S O M E A S P E C T S O F T H E E M B R Y O L O G Y O F TETRASTICHUS P Y R I L L A E C R A W . ( E U L O P H I D A E : H Y M E N O P T E R A ) *

Part I. Organization of Egg

BY KUNDAN LAL, M.Sc. (HONS.), PH.D. [ Retired Scientist, Entomology Division, Ina~an Agricultural Research Institute, New Delhi (India)]

Received October 8, 1965 (Communicated by Dr. B. It. Subba Itao, F.A.se.)

ABSTRACT

Some aspects of the embryology of the parasite Tetrastichus pyrillae, an egg parasite of Pyrilla perpusilla Walk., have been studied with a view to find out some of the complexities of development of parasitic insects.

In the organisation of the egg of this parasite it has been observed that Tetrastichu~ pyrillae does not conform to the law of orientation enunciated by Hallez (1886). It has been found during this investigation that the slightly tapering end of the parasite egg which is directed towards the head of female is definitely the anterior end bearing the micropylar opening. During development of Tetrastichus pyrillae egg it has been observed that the head of the embryo is always formed towards the broader end of the egg(Posterior). The presence of germ cell determi- nant at the posterior end of the egg adds to the interest of the study of this form.

(A) INTRODUCTION

IN the published literature we have a large number of fine contributions on insect embryology. Yet there are many problems on which there has been no unanimity amongst the workers. For instance, there is a gap in our knowledge of the formation of gastrulation, the origin of the midgut epithelium and the numOer of segments found in the head region, etc. Also, in addition to having a clear understanding of the phylogenic evolu- tion of an organism, it is of the utmost importance that we have a clear knowledge of its embryonic development for, now it is an established bio- logical fact that in the embryonic development a living organism repeats

* Part of the Thesis approved for award of Ph.D. of the Panjab University. 38

Embryology of Tetrastichus pyrillae Craw. 39

and recounts its ancestral story to a great extent though often times we miss a link here and there. In an organism like the insect which is not so highly evolved as the other higher vertebrates, the ontogeny may give us all the information that we may want. The embryology of an insect is one of the most fascinating problems in histology and the embryology of parasitic insect with its complexities of development is one that calls for sustained work. In these investigations some aspects of the embryology of Tetra- stichus pyrillae has been studied with a view to find out some of these complexities.

Tetrastichus pyrillae egg is very minute, only 0.25 mm. long on an average, and is contained in the egg of Pyrilla which is as long as 0.90 mm. Thus the study of such an egg is beset with unusual difficulties. Further, it may be pointed out that externally there is no indication that the host egg contains the parasite egg. New techniques had to be evolved to make suitable whole mount of the parasite egg because in all the well-known cleating media it shrank.

Hundreds of host eggs were fixed, dehydrated, cleared, embedded, sectioned and stained before any embryonic stage was got. The fecundity of the parasite being very low, failure to get any embryonic stage was com- mon. Thus the study of the embryology of a parasite is a difficult task when so minute an egg of a parasite is contained in another egg. The greatest difficulty was experienced as hundreds of Pyrilla eggs had to be sectioned before one could get a parasitised egg. It was more difficult to obtain the segmentation stages of the parasite egg.

This is probably one of the reasons why the embryology of any egg parasite has not been studied in detail so far.

(B) TECHNIQUE The descriptive embryology has been studied with two techniques, i.e.,

the study of the entire egg as a whole mount and serial sections of the egg of parasite which is (0.25 mm. long) contained in the host egg which is 0.9 ram. long. Briefly, the eggs were fixed in situ within the host egg. Then the eggs were dehydrated in alcohol series, kept in cedarwood oil and then passed through benzene and embedded in paraffin. The eggs were then cut along with host egg into serial sections of 6-8/~ stained and mounted. Critical stages both early and late were stained with Delafield's haemato- xylin without counterstain as the variety of stains such as eosine, acid fuchsin, etc,, do not give good results, The treatment of the larvae and

40 KUNDAN LAL

pupae contained in the host egg and adult parasites prepared for serial sectioning was similar to those of eggs mentioned above except that the penetration of paraffin was continued over for much longer period.

Some remarks on the size of egg o f parasite.--In Figs. 1, 2, 3 and 6 eggs are drawn to the same scale. It will be noticed that their sizes differ often quite considerably. In Figs. 1, 3 and 6 eggs are drawn from the widest part of the egg. Here also wide differences in size occur. The germ cell deter- minant is shown in Fig. 6. But it was observed that its shape varies in different eggs. It has been observed that all the eggs in the ovary of an insect are graded in size, the lowermost eggs being the largest, while those higher up in the ovarian tube smaller. Gatenby (1917) also observed wide differences in the size of the eggs of Trichogramma evanescens. For earlier stages, the age of the egg was known within hours. For later stages, their ages were not quite accurately known in all cases. Thus the later stages of eggs of the parasite contained in the host egg were incubated under con- stant temperature i.e., 18 ° C. temperature and 80% relative humidity for varying length of time from 2 hours to 2 days. This was considered very important for it is known that temperature and humidity greatly affect the rate of development. Even under conditions of constant temperature and humidity there was slight individual variation in the time taken to develop a given stage. The duration of embryonic period of control under the conditions was approximately 2 days. There was a slight variation in time taken to hatch but it was never longer than 2 days for the eggs to hatch successfully.

(C) ORGANIZATION OF

The parasite lays its egg in any place by piercing the dorsal side of the host egg. It lies inside the host egg sometimes in the same place as the host egg. In other cases, it lies at right angle to that of the host egg as revealed by sectioning of the host egg containing the parasite egg. When the egg of Pyrilla is dissected the egg of the parasite is dull white in appear- ance. It is not attached to any part of the host egg but it lies embedded in the contents of host egg. When mounted in glycerine the egg is slightly Curved and tapers towards the anterior end (Fig. 6). In length it varies from 0.242 to 0.262 ram. and 0.075 to 0.08 nun. in breadth across the middle. At its largest breadth point the egg has an average diameter 0.10 ram. or approximately the total length of the egg is 2.5 times that of the diameter. The difference between the size of these two ends is common among the eggs of insects in general and always bears a distinct and constant

Embryology of Tetrastichus pyriUae Craw. 41

relation to the position of future embryo. In the egg of Tetrastichus pyrillae Craw. with reference to the part of the embryo the larger of the two ends is cephalic (posterio0 the opposite . . . . . . . . (anterior). The convex side is dorsal. The concave side is ventral.

t

S

A

L

3

6

T /~""~------ An.&

?oo I

FIGS. 1-6. Fig. 1. Whole mount of egg--26 hours old (A, Amnion; H.L, Head lobe; S, Serosa). Fig. 2. Whole mount of egg----45 hours old (H.L, Head lobe; S, Serosa. Fig. 3. Whole mount of egg--23 hours old. (A, Amnion; H.L, Head lobe; S, Sexosa). Fig. 4. L.S. through thorax and abdomen of adult parasite. (Ab~ Abdomen; T, Thorax). Fig. 5. L.S. through abdomen of adult parasite (Ab, Abdomen; An.e, Anterior end; Post. e, Posterior end). Fig. 6. Egg from overy (C, Chorion; G, Germ-cell determinant; N, Nucleus; V, Vitelline membrane).

Nelson (1915) in dealing with the embryology of the honey-bee has said, "In many other insects it is known, moreover, that the egg before deposition lies in the ovary of the mother in such a position that the parts of the future embryo are directed or oriented coincidently with those of the mother,

42 KUNDAN LAL

That is, the cephalic pole of the egg is turned towards head of mother. This is known as law of orientation of Hallez, named from its discoverer (1886)." Nelson (1915) in his embryology of honey-bee has further said, " In case of egg of honey-bee the future caudal pole of the embryo would correspond with the caudal end of the queen and since in the ovary of the queen the eggs are deposited parallel to her long axis, the law of Hallez doubtless applies to the honey-bee." Thus generally this disposition, i.e., cephalic end anterior and caudal end posterior, is found in insects. Imms (1934) has said, " I n the eggs of most insects there is always a distinction between anterior and posterior pole which bears a definite relation to the position of future embryo. The eggs are located in the overioles in such a position that the cephalic pole of each is directed towards the head of parent, also the dorsal and ventral aspects of the eggs correspond with those of the parent and the future embryo (Hallez, 1886)". In the Embryology of Insects and Myriapods Johannsen and Butt (1941) have said, "Hallez" law of orientation; this law formulated by Hallez (1886) is based on the observation that the mature eggs within the ovary lies in such a position that all the three axes of the presumptive embryo are oriented coinci- dently with those of the mother. From this it follows that embryonic axes are determined in the egg before laying. Although this is a general rule, it is not universal. Also, there are certain insects, e.g., Melanoplus in which it is reported by Silfer (1932) that the embryo begins to develop in the reverse position with the head towards the micropylar opening at the posterior end of the egg but that the normal position is attained by later reversal of the embryo during blastokinesis. In the radially symmetrical egg of the bug Pyrrhochoris Seidal (1924) reports that the longitudinal embryonic axis may vary from being coincident with the longitudinal axis of the egg to being transverse to it. Pteronareys proteus, likewise according to Miller (1936), does not conform to the law, the prospective position of the embryo being at right angle to the longitudinal axis of the ovariole."

But in Tetrastichus pyrillae Craw. by dissection of the female as well as sectioning the adult female (Figs. 4 and 5) it has been found that the slightly tapering end of the parasite egg Which is directed towards the head of female is definitely the anterior end and it bears the micropylar opening. But it has been found during development" of Tetrastichus pyrillae egg that the head of the embryo in this case is always formed towards the broader end of the egg (posterior) (Figs. 1 and 2). Thus the law of HaUez doubtless does not apply to Tetrastichus pyrillae Craw. This observation is one of the exceptions which has been brought to light for the first time in the case of Tetrastichus genus,

Embryology of Tetrastiehus pyrillae Craw. 43

Two membranes cover the external surface of the egg. The outer of these chorion (Fig. 6) is very thin and transparent though tough and dense and completely surrounds the eggs and protects it from direct contact with the atmosphere. It is smooth. In other insects the micropyle is a performation or system of performation of the chorion permitting the entrance of spermatozoa into the substance of the egg. It is always fre- quently situated at the cephalic pole of the egg. But only micropylar per- formation was observed at the tapering end of the Tetrastichus pyrillae egg. In the eggs taken from the ovary of a fertilized female, spermatozoa has been seen entering through the tapering end. It may be added that the micropyle in this case is not situated at the cephalic pole of the egg. The vitelline membrane the second of the two membranes is thinner than the chorion and apparently stuctureless (Fig. 6). It has recently been observed in some insect eggs by Rattan Lal (1958) that there is an oily layer in between the chorion and vitelline membrane. But in the case of Tetrastichus pyrillae no such layer was observed. Most probably due to the fact that the parasite egg is embedded in the host egg and it does not require such layer to ward off atmospheric moisture, etc. The content of the egg broadly speaking is made up of two portions, namely protoplasm --the so-called formative yolk--the material basis of the all life and part of ovum immediately concerned in the development and deutoplasm--a store of food material destined to be consumed by the developing embryo. Turning to the fixed and stained egg from ovary, the egg is seen to be filled with a network of stained protoplasm enclosing irregular spaces. With- in the spaces of the network are scattered round bodies, spherical to ovoid in form more or less densely stained. They are vitelline bodies. The vitelline bodies are enclosed by large transparent spheres of colourless fluid and they are called vitelline spheres. The latter components constitute the deutoplasm and this makes up for the greater volume of the substance of the egg. The nucleus is situated away from the centre towards the anterior end of the egg (Fig. 6). Besides this it may be added that in unfertilized egg the protoplasm and deutoplasm fill the whole of the egg from the anterior up to the posterior end of the egg enclosed by the vitelline membrane. But in the fertilized egg the yolk and protoplasm do not touch the anterior and posterior end of chorion. It means that when the egg has been fertilized a kind of stimulus is given to the contents of the egg for further stoppage of sperms and thus this contraction in the egg occurs. Besides this the presence of germ cell determinant at the posterior end of the eggs adds to the interest of the study of this form (Fig. 6).

44 KUNDAN LAL

(D) ACKNOWL~GEML~NT

I take this opportunity of expressing my deep sense of gratitude to Dr. E. S. Narayanan, F.A.SC., formerly Head of the Division of Entomology, Indian Agricultural Research Institute, New Delhi, for providing me all necessary facilities to carry out this piece of research work and guidance throughout the course of these investigations.

(E) R~FEP.L~CES

Gatenby, J.B. .. "The segregation of germ cells in Trichogramma evanescens,'" Quart. J. Micro. Sci., 1917, 63, 161-73.

Nelson, J .A. . . Embryology of Honey-bee, Princeton University Press, Prin. ceton, Humphrey Millford, Oxford University Press, London.

Imms, A.D. . . Gen. Text. Book Ent. Metheun & Co., Ltd., 36, Essex Street, W. C. London. 1934, pp. 170--86.

Johannsen, O. A. and Butt, E .H. Embryology_ of Insects and Myriapods, Mcgraw Hill Book Company, Inc., New York and London. 1941.

Rattan Lal . . "Chemical composition of the shell of external and internal parasitic eggs (Hymenoptera) and their host eggs and penetration of chemicals through them," Proc. nat. Inst. Sci. India, 1958, 24(1)B, 1-18.