Russian Entomol. J. 18(3): 157–164 © RUSSIAN ENTOMOLOGICAL JOURNAL, 2009

Embryonic Molt Numbers in Hemimetabolous and Holometabolous Insects

×èñëî ýìáðèîíàëüíûõ ëèíåê ó íàñåêîìûõ ñ íåïîëíûì (Hemimetabola) è ïîëíûì (Holometabola) ïðåâðàùåíèåì

S.Yu. Chaika1, E.Yu. Orlova2 Ñ.Þ. ×àéêà1, Å.Þ. Îðëîâà2

Moscow State University, Faculty of Biology, Department of Entomology, Vorobievy Gory, Moscow 119899, Russia. E-mail: [email protected]; [email protected] Ìîñêîâñêèé Ãîñóäàðñòâåííûé Óíèâåðñèòåò, Áèîëîãè÷åñêèé ôàêóëüòåò, êàôåäðà ýíòîìîëîãèè, Âîðîáü¸âû Ãîðû, Ìîñêâà 119899, Ðîññèÿ.

KEY WORDS: metamorphosis, Hemimetabola, Holometabola, pronymph, embryonic development, cuticle, ontogenesis. ÊËÞ×ÅÂÛÅ ÑËÎÂÀ: ìåòàìîðôîç, Hemimetabola, Holometabola, ïðîíèìôà, ýìáðèîíàëüíîå ðàçâèòèå, êóòèêóëà, îíòîãåíåç

ABSTRACT. Embryonic molts reflect free-living Based on how radical such metamorphosis is, two ancestral stages and facilitate the study of embryoniza- major groups are recognized among the Pterygota: tion, the compressing of post-embryonic stages into the Hemimetabola (with incomplete metamorphosis) and egg. In Hemimetabola, three cuticles are formed during Holometabola (complete metamorphosis) [Ivanova- embryonic development. Cuticles 1 and 2 are purely Kazas, 1981]. embryonic, while cuticle 3, also formed in ovum, be- In the promising model developed by Truman and comes the first-instar nymphal cuticle. Holometabolism Riddiford [1999], the holometabolous larva is homolo- involves a single embryonic cuticle, same as the first- gous to the hemimetabolous pronymph (pre-larva) that instar cuticle. may show various degrees of embryonization and, there- fore, various free-living times. The hemimetabolous ÐÅÇÞÌÅ. Èçó÷åíèå ýìáðèîíàëüíûõ ëèíåê, îò- nymph should be thus considered homologous to the ðàæàþùèõ îñîáåííîñòè ñâîáîäíîæèâóùèõ ïðåä- holometabolous pupal stage. This model is supported êîâûõ ñòàäèé, äà¸ò êëþ÷ ê ïîíèìàíèþ ïðîöåññîâ by the findings of developmental biology, metamorpho- ýìáðèîíèçàöèè, ò.å. ïðîõîæäåíèÿ îòäåëüíûõ ñòà- sis patterns and the presence of a pronymphal stage in äèé ïîñòýìáðèîíàëüíîãî ðàçâèòèÿ ïîä çàùèòîé Hemimetabola. ÿéöåâûõ îáîëî÷åê. Ó Hemimetabola â ïðîöåññå ýì- The degree of embryonization in a taxon may be áðèîíàëüíîãî ðàçâèòèÿ êóòèêóëÿðíûé ïîêðîâ ôîð- established based on the outcome of embryogenesis and ìèðóåòñÿ òðèæäû. Ïðè ýòîì ïåðâàÿ è âòîðàÿ êóòè- the presence of embryonic cuticles/molts [Polivanova, êóëà ÿâëÿþòñÿ èñêëþ÷èòåëüíî ýìáðèîíàëüíûìè, à 1979]. We studied embryonic molting and integuments òðåòüÿ, íåñìîòðÿ íà òî, ÷òî òàêæå ôîðìèðóåòñÿ in in the embryo and free-living stages of representative ovum, ïðåäñòàâëÿåò ñîáîé ïîêðîâû íèìôû ïåðâî- members of Holometabola and Hemimetabola. ãî âîçðàñòà. Ó Holometabola ôîðìèðóåòñÿ òîëüêî îäíà ýìáðèîíàëüíàÿ êóòèêóëà, êîòîðàÿ îäíîâðå- ìåííî ÿâëÿåòñÿ êóòèêóëîé ëè÷èíêè ïåðâîãî âîçðà- Materials and Methods ñòà. Two species, one hemimetabolous and one holom- Introduction etabolous, were studied: (i) Locusta migratoria L. (Hemimetabola: Orthoptera: Acrididae), the migrato- Insect ontogeny studies are driven by the quest for ry locust, whose life cycle includes a vermiform larva the origins of the incomplete vs. complete metamorpho- representing a pronymphal stage; and (ii) Lucilia cae- sis that goes back to the Antiquity and by the desire to sar L. (Holometabola: Diptera: Calliphoridae), the correlate the developmental stages between hemi- and greenbottle fly — an advanced, less embryonized in- holometabolous insects [Yezhikov, 1929; Chaika, 2003]. sect. In the winged insects (Pterygota), the various de- For electron microscopy, the samples were kept in velopmental stages differ greatly, which means their 2.5% glutaraldehyde in phosphate buffer (pH 7.3) for postembryonic development involves metamorphosis. 4 hours, then in 2% osmium tetroxide for 2 hours, then 158 Chaika S.Yu., Orlova E.Yu. dehydrated in a battery of EtOH concentrations. The Results samples were opacified in saturated uranyl acetate in 70% EtOH and, after complete dehydration, embed- Locusta migratoria ded in Epon following the standard protocol. Ultrathin The locust produces three cuticles in the course of slices stained using the Reynolds technique were ex- embryonic development: embryonic cuticle 1 (EC1), amined under JEM-1011 transmission electronic mi- embryonic cuticle 2 (EC2), and the third cuticle that croscope with Gatan ES800W digital cell. eventually becomes the first-instar integument.

ec1

ec1

1 2

msc.

nuc.

ec2

ec2

3 4

ec2

ec2

5 6

Figs 1–6. Embryonic cuticles of Locusta migratoria: 1–2 — first embryonic cuticle; 3–6 — second embryonic cuticle. Ðèñ. 1–6. Ýìáðèîíàëüíûå êóòèêóëû ñàðàí÷è Locusta migratoria: 1–2 — ïåðâàÿ ýìáðèîíàëüíàÿ êóòèêóëà; 3–6 — âòîðàÿ ýìáðèîíàëüíàÿ êóòèêóëà. Embryonic molt numbers in Hemimetabolous and Holometabolous Insects 159

The EC1 appears at the limb formation stage as a 0.02 During the EC2 formation, the embryonic integument to 0.03 ìm thick single-layer high electron density homo- is distinct and consists of three layers: the hypoderm, the geneous cuticle (Fig. 1). The high electron density sug- basal membrane, and the cuticle. Individual hypodermal gests its epicuticular nature — most likely, the cuticuline cells are barely distinguishable, but membrane structures layer. The EC1 shows no aciculae or other outgrowths in the cytoplasm are distinct (Fig. 3). The cells are short, (Fig. 2) and disappears after the EC2 is fully formed. slightly over 2 ìm maximum height (Fig. 4), and contain

ec1 ec1 ec2

ec2

7 8

ec1

p.c. ec2 ec1

9 10

p.t. msc.fasc.

11 12

Figs 7–12. Second embryonic cuticle of Locusta migratoria: 7–8 — formation of the second embryonic cuticle; 9–11 — pore canals; 12 – muscle bundles. Ðèñ. 7–12. Âòîðàÿ ýìáðèîíàëüíàÿ êóòèêóëà ñàðàí÷è Locusta migratoria: 7–8 — ôîðìèðîâàíèå âòîðîé ýìáðèîíàëüíîé êóòèêóëû; 9–11 — ïîðîâûå êàíàëû; 12 — ïó÷êè ìûøö. 160 Chaika S.Yu., Orlova E.Yu. large oblong nuclei up to 4 ìm long. The cuticle is 0.8–1.5 lower density sublayers and can be interpreted as procu- ìm thick, with two clearly distinguishable layers (Fig. 5). ticle (Figs 6–8). Pore canals, best visible in its outer part, The outer, higher electron density layer is about 0.1 ìm are a typical feature of the procuticle (Figs 9–11). The thick and is presumably homologous to the adult epicu- integument has muscles attached to it (Fig. 12). ticle. The second layer, less electron dense and up to 1.5 Once the embryonic development is completed, a ìm thick in some areas, shows several alternating higher/ vermiform larva (=pronymph) hatches.

nuc. msc.fib. msc.fib.

nuc.

13 14

b.lab.

p.g.

cut. cut.

15 16

encut. encut.

f.encut.

p.c.

17 18

Figs 13–18. Structure of integument of instar I nymph of Locusta migratoria: 13–14 — ultrastructure of hypodermal cells; 15–16 — ultrastructure of integument; 17–18 — ultrastructure of endocuticle. Ðèñ. 13–18. Ñòðîåíèå ïîêðîâà íèìôû I âîçðàñòà ñàðàí÷è Locusta migratoria: 13–14 — óëüòðàñòðóêòóðà ãèïîäåðìàëüíûõ êëåòîê; 15–16 — óëüòðàñòðóêòóðà ïîêðîâà; 17–18 — óëüòðàñòðóêòóðà ýíäîêóòèêóëû. Embryonic molt numbers in Hemimetabolous and Holometabolous Insects 161

Notably, the larva makes it to the surface fully cov- mediate molt’) and turns into a first-instar nymph. ered with embryonic cuticle sculptured on the inside in Unlike the embryonic cuticle, the first-instar cuticle a pattern matching (as a mould) the pattern of the un- shows a more elaborate pattern of subpentagonal cells, derlying first-instar nymphal cuticle. each with 2–5 small tubercles in the center. The entire The vermiform phase lasts 2 to 5 minutes only; once body surface is covered with sensory organs (sensilla). in the open, the larva loses its embryonic cuticle (‘inter- The thoracic integument consists of an epithelial layer, a

encut.

f.encut. p.c.

p.c.

19 20

p.c.

encut.

21 22

f.encut. p.t.

p.c. p.t. p.c.

23 24

Figs 19–24. Structure of integument of instar I nymph of Locusta migratoria: 19–22 — ultrastructure of cuticle; 23–24 — ultrastructure of cuticle and apical part of hypodermal cells. Ðèñ. 19–24. Ñòðîåíèå ïîêðîâà íèìôû I âîçðàñòà ñàðàí÷è Locusta migratoria: 19–22 — óëüòðàñòðóêòóðà êóòèêóëû; 23–24 — óëüòðàñòðóêòóðà êóòèêóëû è àïèêàëüíîãî îòäåëà ãèïîäåðìàëüíûõ êëåòîê. 162 Chaika S.Yu., Orlova E.Yu. basal membrane, and the cuticle. The epithelial cell, ca. 8 larger size. In the apical area, the hypodermal cells ìm high, harbors a chromatin-containing nucleus located feature lysosome-like subglobular membranous ele- usually at the bottom (sometimes centrally or apically). ments. Besides the usual mitochondria, microtubules, vac- Some cells contain long fibers running from the base uoles, and the granular endoplasmic reticulum, the cyto- to apex (Figs 13–14). Pore canals containing high- plasm contains high electron density globular granules density rods strongly suggest that these are probably (0.4–0.5 ìm) and lower-density granules of similar or tonofibrillae connecting muscles with the cuticle.

ex.ch. trab. ex.ch.

trab.

cut. end.ch.

25 26

ch.

cut.

27 28

b.m.

cut.

sens. nuc.

cut.

29 30

Figs 25–30. Ultrastructure of coatings of Lucilia caesar: 25–26 — ultrastructure of chorion; 27 — cross slice of an egg; 28 — forming cuticle; 29 — formed cuticle; 30 — formation of sensilla. Ðèñ. 25–30. Óëüòðàñòðóêòóðà ïîêðîâîâ Lucilia caesar: 25–26 — óëüòðàñòðóêòóðà õîðèîíà; 27 — ïîïåðå÷íûé ñðåç ÿéöà; 28 — ôîðìèðóþùàÿñÿ êóòèêóëà; 29 — ñôîðìèðîâàííàÿ êóòèêóëà; 30 — ôîðìèðîâàíèå ñåíñèëëû. Embryonic molt numbers in Hemimetabolous and Holometabolous Insects 163

Cell apices and bases feature irregularly located pro- show regular density. The procuticular layer appears at jections or invaginations of cell membrane. Such invag- this stage not fully formed and thus has to be still inations are often well developed at the cell base and growing. form the basal labyrinth (Figs 15–16). Lucilia caesar Secretory cells scattered among the hypodermal cells are easily recognizable by their rather rounded In Lucilia caesar, the 3.2 ìm thick chorion is subdi- shape and basal parts protruding beyond the hypoder- vided into a denser exochorion, a less dense endochori- mal layer. Their cytoplasm is rich in inclusions ca. 2 ìm on, and clearly visible trabeculae (Figs 25–26). Fixed in diameter. The 0.2 ìm thick basal membrane appears preparations show a 3.6 ìm average gap between the homogenous, fine-grained, and of medium electron den- embryo and the chorion (Fig. 27). A single embryonic sity. cuticle is formed to eventually become the first-instar The nymphal cuticle varies in thickness from 2 to 5 cuticle (Fig. 28). As the embryo develops, this cuticle ìm and has two easily visible layers, epicuticle and changes. In freshly laid eggs, the cuticle is 0.25 ìm procuticle. The epicuticle is more electron dense, ap- thick, with no visible layers; the hypoderm, still under- pears homogeneous and is 0.1–0.2 ìm thick or up to 0.5 developed, looks like a higher electron density (com- ìm where associated with cuticular projections. Procu- pared to other tissues) cell layer underlying the cuticle; ticle (Figs 17–19) has a layered structure, with nine 12 hours thereafter, the embryo is covered with a thin layers in average, and conspicuous irregularly scattered 0.8 ìm cuticle (Fig. 29) that thickens as the embryo pore canals, many with high-density muscle attachment grows to reach 1.25 ìm 24 hours after oviposition, at rods (Figs 20–22). which point the basal membrane is clearly visible and Typical of this stage is a thin (0.2–0.3 ìm) promi- closely underlies the hypoderm, with 3.2 ìm gap be- nent granular layer between the bottom of the procuticle tween it and the embryonic cuticle. The cuticle has pore and the apical epithelial microvilli that appears on elec- canals. At this stage, the sensory elements emerge at the tron micrographs homogeneous and much darker than surface of the cuticle (Fig. 30). the procuticle (Figs 23–24). In areas where new cuticle layers are being formed, this layer appears transformed into a typical endocuticle with the helicoid lamellar Discussion arrangement. These areas can be seen only where the procuticle has been fully formed and the material is used In the Hemimetabola, three embryonic cuticles are to produce new procuticular layers, not the epicuticle. produced, of which the third one becomes the cuticle of The tips of the microvilli touching this layer are high the first-instar nymph. The three cuticles differ greatly electron density — perhaps because this is where the in structure. The thin single-layer EC1 can be consid- cuticular material gets released, since there is no such ered epicuticle based both on its high electron density material between the microvilli where the membranes and the extensive electron microscopic studies suggest-

ABBREVIATION ÓÑËÎÂÍÛÅ ÎÁÎÇÍÀ×ÅÍÈß b.lab. — basal labyrinth b.lab. — áàçàëüíûé ëàáèðèíò b.m. — basal membrane b.m. — áàçàëüíàÿ ìåìáðàíà cut. — cuticle cut. — êóòèêóëà ec1 — first embryonic cuticle ec1 — ïåðâàÿ ýìáðèîíàëüíàÿ êóòèêóëà ec2 — second embryonic cuticle ec2 — âòîðàÿ ýìáðèîíàëüíàÿ êóòèêóëà ec3 — third embryonic cuticle ec3 — òðåòüÿ ýìáðèîíàëüíàÿ êóòèêóëà msc. — muscles msc. — ìûøöû msc.fasc. — muscular fascicles msc.fasc. — ïó÷êè ìûøö msc.fib. — muscle fibrillae msc.fib. — ìûøå÷íûå ôèáðèëëû p.g. — pigment granules p.g. — ïèãìåíòíûé ãðàíóëû p.c. — pore canal p.c. — ïîðîâûå êàíàëû p.t. — pore tubes p.t. — ïîðîâûå òðóáêè sens. — sensilla sens. — ñåíñèëëû r.p.c. — rod of pore canal r.p.ch. — ñòåðæåíü ïîðîâîãî êàíàëà trab. — trabecules trab. — òðàáåêóëû f.encut. — forming endocuticle f.encut. — ôîðìèðóþùàÿñÿ ýíäîêóòèêóëà ch. — chorion ch. — õîðèîí ex.ch. — exochorion ex.ch. — ýêçîõîðèîí encut. — endocuticle encut. — ýíäîêóòèêóëà end.ch. — endochorion end.ch. — ýíäîõîðèîí nuc. — nucleus nuc. — ÿäðî 164 Chaika S.Yu., Orlova E.Yu. ing that chitin-containing procuticle always exhibits How does the number of embryonic cuticles com- lower electron density. pare in Hemimetabola vs. Holometabola? This would Unlike EC1, the EC2 consists of two distinct layers, require to find out how many embryonic cuticles the homologous to the adult epicuticle and procuticle, re- holometabolans have. spectively. The procuticle is further divided into sublay- In Lucilia caesar, a higher dipteran, a single embry- ers and is pierced by numerous pore canals. Notably, the onic cuticle is formed to become the first-instar cuticle. EC2 integument has muscles attached to it — the con- As the embryo develops, the cuticle grows thicker, more nection that does not occur before the EC2 had been elaborate, and produces the cuticular parts of the sensilla. fully formed. This is especially significant, since it is the EC2-stage embryo that is arguably homologous to the References embryonized free-living ancestral larva. The third embryonic cuticle, i.e. the larval cuticle, is Chaika S.Yu. 2003. Insect origins and segmentation: A Text- about twice as thick as the EC2 and is also bi-layered, book. Moscow: MAKS Press. 92 pp. [in Russian]. consisting of an epicuticle of higher electron density Ivanova-Kazas O.M. 1981. The comparative embryology of inverte- and a laminated (ca. nine-layered) procuticle. The pro- brates. Atelocerata. Moscow: Nauka. 207 pp. [in Russian]. Polivanova E.N. 1979. Embryonization of onthogenesis, origin of cuticle is characterized by prominent pore canals with embryonic molts and types of development in insects // Zool. Zh. high electron density rods for muscle attachment. Un- Vol.58. P.1269–1280 [in Russian]. like EC2, the third embryonic cuticle produces sensory Truman J.W. & Riddiford L.M. 1999. The origins of insect metamor- cuticular elements of both mechano- and chemorecep- phosis // Nature. Vol.401. P.447–452. tors. Yezhikov I.I. 1929. [Insect metamorphosis]. Moscow: Timirya- zev State Research Institute Publishers. 52 pp. [in Russian].