Russian Entomol. J. 11 (3): 307–310 © RUSSIAN ENTOMOLOGICAL JOURNAL, 2002

The chorionic architecture and shell structure of Diaphania pulverulentalis (Hampson) (Lepidoptera: Pyralidae) eggs

Àðõèòåêòóðà õîðèîíà è ñòðîåíèå îáîëî÷êè ÿéöà îãí¸âêè Diaphania pulverulentalis (Hampson) (Lepidoptera: Pyralidae)

Vineet Kumar*, Virendra Kumar, S. Rajadurai, A.M. Babu, R.L. Katiyar, B.K. Kariappa, V. Thiagarajan and K.P. Jayaswal Âèíèò Êóìàð*, Âèðåíäðà Êóìàð, Ñ. Ðàäæàäóðàè, À.Ì. Áàáó, Ð.Ë. Êàòüÿð, Á.Ê. Êàðèàïïà, Â. Òèàãàðàäæàí, Ê.Ï. Äæàÿñâàë

Central Sericultural Research and Training Institute, Srirampura, Mysore 570 008, India. * Corresponding author ([email protected])

KEY WORDS: chorionic architecture, shell structure, eggshell, Diaphania pulverulentalis, SEM. ÊËÞ×ÅÂÛÅ ÑËÎÂÀ: àðõèòåêòóðà õîðèîíà, ñòðóêòóðà îáîëî÷êè, îáîëî÷êà ÿéöà, Diaphania pulverulen- talis, ÑÝÌ.

ABSTRACT: The eggshell of Diaphania pulverulen- Introduction talis (Hampson) was investigated by scanning electron microscopy. Surface of the pyralid egg shows structural First studies of the structure and function of insect elements, viz., aeropyles and micropylar rosette around the eggshell have been done by Leuckart [1855] and Kor- micropyles. Eggs are longish-oval, 332.80±4.32 µm long, schelt [1887]. Light microscopic (LM) and physiologi- and 218.60±4.71 µm wide, marked by a reticulate pattern cal studies of gas exchange and permeability were fol- of rectangular and pentangular cells. Micropylar struc- lowed by research [Beament, 1948; Wigglesworth and tures are situated at the anterior pole of the egg opposite Beament, 1950] using transmission and scanning elec- to its point of attachment to the host plant leaf. Micropylar tron microscopy (TEM and SEM). Since that time, fine area has eight unequal petal-shaped cells occupying total structure of a number of lepidopteran eggs has been length of 27.76±0.10 µm. Micropylar pit having 2.34±0.78 described, with a particular emphasis on surface sculp- µm in diameter is at the centre of an asymmetrical rosette ture [Matheny & Heinrichs, 1972; Mazzini, 1974; at the anterior pole. Aeropyles are annular, slightly raised Downey & Allyn, 1980; Hill, 1982; Salkeld, 1983, above the chorionic surface. They have 0.32±0.005 µm in 1984; Kumar et al., 1999]. A taxonomic key of the diameter, being surrounded by cell ridges. Total number family Pyralidae based on characters of egg chorion and of aeropyles varies from 15 to 18 per egg. its architecture in 15 species of sod worm moths has been created [Matheny & Heinrichs, 1972]. Due to small eggs ÐÅÇÞÌÅ: Îáîëî÷êà ÿéöà Diaphania pulverulen- size in the subfamily Crambinae it is difficult to employ talis (Hampson) èññëåäîâàíà ïðè ïîìîùè ñêàíèðóþ- conventional techniques for preparing slides and study- ùåé ýëåêòðîííîé ìèêðîñêîïèè. Ïîâåðõíîñòü ÿèö ing chorion structure. High resolution of SEM studies îãí¸âîê èìååò ñòðóêòóðíûå ýëåìåíòû, òàêèå êàê makes them an invaluable tool for examining diagnostic àýðîïèëå è ìèêðîïèëÿðíóþ ðîçåòêó âîêðóã ìèêðî- characters in insects. Hinton [1969] used scanning elec- ïèëå. ßéöà óäëèí¸ííî-îâàëüíûå, 332,80±4,32 ìêì tron micrographs to illustrate respiratory systems of äëèíîé è 218,60±4,71 ìêì øèðèíîé, ñ ñåò÷àòûì various insect eggshells. ðèñóíêîì èç ïðÿìîóãîëüíûõ è ïÿòèóãîëüíûõ ÿ÷ååê. Mulberry (Morus spp., family Moraceae), the sole Ìèêðîïèëÿðíûå ñòðóêòóðû ðàñïîëîæåíû íà ïåðå- food plant of silkworm, Bombyx mori L., an economical- äíåì ïîëþñå ÿéöà íàïðîòèâ òî÷êè ïðèñîåäèíåíèÿ ly important insect used in sericulture, is also prone to ÿéöà ê ëèñòó ðàñòåíèÿ-õîçÿèíà. Ìèêðîïèëÿðíàÿ îá- attack by various insect pests in different seasons of the ëàñòü èìååò âîñåìü íåðàâíûõ ëåïåñòêîâèäíûõ ÿ÷å- year. Recently, a new pest, leaf roller Diaphania pulver- åê, çàíèìàþùèõ îáùóþ ïëîùàäü 27,76±0,10 ìêì. ulentalis (Hampson), becomes a growing threat to mul- Ìèêðîïèëÿðíàÿ ÿìêà äèàìåòðîì 2,34±0,78 ìêì ðàñ- berry in the southern sericultural states of India viz., ïîëîæåíà â öåíòðå àñèììåòðè÷íîé ðîçåòêè çàäíåãî Karnataka, Andhra Pradesh and Tamil Nadu with an ïîëþñà. Àýðîïèëå êîëüöåâûå, ñëåãêà ïðèïîäíÿòûå average occurence of 21.77% [Rajadurai et al., 1999]. êàä ïîâåðõíîñòüþ õîðèîíà, 0,32±0,005 ìêì â äèà- D. pulverulentalis feeds on young leaves of mulberry ìåòðå, îêðóæåíû ãðåáíÿìè ÿ÷ååê. Îáùåå ÷èñëî àýðî- (Chawki leaves), thus being a serious pest and causing a ïèëå âàðüèðóåò îò 15 äî 18 íà ÿéöî. quantitative crop loss. Consequently, it has a consider- 308 Vineet Kumar, Virendra Kumar, S. Rajadurai, et al.

Figs. 1–8. Scanning electron micrographs of eggshell micropyle and aeropyles of Diaphania pulverulentalis: 1 — single egg (Eg) attached (arrow) between the leaf veins (Lv) on the adaxial surface (Ads) of mulberry leaf; 2 — lateral side of the egg (Eg) covered by leaf trichomes (Tr) of the host plant leaf; 3–4 — top view of egg pole showing micropylar region (Mp) surrounded by eight petal- shaped cells (O); 5 — magnified view of the micropyle (Mp); 6–7 — egg chorion with shell imprints (Si) and aeropyles; 8 — a single protruded aeropyle (Ap) on the shell imprints. Scale bars: 0.5 µm (8), 1.2 µm (5), 2.75 µm (7), 3 µm (3), 4 µm (4), 10 µm (6), 40 µm (2), 50 µm (1). Diaphania pulverulentalis (Lepidoptera: Pyralidae) eggs 309 able impact on Indian sericulture. To our knowledge, eggs were 419±0.008 µm long, 295±0.004 µm wide and little research of various stages of D. pulverulentalis has 582±0.012 µm long, 413±0.014 µm wide respectively. been carried out. The present study of the chorionic Eggs of the minimum size 396±0.006 µm long and architecture and eggshell structure of this pest may help 315±0.006 µm wide were observed in Crambus alb- in establishing its effective control. oclavellus. Those dimensions are very close to the egg size of D. pulverulentalis. Arbogast et al. [1989] studied Materials and Methods eggs of three moths under scanning electron microscope and found that eggs of Niditinea fuscella, Tinea palles- Laboratory culture of D. pulverulentalis has been centella and Tinea occidentella were subcylindrical, established using freshly emerged adults. They were having been 450, 640 and 620 µm long and 280, 330 and released in an insect cage (28 x 18 cm) and provided with 360 µm wide respectively. The micropylar area is slight- 10% honey solution for feeding and fresh mulberry ly raised at the anterior pole of the egg, and contains branches for egg-laying. After mating, females laid eggs eight unequal petal-shaped primary cells (Figs. 3–4). on the abaxial leaf surface. Eggs were then gently Hinton [1981] found that micropyles are always situated removed with a fine paintbrush. For a SEM study, eggs at the anterior pole of lepidopteran eggs. However, were fixed for two hours at room temperature in 2.5% unusual position of the micropylar structure on the lower glutaraldehyde solution in 0.2M cacodylate buffer (pH surface of the egg of Cydia pomonella was observed 7.2), then dehydrated in a graded alcohol-acetone series, [Fehrenbach et al., 1987]. The micropylar area with and dried in a critical point dryer (EMS – 850) using CO eight unequal petal-shaped cells is 27.76±0.10 µm wide. 2 as a transition fluid. Dried eggs were mounted onto It forms an asymmetrical rosette at the anterior pole of copper stubs in different positions. Mounted stubs were the egg of D. pulverulentalis with a central micropylar gold coated (20 nm thick) in a sputter coater (EMS – pit (2.34±0.78 µm in diameter) (Figs. 3–4). Similar 550) and examined using a JEOL 100 CX II ASID 4D rosettes of unequal petal-shaped cells were studied in scanning electron microscope at 20 kV. some other lepidopteran species,Plusia orichalcea[Goel & Kumar, 1987]; T. pallescentella, T. occidentella, N. Results and Discussion fuscella [Arbogast et al., 1989] and Spilarctia obliqua [Kumar et al., 1999]. There are seven primary cells Mated females of D. pulverulentalis laid eggs on the around the micropylar pit in P. orichalcea, 5–8 in T. lower surface of mulberry leaves between the midribs occidentella, 6–9 in N. fuscella and 10–14 in S. obliqua. and sub-veins (Figs. 1-2). A number of authors also However, primary cells were of subequal lengths in describe that most of the moths lay their eggs on the some eggs of N. fuscella, thus forming symmetrical lower surface of host plant leaves [Hinton, 1981, Goel & rosettes. Fehrenbach et al. [1987] examined fine struc- Kumar, 1987; Kumar et al., 1999]. Fresh eggs of D. ture of eggs of three lepidopteran species. They found pulverulentalis are creamy white, getting a pink shade that the micropylar rosette was composed of a rosette of before eclosion. Matheny and Heinrichs [1972] de- 13–15 petal-shaped cells in Heliothis virescens, where- scribed chorion features of 15 pyralid species. Most of as in Spodoptera littoris the number of rosette petals the eggs are creamy white or light yellow, turning yellow varied from 6 to 11, with 7 and 8 having been the most orange or deeper yellow before eclosion. Females of D. frequent. In Cydia pomonella the micropylar rosette is pulverulentalis laid single eggs amidst leaf trichomes to composed of 8 or 9 rosette petals. Matheny and Hein- protect the developing embryo (Fig. 2). Eggs are at- richs [1972] have used chorion characteristics of 15 tached to the leaf on their flat side and adhere to the species to prepare a taxonomic key of pyralid eggs of the substrate by a glue-like secretion. Hinton (1981) wrote subfamily Crambinae. They observed that the immedi- that flat eggs are characteristic of families of the former ate micropylar region is surrounded by 5–8 cells in Microlepidoptera — Tineidae, Gelechiidae, Gracil- Chrysoteuchia topiaria, 5–9 cells in Pediasia trisecta lariidae, Pterophoridae, Pyralidae etc. This author also and by a minimum of 4–6 cells in Pediasia mutabilis, pointed out that size and shape of the eggs of the tortricid Crambus alboclavellus, Pediasia luteotella and Cram- Rhyacionia duplana slightly varied according to the bus decorellus. pine species on which they were laid. Eggs of D. pulver- In D. pulverulentalis, the rosette of a single row of ulentalis are longish-oval, 332.80±4.32 µm long and eight petal-shaped cells at the anterior pole of the egg is 218.60±4.71µm at their maximum width, bluntly round- followed by densely ridged shell imprints, covering the ed at both ends (Figs. 1–2). Matheny and Heinrichs rest of egg surface (Figs. 1, 2, 6, 7). These imprints are [1972] described longish-oval eggs in other pyralids, represented by rectangular and pentangular cells of Pediasia luteolella and Crambus decorellus. Those variable size, 13.56±0.51 µm long and 8.61±0.22 µm

Ðèñ. 1–8. ÑÝÌ-ôîòîãðàôèè ìèêðîïèëå è àýðîïèëå îáîëî÷êè ÿéöà Diaphania pulverulentalis: 1 — îäèíî÷íîå ÿéöî (Eg), ïðèêðåïë¸ííîå (ñòðåëêà) ìåæäó æèëêàìè (Lv) íà àäàêñèàëüíîé ïîâåðõíîñòè (Ads) ëèñòà øåëêîâèöû; 2 — áîêîâàÿ ñòîðîíà ÿéöà (Eg), ïîêðûòàÿ âîëîñêàìè (Tr) ëèñòà ðàñòåíèÿ-õîçÿèíà; 3–4 — ïîëþñ ÿéöà ñ ìèêðîïèëÿðíîé îáëàñòüþ (Mp), îêðóæ¸ííîé âîñåìüþ ëåïåñòêîâèäíûìè ÿ÷åéêàìè (O), âèä ñâåðõó; 5 — ìèêðîïèëå (Mp), óâåëè÷åíî; 6–7 — õîðèîí ÿéöà ñ îòïå÷àòêàìè îáîëî÷êè (Si) è àýðîïèëå; 8 — îäèíî÷íîå àýðîïèëå (Ap) íà îòïå÷àòêå îáîëî÷êè. Ìàñøòàá: 0,5 ìêì (8), 1,2 ìêì (5), 2,75 ìêì (7), 3 ìêì (3), 4 ìêì (4), 10 ìêì (6), 40 ìêì (2), 50 ìêì (1). 310 Vineet Kumar, Virendra Kumar, S. Rajadurai, et al. wide (Figs. 7, 8). Fehrenbach et al. [1987] studied fine [1999] each aeropyle is surrounded by a well-defined structure of eggshells of lepidopterous pests. They found collar in D. pulverulentalis. Aeropyles were also sur- that the network of the flanks usually consisted of rounded by collars in T. occidentella and N. fuscella rectangular plates ca. 35 µm long and ca. 25 µm wide [Arbogast et al., 1989]. which is about three times more than the corresponding panels of the eggs of D. pulverulentalis. Arbogast et al. References [1989] examined external morphology of the eggs of three lepidopteran species using SEM. They found that Arbogast R.T., Brower J.H., Strong R.G. 1989. External morpho- egg chorion was marked by a reticulate pattern of polyg- logy of the eggs of Tinea pallescentella Stainton, Tinea occidentella Chambers, and Niditinea fuscella (L.) (Lepi- onal (mostly hexagonal) cells in T. occidentella whereas doptera: Tineidae) // Int. J. Insect Morphol. & Embryol. in N. fuscella it was marked by bold pattern of strongly Vol.18. No.5/6. P.321–328. depressed polygonal (mostly hexagonal) cells outlined Beament J.W.L. 1948. The penetration of insect egg-shells. I by heavy ridges. Recently, Kumar et al. [1999] wrote Penetration of the chorion of Rhodnius prolixus, Stal // Bull. that the cell imprints of the eggs of S. obliqua were Entomol. Res. Vol.39. P.359–383. Downey J.C., Allyn A.C. 1980. Eggs of Riodinidae // J. Lepi- mostly pentagonal or hexagonal and the aeropyles were dopterol. Soc. Vol.34. P.133–145. situated on their ridges. Similarly to results obtained by Fehrenbach H., Dittrich V., Zissler D. 1987. Eggshell fine Fehrenbach et al. (1987), and Kumar et al., [1999], structure of three lepidopteran pests: Cydia pomonella (L.) aeropyles were not observed on the first few rows of (Tortricidae), Heliothis virescens (Fabr.), and Spodoptera littorilis (Boisd.) (Noctuidae) // Int. J. Insect Morphol. & cells at the micropylar zone of the eggs of D. pulverulen- Embryol. Vol.16. No.3/4. P.201–219. talis. Aeropyles of the eggs of D. pulverulentalis are Goel S.C., Kumar V. 1987. Life history of a noctuid Plusia annular, slightly raised above the chorionic surface, orichalcea Fabr. on sunflower // Geobios News Reports. having 0.32±0.005 µm in diameter. An average distance Vol.6. P.111–114. Hill L. 1982. Eggs of some Tasmanian Noctuidae (Lepidoptera) between the two aeropyles is 22.74±0.19 µm. Matheny // Aust. Entomol. Mag. Vol.9. P.49–59. and Heinrichs [1972] also found annular and slightly Hinton H.E. 1969. Respiratory system of insect eggshells // protruded aeropyles on the egg surface of C. topiaries, Annu. Rev. Entomol. Vol.14. P.343–368. M. elegans and C. pascuellus floridus. However, diam- Hinton H.E. 1981. Biology of Insect Eggs. Oxford: Pergamon Press.Vol.2. P.475–778. eters of the aeropyles in these species, 3.33±0.35 µm, Korschelt E. 1887. Zur Bildung der Eihullen, der Mikropylen und 2.56±0.17 µm and 2.14±0.15 µm were much larger than Chorionanhange bei der Insekten // Nova Acta Ksl. Leop. those structures observed in D. pulverulentalis. Arbo- –Carol. Dtsch. Akad. Naturforsch. Bd.51. S.185–252. gast et al. [1989] found that the diameter of aeropyle Kumar V., Babu A.M., Kariappa B.K., Jayaswal K.P., Katiyar R.L., Datta R.K. 1999. Surface ultrastructure of the egg chorion of opening was in T. occidentella 0.55 to 0.93 µm Spilarctia obliqua Walker (Lepidoptera Arctiidae) // Redia. (0.77±0.15), in T. pallescentella — 0.667 to 1.20 µm Vol.82. P.137–143. (0.94±0.15), and in N. fuscella — 0.65 to 1.50 µm Leuckart R. 1855. Ueber die Micropyle und der feineren Bau der (1.10±0.36) for anterior aeropyles and 0.55 to 0.75 µm Schalenhaut beiden Insekteneiern. Zugleich ein Beitrag zur Lehre von der Befruchtung // Arch. Anat. Physiol. Wiss. (0.65±0.07) for posterior ones. Moreover, Fehrenbach Medizin. S.90–264. et al. [1987] wrote that the approximate diameter of Matheny E.L., Heinrichs E.A. 1972. Chorion characteristics of sod aeropylar openings in H. virescens was 1.9 µm and in C. webworm eggs // Ann. Ent. Soc. Am. Vol.65. No.1. P.238– pomonella — 0.6 µm. The number of aeropyles in D. 246. Mazzini M. 1974. Sulla fine struttura del micropilo negli insetti pulverulentalis was substanially smaller, i.e. from 15 to // Redia. Vol.55. P.343–372. 18 per egg, those structures having been found only at Rajadurai S., Manjunath D., Katiyar R.L., Prasad K.S., Sen A.K., the anterior pole. Fehrenbach et al. [1987] reported that Shekhar M.A. Ahsan M.M., Datta R.K. 1999. Leaf roller — A there were only 50 aeropyles restricted to the upper region serious pest of mulberry // Indian Silk. Vol.37. P.9–11. Salkeld E.H. 1983. A catalogue of the eggs of some Canadian of the egg of H. virescens, whilst there were about 400 Geometridae (Lepidoptera), with comments // Mem. Ento- apertures per egg in S. littoralis and about 140 ones — mol. Soc. Lon. Vol.126. P.3–271. in C. pomonella. Recently, Kumar et al. [1999] found Salkeld E.H. 1984. A catalogue of the eggs of some Canadian that the number of aeropyles in S. obliqua varied from 18 Noctuidae (Lepidoptera), with comments // Mem. Entomol. to 47 per shell imprint. These authors also wrote that the Soc. Lon. Vol.127. P.1–167. Wigglesworth V.B., Beament J.W.L. 1950. The respiratory mech- number of aeropyles for the whole egg was too high to anisms of some insect eggs. // Q. J. Microsc. Sci. Vol.91. be counted. Similarly to observations by Kumar et al. P.429–452.