Ultramorphology of the mature larvae of Sericinus montela Grey (Lepidoptera: Papilionidae), with descriptions of osmeterium using a novel method of larval preservation

Abstract

Papilionidae, swallow-tailed butterflies, attract the attention of biologists for the importance of their ecological and evolutionary study. Papilionidae are peculiar among Lepidoptera for their larvae bearing a Y-shaped eversible osmeterium on the prothorax. However, morphological study of the larvae of Papilionidae, especially in terms of the osmeteria, are far from satisfactory. In this study, larval ultramorphology of Sericinus montela was described using scanning electron microscopy after a novel method of sample preservation. Larvae of S. montela are peculiar for the 15 teeth on the incisor, the triordinal mesoseries plus biordinal lateroseries of crochets on the anal prolegs, the urticating acanthae on secondary setae, and the micro openings and surrounding microtrichia on the osmeterium. The method of sample preservation, larval morphological characters and correlated defensive strategies are compared and briefly discussed.

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Ultramorphology of the mature larvae of Sericinus
montela Grey (Lepidoptera: Papilionidae), with
descriptions of osmeterium using a novel method
of larval preservation
Zi-Hao Wang & Lu Jiang
To cite this article: Zi-Hao Wang & Lu Jiang (2023) Ultramorphology of the mature larvae
of Sericinus�montela Grey (Lepidoptera: Papilionidae), with descriptions of osmeterium
using a novel method of larval preservation, Journal of Natural History, 57:1-4, 38-53, DOI:
10.1080/00222933.2023.2167620
To link to this article: https://doi.org/10.1080/00222933.2023.2167620
Published online: 07 Feb 2023.
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Ultramorphology of the mature larvae of Sericinus montela
Grey (Lepidoptera: Papilionidae), with descriptions of
osmeterium using a novel method of larval preservation
Zi-Hao Wang and Lu Jiang
Key Laboratory of Economic and Applied Entomology of Liaoning Province, College of Plant Protection,
Shenyang Agricultural University, Shenyang, Liaoning, China
ABSTRACT
Papilionidae, swallow-tailed butteries, attract the attention of
biologists for the importance of their ecological and evolutionary
study. Papilionidae are peculiar among Lepidoptera for their larvae
bearing a Y-shaped eversible osmeterium on the prothorax.
However, morphological study of the larvae of Papilionidae, espe-
cially in terms of the osmeteria, are far from satisfactory. In this study,
larval ultramorphology of Sericinus montela was described using
scanning electron microscopy after a novel method of sample pre-
servation. Larvae of S. montela are peculiar for the 15 teeth on the
incisor, the triordinal mesoseries plus biordinal lateroseries of cro-
chets on the anal prolegs, the urticating acanthae on secondary
setae, and the micro openings and surrounding microtrichia on the
osmeterium. The method of sample preservation, larval morphologi-
cal characters and correlated defensive strategies are compared and
briey discussed.
ARTICLE HISTORY
Received 9 May 2022
Accepted 9 January 2023
KEYWORDS
butterfly; caterpillar;
osmeteria; sensillum; crochet
Introduction
Larvae occupy a signicant period in the life history of endopterygote insects
(Kristensen 1999; Gullan and Cranston 2010), contribute important nutrient accumu-
lation and face increasing challenges in multiple ecosystems (Wagner 2005; Meier
and Lim 2009). On one hand, larvae are considered feeding devices that turn smaller
embryos into larger individuals (Zacharuk and Shields 1991; Hall and Wake 1999). On
the other hand, larvae exhibit diverse relationships with host plants, natural enemies
and ecological factors in their relatively longer living periods (Ruxton et al. 2005;
Jiang et al. 2019). In fact, in peculiar situations, larvae are more likely to attract
attention from biologists than are the corresponding adults, even in the most famous
insect lineage, butteries (Igrashi 1984; Stehr 1987; Hasenfuss 1999; Casagrande and
Mielke 2005).
Papilionidae are commonly called swallow-tailed butteries (Chou 1998), and
comprise an estimated 570 species (Simonsen et al. 2011 van Nieukerken et al. 2011;
Condamine et al. 2018) assigned to seven tribes within three subfamilies (Aubert et al.
1999). Although Papilionidae attract the attention of biologists for the importance of
CONTACT Lu Jiang jianglu@syau.edu.cn
JOURNAL OF NATURAL HISTORY
2023, VOL. 57, NOS. 1–4, 38–53
https://doi.org/10.1080/00222933.2023.2167620
© 2023 Informa UK Limited, trading as Taylor & Francis Group
Published online 07 Feb 2023

their ecology and evolution (Silva–Brandao et al. 2005), the phylogenetic relationships
in this superfamily are still ambiguous, whether based on morphological or molecular
evidence (Munroe 1960; Common 1975; Hancock 1983; Igrashi 1984; Aubert et al. 1999;
Kondo et al. 2003; Espeland et al. 2018). A possible reason for this ambiguity is the
relative abundance of data sets with immature and adult characters (Nazari et al. 2007;
Meier and Lim 2009).
The osmeterium, an eversible Y-shaped exocrine organ on the larval prothorax, is
an autapomorphy characterising the Papilionidae (Miller 1987; Vegliante and
Hasenfuss 2012). When larvae are disturbed, the osmeterium can evert sponta-
neously and secrete chemicals as a defensive weapon to repel ants or other enemies
(Honda 1983; Ômura et al. 2006). However, detailed morphological characters of
osmeteria are inadequately studied, because the retracted organs can hardly be
observed in normal specimens (Damman 1986; Stehr 1987; Wagner 2005). More
methods of sample preservation are required for better observing this protrusible
organ.
Sericinus montela Gray, the so-called dragon swallowtail, is the only species of Sericinus
of Zerynthiini in Parnassiinae, which is distributed only in East Asia (Li and Zhu 1992; Chou
1998; Igarashi 2003). As an introduced species, S. montela are highly susceptible to climate
change and anthropogenic action (Tani 1994), and their larvae are reported to feed on
Aristolochia debilis or A. contorta (Ji et al. 2012). Accurate and rapid larval identication is
of great signicance for species protection and biodiversity studies (van Emden 1957).
However, knowledge of the the larval morphology of S. montela is highly unsatisfactory,
especially on an ultramorphological level. This study describes the ultramorphology of
larval S. montela, to provide novel characters for larval taxonomy and to analyse the
defence strategies of Papilionidae.
Materials and methods
Insect collection and rearing
A cluster of eggs were collected soon after being deposited by a single female Sericinus
montela, in the botanical garden of Shenyang Agriculture University (123.633°N, 41.850°
E), Shenyang, Liaoning Province of north-east China, in early May in 2019. The eggs
together with the host plants were transferred into plastic boxes which were then
covered with absorbent paper to keep them moist at room temperature (around
25°C). Fresh leaves of Aristolochia contorta Bunge were provided daily as food items
for the larvae.
Morphological observation
For morphological observations, the larvae were xed in Dietrich’s solution (forma-
lin:95% ethanol:glacial acetic acid:distilled water = 6:15:1:80, v/v), which was heated
to 70°C, and then they were left for 12 h under a hood before being preserved in
75% ethanol (Jiang and Hua 2015b). The xed larvae were dissected under a Leica
EZ4HD stereoscopic microscope. Photographs were taken using a Nikon D90 digital
camera (Nikon, Tokyo, Japan). For scanning electron microscopy (SEM), a total of 10
JOURNAL OF NATURAL HISTORY 39

xed larvae were serially dehydrated in a graded ethanol series (80, 90, 95, 100 and
100%) for 30 min at each step. Then the solution was replaced with graded mixtures
of ethanol and tertiary butanol (3:1, 1:1 and 1:3, v/v) for 15 min and pure tertiary
butanol for 30 min twice. After freeze-drying for 2 h, the samples were sputter-
coated with gold, and examined under a Hitachi S-3400N scanning electron micro-
scope (Hitachi, Tokyo, Japan) at 5 kV, following Qu et al. (2019). Larval morphological
nomenclature follows Stehr (1987).
Results
General larval morphology
The nal-instar larvae of Sericinus montela are of typical eruciform. The cylindrical trunk
bears three pairs of thoracic legs, four pairs of abdominal prolegs and a pair of anal
prolegs (Figure 1). The head is orthognathus, with the mouthparts directed ventrally. The
Figure 1. General larval morphology of Sericinus montela. (a) Live mature larva, habitus; (b) mature
larva specimen. abp, abdominal proleg; anp, anal proleg; os, osmeterium; pf, protuberant filament; tl,
thoracic leg; vr, verruca.
40 Z.-H. WANG AND L. JIANG

larval trunk is furnished with a pair of prominent laments (PF) and a Y-shaped eversible
osmeterium (Os) on the prothorax, and three rows of verrucae (Vr) arranged on the dorsal,
lateral and subventral surface, respectively (Figure 1(b)).
Head
The head capsule is sclerotised, bearing a pair of antennae, six pairs of stemmata and a group
of chewing mouthparts directed ventrally. The head capsule is black and hirsute (Figure 1(b)).
The number and position of cranial secondary setae vary greatly among specimens and may
even be asymmetrical between sides on a single individual (Figure 2(a,b)). The frontoclypeus is
approximately triangular, bearing numerous secondary setae. The anteclypeus is subtrape-
zoidal and membranous, and bears six pairs of asymmetrical setae.
The larvae have six pairs of stemmata (Figure 2(c)), which are generally equal in size.
Stemmata 1–4 and 6 are protuberant and arranged in a semicircle. Stemma 5 is widely
separated from the semicircle and located anteriorly near the antennal base.
Figure 2. Heads of larvae of Sericinus montela. (a) Dorsal view; (b) ventral view; (c) lateral view; (d)
frontal view. an, antenna; el, ecdysial line; lr, labrum; lp, labial palp; md, mandible; ; Mx, maxilla; sp,
spinneret; s1–6, stemma.
JOURNAL OF NATURAL HISTORY 41

Each antenna is located between stemma 5 and the mandible (Figure 2(d)). The
antenna is cylindrical and three-segmented, with the second segment being the longest
(Figure 3(a)). The second segment bears distally two sensilla chaetica (SC1 and SC2) and
two sensilla basiconica (SB1 and SB2). The sensilla chaetica varied signicantly in length,
with SC1 being much longer than SC2. The third segment of the antenna is very short,
bearing a sensillum basiconicum distally (SB3).
Figure 3. Antennae and mouthparts of larvae of Sericinus montela. (a) Antenna; (b) epipharynx; (c)
internal surface of the left mandible; (d) external surface of the right mandible; (e) magnification of
maxilla, insert showing magnification of distal joint of maxillary palp; (f) magnification of spinneret. an,
1–3 antenna; lp, labial palp; mp, maxillary palp; mx, maxilla; sb1–3, sensillum basiconicum; sc1–2,
sensillum chaeticum; scf, sensillum campaniformium; ss, sensillum styloconicum.
42 Z.-H. WANG AND L. JIANG

Larval mouthparts
The mouthparts are mandibulate, comprising a labrum, a pair of mandibles, a pair of
maxillae and labia, and a tubular spinneret.
The labrum is trapezoidal proling a V-shape notch, and equipped with 10 pairs
of setae (Figure 2(d)). The epipharynx has a pair of attened sensilla digitiformia
located on the lateral side, three pairs of sensilla chaetica at the anterior margin,
and two pairs of sensilla campaniformia on the central area. In addition, the
epipharynx is furnished with inconspicuous microtrichia on the proximal region
(Figure 3(b)).
The mandible is attened and heavily sclerotised, forming 15 cusps (distal teeth) on the
apical margin (Figure 3(c,d)). The cusps are more prominent in the middle and gradually
diminish to each side. The mandible bears two setae on the external surface (Figures 2(d)
and 3(d)).
The maxilla possesses two sensilla styloconica and three sensilla chaetica on the mesal
lobe (Figure 3(e)). The maxillary palp is two-segmented, and furnished with a sensillum
digitiformium, two sensilla campaniformia (inset of Figure 3(e)), and 10 sensilla basiconica
surrounding a styloconic sensilla basiconica at the apex (Figure 3(e)).
The labium has a pair of labial palps and an elongated tubular spinneret (Figure 3(f)).
Each labial palp is furnished with a campaniform sensillum (SCf) basally, and a sensillum
basiconicum (SB) and a sensillum styloconicum (SS) distally. The spinneret is tubular and
protrudes ventrally.
Thoracic legs
The thoracic legs are ve-segmented: each consists of a coxa, a femur, a tibia, a tarsus and
a single claw on the apex (Figure 4(a)). The coxa is furnished with plentiful microtrichia on
the lateral and mesal surfaces. The femur, tibia and tarsus are sclerotised and bear dense
setae on the mesal surface and sparse setae laterally. The claw is heavily sclerotised, with
a sharp apex (Figure 4(b)).
Abdominal and anal prolegs
The unsegmented proleg consists of a proximal base and a distal planta where the
crochets arise (Figure 4(c)). The proximal base bears abundant setae on the lateral and
mesal surface. The planta is furnished with numerous microtrichia mesally (Figure 4(c)).
The crochets of the abdominal prolegs are triordinal mesopenellipse arranged in an
incomplete circle (Figure 4(d)). The crochets of the anal prolegs are remarkable for
possessing combined mesal triordinal penellipse and lateral uniordinal series
(Figure 4(e)).
Osmeterium
The osmeterium is normally contracted into the prothorax (Figure 1(a)), and stretches out
as a yellowish Y-shaped gland if the larva is disturbed (Figures 1(b) and 5(a)). When fully
stretched, the osmeterium can be further subdivided into a glabrous basal stem and
JOURNAL OF NATURAL HISTORY 43

paired distal arms (Figure 5(a)). The tubular arms of the osmeterium are hirsute distally
(Figure 5(b)), and furnished with numerous minor openings surrounded by clusters of
microtrichia (Figure 5(c)).
Epidermal derivates on the trunk
The paired protuberant laments arise from the prothorax (Figure 1(b)), and are externally
wrinkled and black (Figures 1(a) and 6(a)). The laments are equipped with numerous
spinous setae (Figure 6(a)), which are internally hollow and furnished with acanthae on
the surface (Figure 6(b)).
Figure 4. Legs of larvae of Sericinus montela. (a) Thoracic leg; (b) magnification of the apical claw; (c)
proleg, lateral view, inset showing the magnification of the planta; (d) proleg crochets, ventral view;
(e) ventral view of segments A8–9; (f) magnification of epiproct. al, anal proleg; cl, claw; cx, coxa; fe,
femur; ta, tarsus; ti, tibia.
44 Z.-H. WANG AND L. JIANG

The verrucae are arranged on the dorsal, lateral and subventral surfaces of the larval
trunk (Figure 1(b)). The verrucae are generally yellow, except for those on the dorsum of
the metathorax (Figure 1(b)), which are blackish terminally and yellowish proximally
(Figure 1(a)). The verrucae on dorsal and lateral surfaces are comparatively more promi-
nent (Figure 6(c)) than those on the subventral surface (Figure 6(d)). All the verrucae are
covered with luxuriant secondary setae (Figure 6(c,d)), which are also furnished with
numerous acanthae.
Spiracles
The respiratory system is of the peripneustic type; the larvae bear a pair of prothoracic
spiracles and eight pairs of spiracles on the rst eight abdominal segments (Figure 1(b)).
Figure 5. Osmeteria of Sericinus montela. (a) General morphology of osmeterium; (b) magnification of
the arm of osmeterim, arrow indicates a micro opening; (c) microtrichia of osmeterium, arrow
indicates a micro opening; (c) filaments on the flank of the larval prothorax. os, osmeterium; pf,
protuberant filament.
JOURNAL OF NATURAL HISTORY 45

The spiracles are elliptical and generally similar in size (Figure 6(e)). The spiracle is
equipped with longer branched trichomes covering the whole orice and shorter micro-
trichia arranged on the exterior ring (Figure 6e).
Discussion
In this study, morphology of mature larvae of S. montela were described for the rst time
using SEM. The larvae exhibit several remarkable characters of the sensilla, mouthparts,
crochets, osmeteria and some epidermal derivates, the combination of which can be used
to distinguish the larvae from larvae of other species.
The osmeterium is a unique defensive organ of Papilionidae larvae (Hallberg and
Poppy 2003; Vegliante and Hasenfuss 2012). Detailed morphology of the osmeterium
has hardly been observed in previous studies, because this organ is extended only if the
larvae are alive (Damman 1986; Leslie and Berenbaum 1990; Dyer 1997). Based on our SEM
observations, the larval osmeterium can be subdivided into a smooth basal stem and
hirsute distal arms, dierent from previous observations of Heraclides thoas (Martínez et al.
2018), Graphium agamemnon (Chattopadhyay 2011) and Papilio demoleus Fruhstorferthe
Figure 6. Protuberant filaments, verrucae and spiracles of Sericinus montela. (a) Protuberant filament
on larval prothorax; (b) magnification of urticating setae of protuberant filament, arrow indicates the
seta hollow; (c) verruca on dorsum of mesothorax; (d) verruca on dorsum of metathorax; (e) spiracle on
prothorax. lfa, long filter apparatus; pe, peritreme; sfa, short filter apparatus.
46 Z.-H. WANG AND L. JIANG

(Lu and Chow 1991). The micro openings and microtrichia were conrmed in S. montela
for the rst time, using a novel method of sample preservation.
Heated Dietrich’s solution was originally used in larval sample preservation in the
study of scorpiony (Jiang and Hua 2015b), and then proved to be eective in the
study of sawy, moths and scarab beetles (Zhao and Hua 2016; Fang et al. 2018;
Xue and Hua 2018; Qu et al. 2019; Zhang et al. 2022). Three advantages of this
method, at least, were recognised for the morphological study of insect larvae.
Firstly, heated formalin is helpful for tissue solidication, sustaining epidermis and
avoiding collapse during dehydration and freeze-drying (Jiang and Hua 2015b; Li
et al. 2019; Dong et al. 2020; Jia et al. 2020). Secondly, the mixture of glacial acetic
acid and ethanol contributes signicantly to the hardening of larval epidermis,
equivalent to an improved Carnoy’s xation solution (Chen and Hua 2011; Liu
et al. 2011; Xue and Hua 2018). Furthermore, the gradual heating process is con-
ducive for organ eversion or protrusion (Jiang et al. 2015; Jiang and Hua 2015b),
retaining the advantages of previous methods (Tan and Hua 2008; Cai and Hua
2009; Chen and Hua 2011).
Mandibles are of primary importance in food handling for insect larvae (Snodgrass
1935; Bernays and Janzen 1988; Chapman 2013). The mandibles, especially cutting
incisors, are morphologically diverse among insect species (Stehr 1987; Jiang and
Hua 2015a). In Lepidoptera, the incisors have eight teeth in pine-feeding Dendrolimus
kikuchii (Men and Wu 2016), six teeth in the leaf-mining Calycopis bellera (Duarte and
Robbins 2009) and the lawn cutworm Spodoptera depravata (Hu et al. 2021), ve
teeth in the polyphagous Helicoverpa armigera (Queiroz-Santos et al. 2018), are fused
into a smooth cutting edge in some leaf-feeding mature larvae of Notodontidae
(Miller 1987), and retain both cutting edge and dentate teeth in some larvae of
Saturniidae (Zhang et al. 2022). In this study, the attened larval mandibles were
found to be furnished with 15 teeth on the mesal cutting margin, very likely suitable
for chewing on both leaves and stems of the host plants, similar to other
Papilionidae larvae (Palma-Onetto et al. 2020). The morphological dierences we
observed not only resulted from evolutionary processes but are also related to
feeding habits peculiar to the host plants.
Sensilla are signicant in allowing larvae to detect temperature, humidity, and
pheromones and other chemicals (Ansebo et al. 2005). The sensilla are diverse in
terms of type, number and arrangement, all of which provides valuable informa-
tion for taxonomy (Zacharuk and Shields 1991). According to previous studies in
Lepidoptera, there are 16 sensilla on larval antennae in Danaus chryszppus, but
fewer than 10 in Methona themisto (Kitching 1985). In this study, the larvae possess
two sensilla basiconica and two sensilla chaetica on each antenna, similar to the
larvae of Noctuidae, Pyralidae, Tortricidae, Zygaenoidea and Carposinidae (Xie
et al. 2006; Liu et al. 2011; Zhi et al. 2012; Ma et al. 2013; Xiang et al. 2016). In
addition, each maxillary palp possesses 11 sensilla basiconica, dierent from most
of the ground plan in Ditrysia (Grimes and Neunzig 1986).
Crochets, small hooks that are serially arranged on larval prolegs, represent signicant
taxonomic characters for the classication of Lepidoptera larvae (Chu 1949; Hasenfuss
JOURNAL OF NATURAL HISTORY 47

1999). The crochets are found in diverse types among families, being uniordinal in mesal
penellipse in some Noctuidae (Wagner et al. 2006; Xue and Hua 2018; Hu et al. 2021),
uniserial in two transverse bands in Sesiidea (Bąkowski 2013), uniordinal in lateral penel-
lipse in Psychidae (Davis et al. 2008), biordinal in Saturniidae (Rougerie and Estradel 2008),
and triordinal mesal penellipse in some Pyralidae (Meijerman and Ulenberg 1996; Piao
et al. 2006). In this study, the larval crochets of S. montela are triordinal mesal penellipse
on the abdominal prolegs, but we found triordinal mesoseries plus biordinal laterseries
crochets on anal prolegs. This character was not usually mentioned in previous studies.
Phytophagous larvae usually employ various defensive strategies when attacked (Gullan
and Cranston 2010; Waldbauer 2012). In this study, larvae of S. montela are peculiar for
bearing rows of yellowish and blackish verrucae on the larval trunk (Figure 1(a)), an eversible
osmeterium on the prothorax (Figure 5(a)), and acanthae on the secondary setae (Figure 6(a,
b)), all of which are involved in larval defensive strategies. Firstly, the intertwined yellowish
and blackish verrucae arranged on the larval trunk are displayed as an aposematic warning
signal, similar to many other caterpillars (Sillén-Tullberg 1988). Furthermore, osmeterium is
a defence organ unique to the larvae of Papilionidae, and it is believed to release chemicals
to prevent the larva from being picked up by predators (Lu and Chow 1991; Vegliante and
Hasenfuss 2012; Martínez et al. 2018). Otherwise, most of the secondary setae on laments
are equipped (indicated by the triangle in Figure 5) with urticating acanthae, which may
cause painful damage to potential predators, consistent with many larvae in
Thaumetopoeidae and Saturniidae (Lamy et al. 1982; Novak et al. 1987). In fact, these hollow
urticating setae are very likely able to release toxic proteins, as found in previous studies
(Gilmer 1925; Battisti et al. 2011). More evidence is needed to fully understand the diverse
defensive strategies in larvae of Lepidoptera.
Acknowledgements
We are grateful for the help of Yun-Long Ma and Yong-Xu Han during our sample collections.
Special thanks are due to Mrs Si-Xian Yang, Mrs Min Lou, and Mr Li-Ping Zhou, for their long-term
support during our study on caterpillars of butteries.
Disclosure statement
No potential conict of interest was reported by the authors.
Funding
This research was nancially supported by the National Key R&D Program of China [grant
no. 2021YFD1400200], China Postdoctoral Science Foundation [grant no. 2020M680982], Natural
Science Foundation of Liaoning Province [2021-MS-230], Scientic Research Project of Education
Department of Liaoning Province [LJKZ0641], Science and Technology Planning Project of Liaoning
Province [grant no. 1618214601077], National Science & Technology Fundamental Resources
Investigation Program of China [grant no. 2018FY100300], and Scientic Research Foundation for
the Introduced Talent of Shenyang Agricultural University [grant no. 880417008].
48 Z.-H. WANG AND L. JIANG

Author contributions
ZHW: writing the original draft; LJ: conceptualisation, project administration, writing and reviewing
the manuscript.
Data availability statement
The data that support the ndings of this study are available from the corresponding author upon
reasonable request.
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