composite default screen larval morphology and development ...messing/papers fred/muratori et al...

Larval morphology and development of Aphidiusrhopalosiphi (Hymenoptera: Braconidae:

Aphidiinae)1 180

Frédéric Muratori2

Unité d’Écologie et Biogéographie, Centre de Recherche sur la Biodiversité, UniversitéCatholique de Louvain, 4–5 Place Croix du sud, B-1348 Louvain-la-Neuve, Belgique

Jo Le Lannic

Centre de Microscopie Electronique à Balayage et Microanalyse, Université de Rennes I,Campus de Beaulieu, Avenue du Général Leclerc, F-35042 Rennes CEDEX, France

Jean-Pierre Nénon

Laboratoire d’Ecobiologie des Parasitoïdes, Université de Rennes I, Campus de Beaulieu,Avenue du Général Leclerc, F-35042 Rennes CEDEX, France

Thierry Hance

Unité d’Écologie et Biogéographie, Centre de Recherche sur la Biodiversité, UniversitéCatholique de Louvain, 4–5 Place Croix du sud, B-1348 Louvain-la-Neuve, Belgique

The Canadian Entomologist 136: 169 – 180 (2004)

Abstract—The aphid parasitoid Aphidius rhopalosiphi is the most abundant spe-cies of Aphidiinae in cereal fields in northern Europe. Although the larval morphol-ogy of other Aphidiinae has been described, the morphology and immaturedevelopment of A. rhopalosiphi remain unknown. Our goal is to relate growth andlarval developmental stages to morphological changes during parasitoid develop-ment, using light and scanning electron microscopy. Aphidius rhopalosiphi developsthrough three larval stages with clear differences in the morphology of the mouth-parts, tegument sculpturing, and respiratory features that can be related to the differ-ent constraints that the larvae have to face. In the first instar, adaptations to physicalcombat with competitors take the form of strong mandibles, active caudae, and dor-sal spines that allow crawling motion. In the third instar, the larva is adapted to teartissues with short hooked mandibles and to face aerial respiration. All instars pos-sess sensory structures. The “three instars” hypothesis is supported here by the ob-servation of larvae in exuviation. No differences were found between ourobservations and descriptions of other Aphidius species, supporting the idea thatspecies of this genus cannot be distinguished by larval morphology but only bymorphometric analysis. Some new features of the genus are presented for the firstand second instars.

Muratori F, Le Lannic J, Nénon J-P, Hance T. 2004. Morphologie et développement larvaired’Aphidius rhopalosiphi (Hymenoptera : Braconidae : Aphidiinae). The Canadian Ento-mologist 136 : 169–180.

Résumé—La morphologie des larves de l'endoparasitoïde de pucerons, Aphidiusrhopalosiphi, n’a jamais été décrite, bien que certains membres de la sous-familledes Aphidiinae aient fait l’objet d’études morphologiques. Cette espèced’Aphidiinae est pourtant la plus abondante dans les champs de céréales en Europedu Nord. Notre but est de mettre en relation la croissance et le développement des

169

1 Publication BRC028 of the Biodiversity Research Center, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.2 Corresponding author (e-mail: [email protected]).

J:\ent\ent13602\N03-057.vpMarch 10, 2004 9:14:21 AM

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larves d’A. rhopalosiphi et les modifications morphologiques observées au micro-scope optique ainsi qu’au microscope électronique à balayage. Aphidius rhopalosi-phi présente trois stades larvaires morphologiquement distincts notamment auniveau des pièces buccales, ornementation du tégument et appareil respiratoire. Cesdifférences morphologiques sont mises en relation avec les contraintes auxquellesdoit faire face la larve au cours de son développement. Grâce à des adaptations tel-les que de puissantes mandibules, une queue mobile ainsi que des épines dorsalespermettant des mouvements de reptations, le premier stade est particulièrementéquipé pour le combat physique avec des larves compétitrices, en cas de superpara-sitisme. Au dernier stade, la larve présente des structures lui permettant de vider lacuticule de son hôte et de respirer par elle-même. Les trois stades larvaires possè-dent des structures sensorielles. Dans ce travail, l’occurrence de trois stades larvai-res est supportée par l’observation d’individu en exuviation. Comparativement auxdonnées disponibles pour d’autres Aphidius, les résultats supportent l’idée qu’ausein des Aphidiinae, les espèces ne peuvent être distinguées sur base de comparai-sons morphologiques mais uniquement par des analyses morphométriques fines. Ce-pendant, des structures originales jamais décrites au sein du genre ont été observéesau premier et second stades larvaires.

Introduction

Aphidiine wasps are aphid endoparasitoids that undergo larval development insidea living host. Some species are currently used as biocontrol agents against aphids.Studies on larval morphology have reported a relative uniformity among Aphidiinae,but the number of larval instars remains unresolved (Stary 1962; Pennacchio andDigilio 1990). On the basis of larval morphology, three instars have been proposed(Schlinger and Hall 1960; Stary 1966; Calvert and Van Den Bosch 1972; O’Donnell1987, 1989; Pennacchio and Digilio 1990). Certain authors, however, favor four instars(Stary 1970; Couchman and King 1977; Hofsvang and Hågvar 1978; Chorney andMackauer 1979; Pare et al. 1979; Chow and Sullivan 1984) and even five instars(Beirne 1942; Vevai 1942; Stary 1962; Van der Hoek 1971). The four-instar hypothesiswas the most accepted, but quantitative studies on 22 species belonging to 10 differentgenera of Aphidiinae support the three-instar hypothesis (O’Donnell 1987) as con-firmed by Pennacchio and Digilio (1990) for Aphidius ervi Haliday. Even if the occur-rence of intermediary instars is doubtful, the first and last instars are well described andidentification keys are available (Capek 1970; O’Donnell 1989; Finlayson 1990).

Aphidius rhopalosiphi De Stefani Perez is a native solitary aphid parasitoid com-mon in cereal fields in Belgium (Langer et al. 1997) with a high potential for biologicalcontrol (Levie et al. 2000). No precise data are currently available on its developmentalbiology. Here, we describe the morphological criteria used to distinguish larval instars,evaluate the sensory and fighting structures of the larvae, and compare our data withdata for other Aphidiinae.

Materials and methods

Aphidius rhopalosiphi was collected in winter wheat fields near Louvain-la-Neuve, Belgium, in the summer of 2000 and reared on Sitobion avenae (Fabricius)maintained on winter wheat (Triticum aestivum (L.) (Poaceae) ‘windsor’). Colonies ofboth A. rhopalosiphi and S. avenae were kept in the laboratory under controlled condi-tions (19.5 ± 0.6 °C, 40%–50% RH, 16L:8D photoperiod).

Second-instar nymphs of S. avenae were individually parasitized byA. rhopalosiphi females. As soon as an oviposition attack was observed, the parasitized

170 THE CANADIAN ENTOMOLOGIST March/April 2004



aphid was removed and placed on a single wheat leaf to avoid superparasitism. TwentyA. rhopalosiphi females were used, each of which parasitized about 30 aphids. Every24 h, about 70 aphids were dissected in Hank’s salt solution (Sigma-Aldrich). Theparasitoid larvae were observed using a differential interference contrast microscope(Reichert, Vienna, Austria) fitted with a Nikon Coolpix 990 digital camera, and about50 larvae per day were transferred into 70% alcohol for scanning electron microscopy(SEM). Larvae were placed in a metal basket between two polyamide filters of 10-µmmesh and then progressively dehydrated in alcohol (70%, 80%, 90%, 100%) and ace-tone. Critical-point drying was performed with a Balzers CPD-010 critical-point dryer(Balzers, Liechtenstein) using CO2. Larvae were then covered with gold–palladium us-ing a JEOL JFC-1100 sputter unit (JEOL Ltd, Tokyo, Japan) and observed with a JEOLSEM-6400 at the Centre de Microscopie Electronique à Balayage et Microanalyse,Rennes. The nomenclature used in this paper is based on O’Donnell (1987, 1989),Finlayson (1990), and Gauld and Bolton (1996).

Results

First instar

Seventy-two hours after oviposition, embryos are still surrounded by a cellulartrophamnion (Tremblay and Caltagirone 1973). Ninety-six hours after oviposition, allfirst-instar larvae had hatched and teratocytes had been released into the hosthemocoele. Three regions of the larva body were distinguishable: the head (h), threethoracic segments (t1–t3), and ten abdominal segments (a1–a10) (Fig. 1A). Larvae wereof the caudate–mandibulate type (Gauld and Bolton 1996). The head had two strongsickle-shaped mandibles, each with a longitudinal groove (Figs. 1B and 1C). Mandiblesmoved rapidly when active but remained behind the prominent maxillae when at rest,with their extremities hidden in the hypopharyngial “tentacular” excrescences (Fig. 1D).As observed by light microscopy, the labrum adopted an active backward and forwardmotion. The epipharyngeal surface of the labrum had two areas each containing threediscoid structures accompanied by small globular projections (Fig. 1E) in half of thespecimens observed. The sensory structures of the head were well developed and ar-ranged in a quite fixed position (Fig. 1F). The edge of the labrum had two pairs oftrichoid sensilla. A pair of roundish palps associated with a pair of trichoid sensillawere found on each maxilla (Fig. 1G). Ventrally, the head displayed two pairs oftrichoid sensilla on two longitudinal folds.

Two cephalic orifices were found on the dorsal side of the head as described forseveral Aphidiinae (O’Donnell 1989), and a pair of latero-ventral orifices was found be-tween the head and the first thoracic segment. Each abdominal segment had atransversal row of 7–20 robust spines (10–15 µm long) accompanied by 5–10 smallerspines (4–7 µm long) projected to the rear of the larva (Figs. 1H and 4A). No spineswere found on the three thoracic segments. The active cauda was carried by the tenthabdominal segment and was covered by spines (Fig. 1I).

Second instar

Second-instar larvae were found 144 h after oviposition. Body segmentation wassimilar to that of the first-instar larvae (Fig. 2A). The second-instar larvae were of thehymenopteriform type (Gauld and Bolton 1996). Mandibles were absent (Fig. 2B), butthe mandibles of the third-instar larvae could be seen late in the development of the sec-ond instar underneath the cuticle (Fig. 2E, arrow) (Pennacchio and Digilio 1990).

Volume 136 THE CANADIAN ENTOMOLOGIST 171




FIGURE 1. First instar of Aphidius rhopalosiphi. General view showing body segmentation (h, head; t1–t3, thoracic segments; a1–a10, abdominal segments) (A). Lateral view of head (B). Mandible withevident longitudinal groove (C). Hypopharyngial tentacular excrescences (D). Trio of discoidstructures on labrum (E). Schematic arrangement of head structures (a, mandibles; b, trichoidsensilla; c, discoid structures; d, maxillary palp; e, longitudinal fold; f, hypopharyngial excrescences)(F). Frontal view of head with mandibles at rest (G). Dorsal view of abdominal segments, each with arow of spines (H). Cauda (I).



Sensory structures were displayed around the oral opening, but this arrangement variedamong specimens. Groups of two (sometimes three) trichoid sensilla, two groups ofthree discoid structures with projections, and two bifid (trifid in two specimens) sensilla(Fig. 2H) were located dorsally above the oral orifice. On the ventral side of the mouth,at least three pairs of trichoid sensilla were present with variable distribution.

As in the first instar, a cephalic orifice was present at the margin between thehead and the first thoracic segment. The tegument of the abdominal segments was char-acterized by areas of aggregated spicules (length less than 3 µm) on the dorsal side(Fig. 4B). The cauda was reduced and covered with spicules (Figs. 2C and 2F).

Moreover, as observed by light microscopy, late second-instar larvae had imaginaldiscs in the thoracic segments (arrows in Fig. 2D) and tracheal arborescence (not com-pletely connected and no spiracles). A specimen in the molting process (Fig. 2G) dem-onstrated the clear difference in tegument sculpturing between the second and thirdinstars.


FIGURE 2. Second instar of Aphidius rhopalosiphi. Early second instar (A–C). Late second instar (D–F).General views (A, D) (arrows indicate imaginal discs). Oral orifice (B, E) (arrows indicate mandiblesof the third instar visible beneath the cuticle). Cauda (C, F). Larvae in ecdysis to third instar (G).Trifid sensilla at the margin of the oral orifice (H).




FIGURE 3. Third instar of Aphidius rhopalosiphi. General view of larva (A). Head with silk press andfirst thoracic segments (arrow indicates location of the first spiracle) (B). Posterior view showingabdominal pair of sensory buds (arrows) (C). Schematic arrangement of head structures (a, antennalsocket; b, trio of discoid structures; c, trichoid sensilla; d, lateral orifice; e, smooth bud; f, mandibles;g, maxillary palps; h, silk press orifice; i, postlabial trichoid sensillum; j, porous bud; k, anteriortentorial pit) (D). Frontal view of head (E). Hook-shaped mandible (F). Spiracle on mesothorax (G).Pseudospiracle on metathorax (H). Discoid structures on inner side of labrum (I). Maxillary palp withevident unique pore, accessory palp, and trichoid sensillum (J). Prominent porous bud of maxilla (K).Lateral smooth bud on the labroclypeus (L).



Third instar

The third instar was found 168 h after oviposition and corresponded to thehymenopteriform type (Fig. 3A) (Gauld and Bolton 1996).

We can describe the following structures on the head, as schematically repre-sented in Figure 3D: (a) one pair of antennal sockets on the dorsal side of the head;(b) two groups of three discoid structures on the labroclypeus (detail in Fig. 3I); (c) sev-eral pairs of trichoid sensilla, often asymmetrically disposed above the oral orifice;(d) one pair of lateral deep orifices; (e) one pair of lateral smooth buds (detail inFig. 3L); (f) one pair of triangular hook-shaped mandibles without teeth (detail inFig. 3F); (g) one pair of maxillary palps associated with a trichoid sensillum and an ac-cessory palp (detail in Fig. 3J); (h) a silk press orifice on the prominent prelabiumabove two labial palps, each labial palp associated with two trichoid sensilla and an ac-cessory palp; (i) two pairs of trichoid sensilla associated with the postlabium; (j) promi-nent porous buds laterally displayed by the maxillae (detail in Fig. 3K); and (k) twoanterior tentorial pits laterally disposed on the labroclypeus.

The integument of the cephalic capsule was smooth dorsally with characteristicsculpturing above the mandibles, on the maxillae, and on the prelabium and thepostlabium. The integumental sculpturing entirely covered the prothorax but only thedorsal part of the rest of the body (Figs. 3A–3C). Each abdominal segment had a lateraland a ventral pair of smooth buds (similar to the lateral head buds) and an area withlongitudinal dorsal folds (Fig. 4C). A cauda was absent. The last abdominal segment(Fig. 3C) had two groups of three trichoid sensilla and a pair of lateral porous buds(similar to the maxillary buds).

The third instar was characterized by nine pairs of spiracles laterally disposed onthe mesothorax from the first to the eighth abdominal segment. Through light micros-copy observations of the metathorax, we found that the second pair of spiracles was notconnected to the tracheal system as described by Hofsvang and Hågvar (1978) forAphidius colemani Viereck. We called these orifices “pseudospiracles”. As observed by


FIGURE 4. Comparison of integumental sculpturing in first- (A), second- (B), and third-instar larvae(C) of Aphidius rhopalosiphi.

Second instar

First instar Early Late Third instar

Mandibles Sickle shaped Absent Absent Hook shapedCauda Present and mobile Reduced Reduced AbsentTracheae Absent Absent Present but not

completely connectedConnected to

spiraclesTegument

sculpturingRow of spines Areas of

spiculesAreas of spicules Globular sculpturing

TABLE 1. Morphological characteristics allowing distinction between larval instars of Aphidiusrhopalosiphi using light microscopy.




Spe

cies

A.

ervi

Hal

iday

A.

nigr

ipes

Ash

mea

dA

.sm

ithi

Sha

rma

etS

ubba

Rao

A.

pulc

her

Bak

erA

.co

lem

ani

Vie

reck

A.

rhop

alos

iphi

De

Ste

fani

Per

ez

Ref

eren

ceP

enna

cchi

oan

dD

igil

io19

90P

are

etal

.19

79C

horn

eyan

dM

acka

uer

1979

Van

der

Hoe

k19

71H

ofsv

ang

and

Håg

var

1978

Thi

sst

udy

Tec

hniq

ue(s

)*S

EM

and

LM

SE

Man

dL

MS

EM

LM

SE

Man

dL

MS

EM

and

LM

Num

ber

ofin

star

s3

44

54

3F

irst

inst

arM

andi

bles

Sic

kle

shap

edF

alci

form

wit

hca

nal

Sic

kle

shap

ed—

†P

rese

ntS

ickl

esh

aped

wit

hca

nal

Hea

dse

nsil

laO

nepa

irof

palp

san

das

soci

ated

seta

eO

nepa

irof

palp

san

das

soci

ated

seta

e—

†—

†—

†T

wo

pair

sof

palp

san

das

soci

ated

seta

eD

orsa

lin

tegu

men

tS

pine

son

abdo

min

alse

gmen

tsS

pine

son

abdo

min

alse

gmen

tsS

pine

son

abdo

min

alse

gmen

tsS

pine

son

all

segm

ents

Row

ofha

irs

onab

dom

inal

segm

ents

Spi

nes

onab

dom

inal

segm

ents

Cau

daC

over

edw

ith

spin

esC

over

edw

ith

spin

esC

over

edw

ith

spic

ules

Cov

ered

wit

hsp

icul

esC

over

edw

ith

spin

esC

over

edw

ith

spin

es

Inte

rmed

iate

inst

ar(s

)M

outh

part

sA

bsen

tA

bsen

tA

bsen

tP

rese

ntin

3rd

and

4th

inst

ars

Pre

sent

in2n

din

star

,ab

sent

in3r

din

star

Abs

ent

Hea

dse

nsil

laA

bsen

tS

ensi

lla

onla

brum

—†

—†

—†

Tri

choi

dse

nsil

la(b

ifid

totr

ifid

)D

orsa

lin

tegu

men

tS

mal

lsp

icul

esS

mal

lsp

icul

esex

cept

onpr

otho

rax

—†

No

spin

esS

pine

sG

roup

sof

spic

ules

TA

BL

E2.

Dat

aav

aila

ble

onla

rval

mor

phol

ogy

ofA

phid

ius

spec

ies.




Cau

daR

educ

ed,

cove

red

wit

hsp

icul

esP

rese

nt,

cove

red

wit

hsp

icul

esR

educ

edR

educ

edor

abse

ntR

educ

edor

abse

ntR

educ

ed

Las

tin

star

Mou

thpa

rts

Sub

tria

ngul

arT

rian

gula

rP

rese

ntP

rese

ntbu

tre

duce

dP

rese

ntH

ook

shap

edw

itho

utte

eth

Hea

dse

nsil

laP

alps

and

sens

illa

Pal

psan

dse

nsil

laV

ario

usse

nsor

yor

gans

—†

—†

Pal

psan

dse

nsil

laD

orsa

lin

tegu

men

tP

apil

lifo

rmsc

ulpt

urin

gS

culp

turi

ng—

†—

†C

over

edw

ith

tube

rcul

esS

culp

turi

ng

Cau

daA

bsen

tA

bsen

t—

†—

†A

bsen

tA

bsen

t

*S

EM

,sc

anni

ngel

ectr

onm

icro

scop

y;L

M,

ligh

tm

icro

scop

y.†

No

data

men

tion

edby

the

auth

ors.



SEM, the connected spiracles (Fig. 3G) had tubular structures pointing to the outside,while unconnected pseudospiracles (Fig. 3H) were deep empty holes.

Discussion and conclusions

Our results support the three-instar hypothesis as proposed by O’Donnell (1987).The morphological characterization of larvae in this study leads us to define three uni-form groups. Early and late second instars are not morphologically different and noth-ing suggests that they are distinct. These two periods can be explained by growth andcould have misled the authors of earlier studies. The three-instar hypothesis is sup-ported by the occurrence of three types of integument (Fig. 4). In addition, we foundlarvae molting between the first and second instars and also between the second andthird instars. We did not find exuviae of larvae inside the aphids. The thinness and (or)rapid degradation of empty cuticle could explain this finding and might be an adapta-tion for waste avoidance within the host. Observation of in vitro reared larvae couldlead us to unequivocally determine the number of instars by counting exuviae(Pennacchio and Digilio 1990). Morphological characteristics easily identifiable withthe aid of a light microscope are listed in Table 1. The placement of some structures(e.g., imaginal discs, tracheal system) is progressive and does not follow the partitioningof larval instars.

In solitary parasitoids, only one larva per host develops to the adult stage. In thecase of superparasitism (more than one egg laid in a single host), competition betweendeveloping larvae for possession of the host must occur (Fisher 1961). Physical combatis one mechanism of elimination of supernumerary larvae (Vinson and Hegazi 1998). Itis generally accepted that the first instar is the one that fights (Chow and Mackauer1984). This idea is well supported by the morphological characteristics of first instars:strong, prominent, mobile mandibles; mobility due to a functional cauda; and body mo-tion and dorsal spines facilitating crawling motion. Moreover, well-developed sensorystructures can be used by larvae to localize or recognize competitor(s), as shown inCardiochiles nigriceps Ashmead (Vinson and Mourad 2000). As suggested byCouchman and King (1977), these morphological characteristics could support thefighting activity of the larvae rather than their feeding activity. Several authors refer tothe integumental absorption of nutrients by first-instar larvae (Arthur 1944; de Eguileoret al. 2001).

Koinobiont parasitoid larvae face a changing environment inside the host and un-dergo hypermetamorphosis to maximize host exploitation (Gauld and Bolton 1996;Chapman 1998). In contrast to the simple molting process, hypermetamorphosis allowssuccessive larval instars to exhibit different morphology. In the first- and third-instarmandibulate larvae, palps are the most exposed structures of the perioral region. Wesuggest that these palps function in recognition of the environment and are related tothe presence of mandibles. For koinobiont species, the host must remain alive and there-fore mandibulate larvae must recognize which tissues they can use as food (vital organsmust be preserved). The second instar is characterized by the absence of mandibles andpalp-like structures. Therefore, it seems that palps and mandibles are associated struc-tures. The appearance of hooked mandibles, a silk press, and functional spiracles in thethird instar is a response to new environmental constraints. Mature larvae will have totear tissues, spin a cocoon, and prepare for aerial respiration.

Concerning the pseudospiracles, our observations agree with those of A. colemaniby Hofsvang and Hågvar (1978). Further studies are needed to elucidate the function ofthe pseudospiracles found in third-instar larvae. These structures are similar in form andposition to the lateral cephalic orifices of first and second instars and could share a yet




unknown function. This is the first time tubular ornamentation of the spiracles has beendescribed.

In this work, we present the first description of the larval instars ofA. rhopalosiphi. Table 2 compares features reported for other Aphidius species withthose of A. rhopalosiphi. New structures reported in this study were probably observedbecause of methodological differences rather than morphological differences betweenspecies. In fact, the genus seems to display a conserved morphological pattern, and inhis work on first-instar morphology, O’Donnell (1989) could not distinguish specieswithin the genus Aphidius. Finlayson (1990) distinguished Aphidius species bymorphometric analysis based on sclerite size and spiracles of the last-instar larvae, butA. rhopalosiphi was not included in her study.

Finally, we have observed a certain level of variability in some structures amongindividuals and, for the same individual, between the left and right sides. This variabil-ity must be quantified to see whether it can be used for fluctuating asymmetry analyses.These kinds of analyses may be useful for comparing development conditions such asartificial in vitro rearing.

Acknowledgments

We thank C. Patty and O. Lebbe for help with the laboratory work. We are alsograteful to G. Boivin, B. Heming, and two anonymous reviewers for their comments onthe manuscript. This study was financed with research funds from the Ministry of Re-search and Technological Development of the Walloon Region, Belgium.

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(Received: 2 June 2003; accepted: 29 October 2003)




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