThe most important points concerning the development of Malpighian

tubules in Orthoptera have already been discussed (Henson, 1944, 1946 a, b;Savage, 1956) and therefore only those aspects of particular importance forCarausius will be considered.

The characteristic feature of the development of Malpighian tubules inOrthoptera is that they arise in a series of generations, each associated witha moult, and hence it has been suggested that their formation is one aspect ofthe inter-ecdysial processes which take place during insect development. InCarausius there are 2 generations of tubules in the embryo and one in eachnymphal instar except the first and last. The occurrence of 2 generations oftubules before hatching is associated with a well-marked embryonic moult;the first generation arises before the moult whilst the second generation arisesimmediately after it and has the same time relations with it as the nymphaltubules have with the post-embryonic moults. The same type of developmentoccurs in Schistocerca and in Locustapardalina and L. migratoria (Jones, 1953,1956 a, b), where it has been shown that the prothoracic glands are associatedwith the embryonic moult and also with the period of active cell-divisionwhich immediately succeeds it for about 3 days. An increase in the numberof Malpighian tubules was also noted at this time. Hence it would appear thatin Locusta as in the closely related form Schistocerca there is a causal relation-ship between the activity of the prothoracic glands, the embryonic moult, andthe initiation of a new generation of tubules.


In Schistocerca and Blatta a new generation of tubules arises at the beginningof each nymphal instar and is probably a response to humoral conditions at thetime of ecdysis. This, at least, would accord with the great amount of workon developmental hormones in insects associated with Rhodnius (Wiggles-worth, 1954) and Platysamia (Williams, 1952). In Carausius, however, notubules are formed in the first and last nymphal instars, and this fact requiressome explanation. Their absence in the last nymphal instar must be due to oneof two conditions; either the humoral balance is inappropriate or the supply oftubule primordia is prematurely exhausted. In support of the latter explana-tion is the fact that tubules often fail to appear in the fifth instar and in anycase are few in number. There is also a considerable reduction in the numberproduced in the last nymphal instar in Schistocerca. This explanation makesit unnecessary to suggest that there is a qualitative difference in the activity ofthe prothoracic glands in relation to tubule initiation whilst other charactersare apparently unaffected.

The absence of tubules in the first instar cannot be explained in terms ofexhaustion of primordia but must be considered in relation to the alternativeof inappropriate humoral balance. The simplest condition of development, asseen in Blatta, is a single embryonic cycle separated by the process of hatchingfrom a first instar in which developmental events are controlled by the activityof the prothoracic glands. In Schistocerca the 2 generations of embryonaltubules are followed by a third, the first nymphal, immediately after hatching.This indicates that 2 complete developmental cycles are passed through in theegg. In Carausius the absence of first instar tubules,coupled with the occurrenceof 2 embryonal generations as in Schistocerca, indicates that the second develop-mental cycle is not completed at hatching but at the end of the first instar.This view is not free from objection since the relationship between hatchingand moulting has not been established, and the behaviour of the prothoracicglands at the time of hatching is unknown.

The developmental processes involved in the histogenesis of an individualtubule include initiation, cell-division, elongation, and functional differentia-tion. These processes occur at definite times within the instars and revealmuch of the underlying pattern of cyclical development.

The growth of each individual tubule is divisible into 2 distinct periods; oneof initiation and cell-division, and one of increase in cell size and differentia-tion. The tubules are formed discontinuously and the period of initiation andcell-division is restricted to the first few days after each moult (or commence-ment of a cycle); it is therefore closely linked with the controlling mechanismof cyclical development. The period of cell enlargement and differentiationcommences immediately after the end of the mitotic phase and continuesindependently of the cycles until the adult stage is reached. It was shown inSchistocerca that the embryonal and nymphal tubules followed the same patternof development, initiation, and cell-division in one instar (cycle) followed byenlargement and differentiation in subsequent instars (cycles). In Carausius,development is more complex since the tubules are differentiated into 3


distinct types. However, it has already been shown that the modes of develop-ment of the tubules are identical until the last stages of differentiation.

In the embryonal tubules of Carausius and all tubules of other describedOrthoptera development takes place as described above. In the nymphaltubules of Carausius there is a significant difference. The nymphal tubules areconcerned with the impregnation of the egg-shell with calcium salts, they donot have an excretory function and hence differ from excretory tubules sincethey do not function until the adult stage. They are initiated in the second tofourth (occasionally fifth) instars, undergo cell enlargement but do not differen-tiate until the sixth (final) nymphal instar. Thus it would appear that herecell enlargement continues independently of cyclical factors until the sixthinstar, when rapid differentiation is due to some humoral factor. The periodof cell enlargement is thus divisible into 2 phases; an earlier one of slowindependent enlargement and a later one of rapid differentiation dependentupon a hormone. It is interesting to note that extirpation of the corpora allatain the adult causes atrophy of certain Malpighian tubules (Pflugfelder, 1938).The differentiation of the nymphal tubules is a modification of the basicdevelopmental plan observed in other Orthoptera, where embryonic andnymphal cycles are identical. In fact this delayed differentiation is like certainaspects of metamorphosis in that it is associated with delayed functioning(e.g. wing-buds in the Holometabola). It may be regarded as a rudimentaryexample of metamorphosis in a specific organ.

My thanks are due to Professor E. A. Spaul for facilities for study and toDr. H. Henson who supervised this work. Material was supplied by Mr.J. Digby Firth and the investigation was made possible by a D.S.I.R. researchgrant.

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1946a. Biol. Rev., 21, 1.19466. Proc. R. ent. Soc. Lond. A, 21, 29.

HEYMONS, R. 1897. Bed. Acad. Sitz., 363.JONES, B. M. 1953. Nature, 172, 551.

1956a. J. exp. Biol., 33, 174.1956b. Ibid., 33, 685.

LEUZINGER, H., WIESMANN, R., and LEHMANN, F. E., 1926. Zur Kenntniss der Anatomie undEntwicklungsgeschichte der Stabheuschrecke, Carausius morosus Sr. Jena.

PFLUGFELDER, O. 1938. Z. wiss. Zool., 151, 149.RAMSAY, J. A. 1955. J. exp. Biol., 32, 183.SAVAGE, A. A. 1956. Quart. J. micr. Sci., 97, 599.

1957. Proc. Leeds phil. lit. Soc, 7, 29.SINETY, R. de. 1901. Cellule, 19, 119.SIRODOT, S. 1858. Ann. Sci. nat. (Zool.), Series 4, 10, 141.THOMAS, A. J. 1936. Quart. J. micr. Sci., 78, 487.WIGCLESWORTH, V. B. 1954. The physiology of insect metamorphosis. Cambridge (University

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