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Uric acid and urate storage in the larva of Chrysopa carnea Stephens (Neuroptera, Chrysopidae)
J. Ins. Physiol., 1962 Vol. 8, pp. 127 to 132. Pergamm Press Ltd.
Printed in Great Britain
URIC ACID AND URATE STORAGE IN THE LARVA OF CHRYSOPA CARNEA STEPHENS (NEUROPTERA, CHRYSOPIDAE) P. E. SPIEGLER Department
of Zoology, The George Washington University, Washington 6, D.C. (Received 6 October 1961)
Abstxact--In this study it has been shown that uric acid is stored as one of the excretory products of nitrogenous metabolism in all areas of the fat body of the third instar larva of Chrysopa canea as either a spherical or an amorphously shaped crystal. Uric acid also appears iu the cells of the Malpighian tubules in small amounts, but it does not appear in the proteinaceous adhesive substance which is produced by the tubules. The sign&awe of the presence of uric acid in the tubules is unknown, but it may be that the uric acid is being stored there or that it may play some role in the formation of the adhesive substance. INTRODUCTION FOR some time the method of excretion and elimination of the excretory product of nitrogenous metabolism in the chrysopid larva has been open to question. It is known that the Malpighian tubules of these forms produce silk during the last larval instar (MCDUNNOUGH, 1909}, and prior to this time the tubules produce an adhesive substance (SPIEGLER,1962). The adhesive substance, containing alpha amino acids, is proteinaceous in nature and serves as an excretory product as well as being used by the larva as an aid in locomotion. It is possible that the silk and adhesive substance are the same material. This substance also appears in other Neuroptera; in the Hemerobiidae (WITHYCOMBE,1925), in the Chrysopidae (KILLINGTON,1936), and in the Berothidae and the Dilaridae (E. G. MACLEOD and P. E. SPIEGLER,unpublished). The question now arises as to whether or not the adhesive substance can account for the total amount of the nitrogenous excretion, by storage or some other means. Since it appears that the only products eliminated by the larval stages are the silk and the adhesive substance, it is most likely that if another form of excretion does exist it would be a storage mechanism. The storage of uric acid is already known in other insects (WIGGLESWORTH, 1947; PATTON,1953). It is therefore the object of this study to see if storage excretion of uric aoid or urates also takes place in the chrysopid larva as a complement to the production of the adhesive substance.
MATERIAL AND METHODS Chrysopa camea Stephens ( = Chrysopa plqabunda Fitch) was selected as an experimental animal because it is already known that in this species the silk and the adhesive substance are produced by the Malpighian tubules (SPIEGLXR,1962). 127
128
P. E. SPIEGLER
The larvae were easily reared in individual glass vials, and they were fed termites, a Reticzllitermessp. The open ends of the 1 dram vials were closed with a pledget of cotton. The first instars were fed the very smallest termites whereas the larger larvae were fed adult termites. Ten mature third instar larvae were fixed and sectioned to be tested for uric acid and urate content. The complete histochemical procedure for the preparation and identification of uric acid and urates was that of Schultz as given by LILLIE (1954). The uric acid standard was purchased from the Fisher Scientific Co. The calcium urate and the uricase were obtained from the Nutritional Biochemical Corp. All of the material was treated by the above technique with but a single modification. This was to leave the material in the methylene blue stain for 5-10 min, which is a 20-fold increase at the maximum. Tissue mat (m.p. SQ-56°C) was used as the embedding medium, and all sections were cut at 10 p* The tissues to be treated with uricase were de&trafhnized in xylene and brought to 96% ethanol. They were then taken directly through four changes of distilled water. The enzyme solution was prepared by adding 1 mg of uricase per ml of a O-2 M borate buffer solution. The buffer solution having a pH of 85 was prepared from the directions of GOMORI (1952). The time of digestion was 30-60 min at room temperature. After digestion the slides were washed in four changes of distilled water and then returned through 95% ethanol to absolute ethanol. The slides were then ready to be stained. The larval material was randomly divided into three groups. The first group, Group I, was tested with the technique of Schultz for uric acid and urate deposition. The second group, Group II, was a control group. It was first digested with uricase and then treated with Schultz’s technique. The third group, Group III, another control group, was treated with Harris’s haematoxylin and eosin (GTJYER, 1934). With each of these groups five slides of both uric acid and calcium urate were treated in the identical manner as the larval material.
OBSERVATIONS
AND RESULTS
Grou. I In the method of Schultz, uric acid crystals appear a deep blue-green, whereas sodium urate gives a light grass-green colour. Nuclei are grey-blue, and the cytoplasm of muscle tissue stains yellow. In the larvae and the chemical standards these results are duplicated in Table 1. In the animals of this group some of the cells of the fat body have large numbers of uric acid crystals. These cells are so packed with the crystals that their boundaries are often obscure. The crystals vary in shape from being small and spherical to being larger with an amorphous shape. The spherical crystals are about 1.2-3.6 y in diameter, and for the most part stain blue-green. The larger, amorphously shaped crystals are about [email protected] y in length, more numerous, and are stained for the most part grass-green in colour. Before treating the slides, the uric acid can be detected in the tissue as dense white material in the fat body. In the sections
CHRYSOPACARNEA
URIC ACID AND IJRATE STORAGEINTHRLARVAOF
129
uric acid is found in all regions of the body except the head. It is found in all portions of the fat body of the abdomen and thorax, even in the fat body extending into the abdominal tubercles. TABLE
~-THE
LOCATION
Area examined
OFURICACIDAND
OTHERCELL
Inclusions present
Colour
INCLUSIONS
Colour after uricase
Meconium
Yes
Blue-green
Green to bhmgreen
Fat body
YeS
Blue-green, grass-green
None
Hypodermis
Yes
Crystalline, red
Same
Rest of cuticle
No
Light green to blue-green
Same
Mesenteron
Yes
Purple-red
Same
Malpighiau tubules
Yes
Blue-green
None
Adhesive substance
No
Light brown
Same
Muscle cytoplasm
No
Yellow
SaIue
Muscle nuclei
No
Blue-grey
Same
Uric acid standard
-
Blue-green
None
Calcium mate standard
-
Grass-green
Same
I In the larva of C. camea the meconium is surrounded by the peritrophic membrane and is kept at the distal end of the mesenteron. The meconium contains various materials, some of which show the typical blue-green colour reaction of uric acid. The other material shows varying reaction to the stain, but there does not appear to be any material having the light grass-green colour of the urate standard. The only other structures containing uric aeid are the Malpighian tubules. Within the cytoplasm of some of the cells secreting adhesive substance, small spherical crystals of a blue-green colour ring the periphery of the cells. These crystals are about l-3 p in diameter, and being few in number are clearly seen. In other secreting cells a diffuse blue-green colour can be seen in the cytoplasm, usually around the nucleus. In other secreting and non-secreting cells of the Malpighian tubules there is no trace of uric acid. whenever the adhesive substance appears in the lumina of the tubules, there is never any blue-green coloration in
130
P. E. SPIEGLER
the substance inany of the larvae. The same result is true of the adhesive substance found in the silk reservoir and the other parts of the hind-gut. As shown in Table 1, the urate and uric acid standards have identical colour reactions with those of Schultz, which was the case for all the controls.
Croup II In this group of controls the slides were first treated with uricase and then with ‘the technique of Schultz. Within 4 hr the five slides with the uric acid standard were completely digested of the acid, but the five slides with the calcium urate standard had not been affected by the uricase. The calcium urate subsequently was stained the same grass-green colour of those in Group IL This would indicate that the grass-green granules of the first group are uric acid. None of the sections treated with uricase showed any blue-green or grass-green coloration in the fat body, a&the fat body was completely devoid of any inclusions similar to those in Group I. It can then be concluded that the blue-green and grass-green inclusions appearing in the fat body of the larvae of Group I are deposits of uric acid. After digestion some of the areas in the cuticle, especially the head region and the large setae, still appeared green to blue in colour. It is then apparent that this coloration in the cuticle of the larvae of Group I is not due to the presence of uric acid. The Malpighian tubules after treatment with uricase showed no blue-green granulation, and the diiuse blue-green coloration in the cytoplasm present in the animals of Group I was now absent. The appearance of the adhesive substance in the tubules also remained unchanged. In the meconium there was no definite change in coloration after treatmentwith uricaae. It appears that there was a slight darkening of the green, but it seems that if there is any uric acid present it is being masked by other material showing the same colour reaction. Group III As a further control of the technique, the chemical standards and the larval material were treated with Harris’s haematoxylin and eosin. In none of the larval tissues was there any blue-green or grass-green coloration. Cell inclusions in the fat body assumed to be uric acid appeared faintly as brown or brownishyellow. The cytoplasm of muscle, fat body and nerve tissue appeared typically eosinophilic, while the nuclei of these tissues had the typical blue colouring of haematoxylin. No crystals at all were to be seen in the Malpighian tubules. In both the slides of the uric acid standard and the calcium urate standard, all of the material took on a tannish-white colour. DISCUSSIum
From the results of the histochemical tests it is shown that uric acid is present and is stored in the fat body of the mature third instar larva of C. cat-ma. The technique as applied to these chrysopids verifies in part the results of Schultz. It is
URIC ACID AND UFL4TE STORAGE IN THE LARVA OF
CHRYSOPA CARNEA
131
evident from the staining reaction of the cuticle and meconium after the treatment of uricase that this method is not specific for uric acid. It is also clear that the colour reaction of the uric acid is different when this substance is within the larva where it stained both blue-green and grass-green. The reaction of the chemical standards used here is identical to that reported by Schultz, but it appears that there may be some factor in the larva affecting the reaction of the uric acid or some factor in the technique as applied to 6. cornea causing the difference in results. The occurrence of uric acid in the Malpighian tubules and not in the adhesive substance, which the tubules produce, is interesting indeed. Since the uric acid per seis not found in the adhesive substance, the question arises as to what is the significance of its presence in the tubules. The fact that the uric acid is found in small amounts in the secreting portions of the tubules in either a difIirse state or as a solid inclusion indicates that it is possible that the uric acid is in a transitory state and may play a role in the synthesis of the adhesive substance. Although there is no evidence here that the purine ring is broken there is still the possibility that the uric acid is undergoing some chemical alteration leading to the synthesis of the adhesive substance. By its presence in the tubules it is possible that the uric acid is being stored there, but this appears doubtful. First, there is little uric acid in the crystalline state, which is hardly the amount to be expected in a mature larva which should have built up a comparable amount to that of the fat body. Secondly, the evidence indicates that the dominant function of the Malpighian tubules is the secretion of silk and adhesive substance. The storing of uric acid in cells differentiated for the production of those substances seems improbable unless the uric acid is being used in some way for the production of those materials. Although the presence of uric acid in the meconium cannot be shown from the results, it is still possible that it is there. C. cmwa is predaceous by nature and .it sucks the haemolymph of its prey. It would seem possible that the uric acid from this source should remain in the meconium. There is no indication that any of the uric acid is to be found in any part of the gut wall as reported in Sitqphilus granmius (L.), a grain-infesting beetle, by GUPTAand SINHA(1960); nor is it possible to state whether uric acid is formed in the meconium or that the meconium has a function of storage excretion. It then appears from the results of this study that the proteinaceous adhesive substance does not account for the total amount of the nitrogenous waste product, but that there is a system of excreting part of the waste product in the form of uric acid stored in the fat body of the larva. REFERENCES GOMORI G. (1952)Microscopic H&ocher&try. University of Chicago Press. GUPTA P. D. and SINHA R. N. (1960) Excretion and its products in some stored-graininfesting beetles. Arm. ent. Sot. Amer. 53, 632-638. GUYERM. F. (1934) Animal Micrology. University of Chicago Press. KILLINGTON F. J. (1936) A Monogr@h of the British Neutopteru, Vol. 1. Rap Society, London.
132
P. E. SPIEGLXR
LILLIE R. D. (1954) Histopathologic Technic and Practical H&ocher&try. Blakiston, New York. MCDTJNNOUGH J. (1909) uber den Bau des Darms und seiner Anhange von Chrysopa perZa L. Arch. Naturgesch. 75, 313-360. PATTON R. L. (1953) Excretion. Insect Physiology (Ed. by K. D. ROEDER),pp. 387-403. Wiley, New York. SPIEGLERP. E. (1962) A study of the origin and nature of the adhesive substance in the genus Chrysopa Leach 1815 (Neuroptera: Chrysopidae). Ann. ent. Sot. Amer. In press. WIGGLE~WORTH V. B. (1947) The Principles of Insect Physiology. Methuen, London. WITWCOMB~ C. L. (1925) Some aspects of the biology and morphology of the Neuroptera with speaial reference to the immature stages and their possible phylogenetic significance. Trans. ent. Sot. Land. (1924), 303-411.
Printed in Great Britain
URIC ACID AND URATE STORAGE IN THE LARVA OF CHRYSOPA CARNEA STEPHENS (NEUROPTERA, CHRYSOPIDAE) P. E. SPIEGLER Department
of Zoology, The George Washington University, Washington 6, D.C. (Received 6 October 1961)
Abstxact--In this study it has been shown that uric acid is stored as one of the excretory products of nitrogenous metabolism in all areas of the fat body of the third instar larva of Chrysopa canea as either a spherical or an amorphously shaped crystal. Uric acid also appears iu the cells of the Malpighian tubules in small amounts, but it does not appear in the proteinaceous adhesive substance which is produced by the tubules. The sign&awe of the presence of uric acid in the tubules is unknown, but it may be that the uric acid is being stored there or that it may play some role in the formation of the adhesive substance. INTRODUCTION FOR some time the method of excretion and elimination of the excretory product of nitrogenous metabolism in the chrysopid larva has been open to question. It is known that the Malpighian tubules of these forms produce silk during the last larval instar (MCDUNNOUGH, 1909}, and prior to this time the tubules produce an adhesive substance (SPIEGLER,1962). The adhesive substance, containing alpha amino acids, is proteinaceous in nature and serves as an excretory product as well as being used by the larva as an aid in locomotion. It is possible that the silk and adhesive substance are the same material. This substance also appears in other Neuroptera; in the Hemerobiidae (WITHYCOMBE,1925), in the Chrysopidae (KILLINGTON,1936), and in the Berothidae and the Dilaridae (E. G. MACLEOD and P. E. SPIEGLER,unpublished). The question now arises as to whether or not the adhesive substance can account for the total amount of the nitrogenous excretion, by storage or some other means. Since it appears that the only products eliminated by the larval stages are the silk and the adhesive substance, it is most likely that if another form of excretion does exist it would be a storage mechanism. The storage of uric acid is already known in other insects (WIGGLESWORTH, 1947; PATTON,1953). It is therefore the object of this study to see if storage excretion of uric aoid or urates also takes place in the chrysopid larva as a complement to the production of the adhesive substance.
MATERIAL AND METHODS Chrysopa camea Stephens ( = Chrysopa plqabunda Fitch) was selected as an experimental animal because it is already known that in this species the silk and the adhesive substance are produced by the Malpighian tubules (SPIEGLXR,1962). 127
128
P. E. SPIEGLER
The larvae were easily reared in individual glass vials, and they were fed termites, a Reticzllitermessp. The open ends of the 1 dram vials were closed with a pledget of cotton. The first instars were fed the very smallest termites whereas the larger larvae were fed adult termites. Ten mature third instar larvae were fixed and sectioned to be tested for uric acid and urate content. The complete histochemical procedure for the preparation and identification of uric acid and urates was that of Schultz as given by LILLIE (1954). The uric acid standard was purchased from the Fisher Scientific Co. The calcium urate and the uricase were obtained from the Nutritional Biochemical Corp. All of the material was treated by the above technique with but a single modification. This was to leave the material in the methylene blue stain for 5-10 min, which is a 20-fold increase at the maximum. Tissue mat (m.p. SQ-56°C) was used as the embedding medium, and all sections were cut at 10 p* The tissues to be treated with uricase were de&trafhnized in xylene and brought to 96% ethanol. They were then taken directly through four changes of distilled water. The enzyme solution was prepared by adding 1 mg of uricase per ml of a O-2 M borate buffer solution. The buffer solution having a pH of 85 was prepared from the directions of GOMORI (1952). The time of digestion was 30-60 min at room temperature. After digestion the slides were washed in four changes of distilled water and then returned through 95% ethanol to absolute ethanol. The slides were then ready to be stained. The larval material was randomly divided into three groups. The first group, Group I, was tested with the technique of Schultz for uric acid and urate deposition. The second group, Group II, was a control group. It was first digested with uricase and then treated with Schultz’s technique. The third group, Group III, another control group, was treated with Harris’s haematoxylin and eosin (GTJYER, 1934). With each of these groups five slides of both uric acid and calcium urate were treated in the identical manner as the larval material.
OBSERVATIONS
AND RESULTS
Grou. I In the method of Schultz, uric acid crystals appear a deep blue-green, whereas sodium urate gives a light grass-green colour. Nuclei are grey-blue, and the cytoplasm of muscle tissue stains yellow. In the larvae and the chemical standards these results are duplicated in Table 1. In the animals of this group some of the cells of the fat body have large numbers of uric acid crystals. These cells are so packed with the crystals that their boundaries are often obscure. The crystals vary in shape from being small and spherical to being larger with an amorphous shape. The spherical crystals are about 1.2-3.6 y in diameter, and for the most part stain blue-green. The larger, amorphously shaped crystals are about [email protected] y in length, more numerous, and are stained for the most part grass-green in colour. Before treating the slides, the uric acid can be detected in the tissue as dense white material in the fat body. In the sections
CHRYSOPACARNEA
URIC ACID AND IJRATE STORAGEINTHRLARVAOF
129
uric acid is found in all regions of the body except the head. It is found in all portions of the fat body of the abdomen and thorax, even in the fat body extending into the abdominal tubercles. TABLE
~-THE
LOCATION
Area examined
OFURICACIDAND
OTHERCELL
Inclusions present
Colour
INCLUSIONS
Colour after uricase
Meconium
Yes
Blue-green
Green to bhmgreen
Fat body
YeS
Blue-green, grass-green
None
Hypodermis
Yes
Crystalline, red
Same
Rest of cuticle
No
Light green to blue-green
Same
Mesenteron
Yes
Purple-red
Same
Malpighiau tubules
Yes
Blue-green
None
Adhesive substance
No
Light brown
Same
Muscle cytoplasm
No
Yellow
SaIue
Muscle nuclei
No
Blue-grey
Same
Uric acid standard
-
Blue-green
None
Calcium mate standard
-
Grass-green
Same
I In the larva of C. camea the meconium is surrounded by the peritrophic membrane and is kept at the distal end of the mesenteron. The meconium contains various materials, some of which show the typical blue-green colour reaction of uric acid. The other material shows varying reaction to the stain, but there does not appear to be any material having the light grass-green colour of the urate standard. The only other structures containing uric aeid are the Malpighian tubules. Within the cytoplasm of some of the cells secreting adhesive substance, small spherical crystals of a blue-green colour ring the periphery of the cells. These crystals are about l-3 p in diameter, and being few in number are clearly seen. In other secreting cells a diffuse blue-green colour can be seen in the cytoplasm, usually around the nucleus. In other secreting and non-secreting cells of the Malpighian tubules there is no trace of uric acid. whenever the adhesive substance appears in the lumina of the tubules, there is never any blue-green coloration in
130
P. E. SPIEGLER
the substance inany of the larvae. The same result is true of the adhesive substance found in the silk reservoir and the other parts of the hind-gut. As shown in Table 1, the urate and uric acid standards have identical colour reactions with those of Schultz, which was the case for all the controls.
Croup II In this group of controls the slides were first treated with uricase and then with ‘the technique of Schultz. Within 4 hr the five slides with the uric acid standard were completely digested of the acid, but the five slides with the calcium urate standard had not been affected by the uricase. The calcium urate subsequently was stained the same grass-green colour of those in Group IL This would indicate that the grass-green granules of the first group are uric acid. None of the sections treated with uricase showed any blue-green or grass-green coloration in the fat body, a&the fat body was completely devoid of any inclusions similar to those in Group I. It can then be concluded that the blue-green and grass-green inclusions appearing in the fat body of the larvae of Group I are deposits of uric acid. After digestion some of the areas in the cuticle, especially the head region and the large setae, still appeared green to blue in colour. It is then apparent that this coloration in the cuticle of the larvae of Group I is not due to the presence of uric acid. The Malpighian tubules after treatment with uricase showed no blue-green granulation, and the diiuse blue-green coloration in the cytoplasm present in the animals of Group I was now absent. The appearance of the adhesive substance in the tubules also remained unchanged. In the meconium there was no definite change in coloration after treatmentwith uricaae. It appears that there was a slight darkening of the green, but it seems that if there is any uric acid present it is being masked by other material showing the same colour reaction. Group III As a further control of the technique, the chemical standards and the larval material were treated with Harris’s haematoxylin and eosin. In none of the larval tissues was there any blue-green or grass-green coloration. Cell inclusions in the fat body assumed to be uric acid appeared faintly as brown or brownishyellow. The cytoplasm of muscle, fat body and nerve tissue appeared typically eosinophilic, while the nuclei of these tissues had the typical blue colouring of haematoxylin. No crystals at all were to be seen in the Malpighian tubules. In both the slides of the uric acid standard and the calcium urate standard, all of the material took on a tannish-white colour. DISCUSSIum
From the results of the histochemical tests it is shown that uric acid is present and is stored in the fat body of the mature third instar larva of C. cat-ma. The technique as applied to these chrysopids verifies in part the results of Schultz. It is
URIC ACID AND UFL4TE STORAGE IN THE LARVA OF
CHRYSOPA CARNEA
131
evident from the staining reaction of the cuticle and meconium after the treatment of uricase that this method is not specific for uric acid. It is also clear that the colour reaction of the uric acid is different when this substance is within the larva where it stained both blue-green and grass-green. The reaction of the chemical standards used here is identical to that reported by Schultz, but it appears that there may be some factor in the larva affecting the reaction of the uric acid or some factor in the technique as applied to 6. cornea causing the difference in results. The occurrence of uric acid in the Malpighian tubules and not in the adhesive substance, which the tubules produce, is interesting indeed. Since the uric acid per seis not found in the adhesive substance, the question arises as to what is the significance of its presence in the tubules. The fact that the uric acid is found in small amounts in the secreting portions of the tubules in either a difIirse state or as a solid inclusion indicates that it is possible that the uric acid is in a transitory state and may play a role in the synthesis of the adhesive substance. Although there is no evidence here that the purine ring is broken there is still the possibility that the uric acid is undergoing some chemical alteration leading to the synthesis of the adhesive substance. By its presence in the tubules it is possible that the uric acid is being stored there, but this appears doubtful. First, there is little uric acid in the crystalline state, which is hardly the amount to be expected in a mature larva which should have built up a comparable amount to that of the fat body. Secondly, the evidence indicates that the dominant function of the Malpighian tubules is the secretion of silk and adhesive substance. The storing of uric acid in cells differentiated for the production of those substances seems improbable unless the uric acid is being used in some way for the production of those materials. Although the presence of uric acid in the meconium cannot be shown from the results, it is still possible that it is there. C. cmwa is predaceous by nature and .it sucks the haemolymph of its prey. It would seem possible that the uric acid from this source should remain in the meconium. There is no indication that any of the uric acid is to be found in any part of the gut wall as reported in Sitqphilus granmius (L.), a grain-infesting beetle, by GUPTAand SINHA(1960); nor is it possible to state whether uric acid is formed in the meconium or that the meconium has a function of storage excretion. It then appears from the results of this study that the proteinaceous adhesive substance does not account for the total amount of the nitrogenous waste product, but that there is a system of excreting part of the waste product in the form of uric acid stored in the fat body of the larva. REFERENCES GOMORI G. (1952)Microscopic H&ocher&try. University of Chicago Press. GUPTA P. D. and SINHA R. N. (1960) Excretion and its products in some stored-graininfesting beetles. Arm. ent. Sot. Amer. 53, 632-638. GUYERM. F. (1934) Animal Micrology. University of Chicago Press. KILLINGTON F. J. (1936) A Monogr@h of the British Neutopteru, Vol. 1. Rap Society, London.
132
P. E. SPIEGLXR
LILLIE R. D. (1954) Histopathologic Technic and Practical H&ocher&try. Blakiston, New York. MCDTJNNOUGH J. (1909) uber den Bau des Darms und seiner Anhange von Chrysopa perZa L. Arch. Naturgesch. 75, 313-360. PATTON R. L. (1953) Excretion. Insect Physiology (Ed. by K. D. ROEDER),pp. 387-403. Wiley, New York. SPIEGLERP. E. (1962) A study of the origin and nature of the adhesive substance in the genus Chrysopa Leach 1815 (Neuroptera: Chrysopidae). Ann. ent. Sot. Amer. In press. WIGGLE~WORTH V. B. (1947) The Principles of Insect Physiology. Methuen, London. WITWCOMB~ C. L. (1925) Some aspects of the biology and morphology of the Neuroptera with speaial reference to the immature stages and their possible phylogenetic significance. Trans. ent. Sot. Land. (1924), 303-411.