Entomologia Experimentalis et Applicata 96: 1–8, 2000.

© 2000 Kluwer Academic Publishers. Printed in the Netherlands.

1

The feeding behavior of Trichogramma brassicae: new evidence for

selective ingestion of solid food

Z. X. Wu1 , A. C. Cohen2 & Donald A. Nordlund2,∗

1 Department of Entomology and Plant Pathology, P.O. Box 9775, Mississippi State University, Mississippi
State, MS 39762-9775, USA; 2 USDA, REE, ARS, MSA, Biological Control and Mass Rearing Research Unit,
P.O. Box 5367, Mississippi State, MS 39762-5367, USA; ∗ Author for correspondence (Phone: (662) 320-7530;
Fax: (662) 320-7571; E-mail: donn@ra.msstate.edu)

Accepted: September 13, 1999

Key words: Hymenoptera, Trichogrammatidae, extra-oral digestion, parasitoids, entomophagous insects

Abstract
A descriptive study of the feeding behavior and structures of Trichogramma brassicae Bezdenko (Hymenoptera:
Trichogrammatidae) was conducted. Based on direct observational and biochemical evidence, larvae feed predom-
inantly on particulate materials, starting ca. 25 h post-oviposition. Feeding lasted for ca. 9 h, at 25 ± 1 ◦ C. During
this feeding period the shape of the larvae changed from vermiform to pyriform and then to sacciform, resulting
in a ca. 40-fold increase in body size. Larvae used elaborate feeding behaviors as they pulled solid food particles
to their oral opening, broke small particles from larger ones, and took the particles into the stomodaeum, which
is a powerful pump. In the stomodaeum, peristaltic movement further macerated the particles, which eventually
passed through the cardiac valve into the midgut. As indicated by changes in fluorescently labeled casein, digestive
enzymes aid in the extra-oral chemical digestion of food. The contents of the gut, during and shortly after feeding,
were almost entirely closely packed solid particles. The behavioral activity of feeding larvae centered almost
exclusively on processing and ingesting solid food particles. The rapid larval growth is much more plausibly
explained by their feeding on the highly concentrated nutrients found in solid foods, rather than the extensive
concentration required if dilute liquids were the principal source of nutrients. The implications of these findings
for the development of practical artificial diets are discussed.

Introduction nutritional balance and special chemicals for pupation

Trichogramma spp. are among our most important
parasitoids for augmentative biological control of lep-
idopteran pests. Larval Trichogramma spp. feed on the
contents of a host egg until they accumulate sufficient
nutrients to become pupae and ultimately adults (Flan-
ders, 1937; Volkoff et al., 1995). Volkoff et al. (1995)
reported that during an 8 h feeding period, the length
of Trichogramma cacoeciae Marchal larvae increased
from 164 µm to 333 µm, a ca. 3-fold increase in vol-
ume. But how Trichogramma spp. larvae ingest food
so rapidly and how the mouthpart structures influence
feeding behavior have not been explained fully.
Interest in developing artificial diets for Tri-
chogramma spp. has led many researchers to focus on
(Wu et al., 1982; Nettles, 1990; Nordlund et al., 1997).
Despite some success in development of artificial di-
ets for Trichogramma spp. high rates of deformities,
manifested primarily as distended abdomens, have
been reported (Wu et al., 1982; Grenier et al., 1995;
Nordlund et al., 1997).
Current artificial diets for Trichogramma spp. are
liquids with diluted components (Wu et al.,1982; Gre-
nier et al., 1995; Xie et al., 1997). Use of ultracen-
trifuged chicken egg yolk in the diet increased the
pupation rate (Xie et al., 1997) and yield of adult of
Trichogramma spp. (Z. X. Wu, unpubl.). These results
prompted questions about the larval feeding process.
In light of evidence that entomophagous arthropods
generally require highly concentrated nutrients avail-
2
able only in solid food (Cohen, 1995, 1998), we were
further prompted to examine the feeding behavior of
Trichogramma spp.
Materials and methods
Insects. T. brassicae were obtained from Beneficial
Insectary (Oak Run, CA) and reared in Helicoverpa
zea (Boddie) eggs at the Biological Control and Mass
Rearing Research Unit, at 23 ± 2 ◦ C with a L14:D10
photoregime. Rearing methods were similar to those
described by Nordlund et al. (1997), for in vivo reared
insects. The H. zea eggs were irradiated at 25 K rad
(Cs-137 source) at 2–24 h of age to prevent further
embryonic development. Trichogramma larvae of a
specific age were obtained by allowing females to
oviposit on the host eggs for 1 h. Parasitoid devel-
opment and all feeding observations were at 25 ±
1 ◦ C.
Larval development. Larval growth was observed
from immediately after hatch to formation of the pre-
pupae. The structure of the digestive system, as it per-
tains to feeding behavior, was observed and recorded
with video recordings, micrographs, and drawings.
For determination of size and growth, 10 samples of
each larval stage were killed with boiling water and
measured with an ocular micrometer and with Image-
Pro Plus1 software (Media Cybernetics, Silver Spring,
MD).
Activity of the larval digestive system and feeding
observation. Observations were focused on the ac-
tive feeding phase. Observations were made using an
inverted stage microscope (Olympus IX 70, Tokyo,
Japan); a video recording system, which consisted
of an Olympus OLY-750 video camera and a Sony
SVO-9500MD videocassette recorder (Montvale, NJ);
a fluorescence microscope (Olympus BX-60, Tokyo,
Japan), and an image analysis system (Image-Pro
Plus). All feeding stages were observed for at least
8 h, and video recordings were made for at least
1 h of constant feeding activity. Feeding was investi-
gated by allowing larvae to feed on host egg contents.
Parasitized H. zea eggs were dissected in a drop of Ri-
naldini salt solution on a depression microscope slide
with fine tipped forceps. One or two drops of Ri-
naldini salt solution were added slowly to cover the
1 Mention of a commercial or proprietary product does not constitute
endorsement by the USDA.
larvae and egg contents, which helped retain the shape
of the host egg particles and facilitated observation
of the feeding behavior. This procedure allowed ob-
servations for more than 1 h without desiccation of
the material. For observations of extra-oral digestion
of proteins, we added fluorescently labeled BODIPY
casein (EnzCheck E-6639, Molecular Probes, Inc. Eu-
gene, OR). The labeled casein, which adhered to the
H. zea egg yolk granules, created particles that were
too large for ingestion (i.e., >2–5 µm). After larvae
had fed for ca. 5 min, the complex of yolk pro-
teins and labeled casein particles were examined using
fluorescence microscopy at 570 nm.
Results
Larval development. Larval development proceeded
through three stages: vermiform, pyriform, and sacci-
form (Clausen, 1940) (Table 1, Figure 1). No molting
was observed during larvae development. The first-
molt cuticle (embryo exuvium) remained attached
to the posterior extremity of the developing larva.
Changes in shape were related primarily to inges-
tion of large quantities of food. At ca. 28 h post-
oviposition, ingested particles had moved to the poste-
rior end of the midgut causing the larvae to take on a
pyriform shape. Ingested particles accumulated in the
anterior of the midgut during the 3–5 h after the onset
of the pyriform stage, causing the larvae to become
sacciform at ca. 34 h, post-oviposition. During the
sacciform stage, the midgut, which occupied most of
the body, was filled with food particles.
No cephalization or segmentation was observed.
However, the cephalic region was evident because
of the conspicuous feeding apparatus, which occu-
pied almost 13 of the body length at the vermiform
stage. The connection between the midgut and the
hindgut was occluded in the period 25–60 h post-
oviposition. After about 60 h, post-oviposition, the
larval bodies became whiter, indicating that the larvae
had digested the food and used the nutrients to proceed
with development. Some organs began to appear in
the cephalic region. After development to the prepu-
pal stage, several whitish, round bodies, presumably
urate (Flanders, 1937; Dahlan & Gordh, 1996), were
evident, scattered over the surface of the midgut at 65–
110 h post-oviposition, suggesting that metabolism
and excretion had occurred.
 3

Figure 1. Photomicrographs of vermiform (A), pyriform (B), and sacciform (C) larvae of Trichogramma brassicae. The dark material present
in the midgut indicates ingestion of solid food particles.

Figure 2. Generalized diagrammatic view of a Trichogramma brassicae larva, showing major internal and external features.
4
Table 1. Time table of the development of Trichogramma brassicae larval feeding at 25±1 ◦ C, indicating
the time of the beginning of each stage, and average size of each stage of development (±S.D. in µm)
Agea Developmental Stage
17.0–18.0 Stomodaeum and midgut formation
in the parasitoid egg
25.0–26.0 Early vermiform larvae
26.0–26.5 Mid- vermiform larvae
26.5–27.0 Late-vermiform larvae
28.0–31.0 Pyriform larvae
34.0–46.0 Sacciform larvae
66.0–68.0 Prepupae
a Hours post oviposition.
Structure of the feeding apparatus. In vermiform lar-
vae, the transparency of the body wall made it possible
to observe structures, the feeding course, and the mus-
cle arrangement in the larvae. The oral opening of
all larval stages was 10–12 µm in diameter and was
located slightly ventral to the anterior extremity of
the vermiform larvae (Figure 2). It appeared to be
a fixed opening with a fine, margin of integument.
Figure 2 shows diagrammatically the morphology of
the feeding apparatus. It summarizes observations of
several larva, showing the oral opening, the roof of
the stomodaeum, dorsal and ventral dilator muscles of
the stomodaeum, mandibles, salivary glands, salivary
ducts, midgut, and the empty hindgut. These structures
were described by Jarjees et al. (1998), except for our
use of the term ‘stomodaeum’ instead of ‘pharynx’.
When the oral opening was viewed, from the anterior,
a valve could be seen to open and close. The valve,
when closed, appeared to seal the anterior end of the
stomodaeum.
The stomodaeum was somewhat oval with 4 dorsal
dilator muscles and 1 ventral dilator muscle, which
appeared to stabilize and slightly depress the floor
of the stomodaeum (Figure 2). The lateral walls of
the stomodaeum were thin and transparent, while the
roof was thick and opaque. The roof formed a press
and bears the attachment of dorsal dilator muscles.
Depression of the roof of the stomodaeum occurs in
an anterior to posterior peristaltic wave (permitted by
the sequential relaxation and contraction of the dor-
sal dilator muscles). Sacciform larvae had the same
mouth structure as the vermiform and pyriform larvae,
though the position was shifted somewhat posteri-
orly of the anterior extremity on the ventral surface.
The mandibles were simple, slightly curved, unjointed
Length Width Volume
(µm) (µm) (µm3 × 105 )
145 ± 10.7 61.9 ± 9.9 2.9
149.5 ± 6.0 73.5 ± 2.1 4.3
284.5 ± 70.8 93.8 ± 9.6 13.2
388.8 ± 84.6 130.6 ± 22.5 34.5
410.2 ± 35.8 159.1 ± 45.9 54.9
479.2 ± 31.3 275.8 ± 31.3 191.5
– – –
structures (Figures 2 and 4). The mandibles remained
ca. 27 µm long, throughout development.
Food ingestion. Larvae started ingestion after hatch-
ing and fed slowly for the first 10 min after which they
began to actively ingest food. The stomodaeum dilated
and constricted 40–58 times per minute, and the larvae
fed continuously. Food could be seen being pumped
back and forth, in the stomodaeum, until the parti-
cles became small enough to pass through the cardiac
valve into the midgut (Figures 3 and 4). The larvae be-
came elongated at ca. 30 min posthatch, then fed more
rapidly as they entered the principal feeding stage. The
cells of the midgut and body wall became thin and
elongated as the gut became distended, until the larvae
reached the sacciform stage. During active feeding,
the larvae were very mobile, bending and stretching
their bodies. Larvae completed active feeding ca. 34 h
post-oviposition, during the sacciform stage. Food
particles retained their original shape within the cen-
ter of the midgut, indicating that little in gut digestion
had taken place up to this point. However, peristaltic
waves passing the full length of the gut continued to
mix the contents. At ca. 36 h post-oviposition, only the
stomodaeum was active with slow movements (10–30
per min) of the roof of the stomodaeum.
Two types of yolk solids were visible in the host
egg, under our observation conditions: smaller parti-
cles (spherical) with diameters of 1–10 µm and larger
particles, many of which were amorphous, exceeding
50 µm in diameter. Ingestion of smaller particles re-
quired less than 20 sec from the oral opening, through
the stomodaeum and cardiac valve into the midgut.
Ingestion of larger particles (Figure 3) required from
5–30 min. Once a particle came into contact with the

Figure 3. A photomicrograph of the cephalic region of a Trichogramma brassicae vermiform larva (ca. 27 h post-oviposition), with small food
particles visible in the stomodaeum.
oral opening, it was partially sucked in and then forced
out of the opening in a constant back and forth move-
ment. This process was repeated 2–5 times, until a
portion of the particle broke off and was retained in
the stomodaeum where it was forced back and forth,
again by peristalsis and anti-peristalsis. A strong neg-
ative pressure is generated in the stomodaeum, which
is 49.5 ± 0.5µm long and 27.5 ± 0.5µm wide, wider
than the oral opening (Figure 2). The negative pressure
in the stomodaeum, amplified by the difference in di-
ameters described, helped to break-up large particles.
Evidently, the peristaltic action generated a cavitation
force that was important in mechanical disruption of
particles, which were simultaneously being attacked
by the chemical action of proteolytic enzymes. After
larvae fed for ca. 5 min on the casein that was labeled
with quenched fluorescent material, bright red fluo-
rescence was observed (indicating un-quenching and
proteolysis) both outside the oral opening and within
the stomodaeum (Figure 5). This fluorescence is com-
pelling evidence of extra-oral digestion by T. brassicae
larvae. The particles in the stomodaeum were further
disintegrated by the roof of the stomodaeum exerting
a positive pressure against them. The cardiac valve is
narrower than the oral opening and provides a second
barrier, restricting the size of particles that enter the
midgut. Nearly all of the contents of the midgut are
solid particles that are up against one-another, leaving
very little room for aqueous liquid between them.
5
Discussion
To avoid controversy concerning the number of instars
in Trichogramma larvae (Clausen, 1940; Dahlan &
Gordh, 1996; Flanders, 1937; Hagen, 1964; Volkoff
et al., 1995), we used shape to describe the devel-
opmental stages. In fact, as only one instar was ob-
served, the shape is a more reasonable developmental
description than instar.
We found that the most active feeding occurred
during the vermiform larval stage, starting 25 h post-
oviposition. The rate of feeding decreased in the pyri-
form and sacciform stages. We described the feeding
apparatus of T. brassicae in general terms, because the
structures were so highly modified that it is difficult
to ascribe them to the categories listed by Snodgrass
(1935). For example, we found no evidence of a ten-
torium, maxillae, labium, or most other mouthparts
that are considered primitive and part of the standard
array of feeding structures. As Snodgrass (1935) dis-
cussed, larval hymenopterans, especially parasitoids,
are highly modified and simplified compared to their
free-living counterparts. Indeed, direct observation of
feeding clearly indicated the simple nature of the life
of a Trichogramma larva. They live in a milieu of
food, and either undertake simple ingestion of mate-
rials small enough to be ingested or mechanically and
chemically attack larger particles until these are small
enough to pass through the oral opening, stomodaeum,
and cardiac valve into the midgut.
6

Figure 4. Photomicrograph of the cephalic region of a Trichogramma brassicae vermiform larva (ca. 27 h post-oviposition) showing the cardiac
valve between the stomodaeum and the midgut. A mandible is clearly visible, near the oral opening.

Figure 5. Photomicrograph of Trichogramma brassicae larvae feeding on fluorescently labeled casein mixed with host egg material. The bright
halo around the larvae is a red fluorescence emanating from the partially hydrolyzed BIODIPY casein. The bright spot in the stomodaeum of
the lower larva indicates ingestion of the hydrolyzed casein. The field was illuminated with 570 nm light.

We found mandibles, an oral opening, and a sto-
modaeum with a valve. This anterior valve may be a
labrum, based on its position at the anterior of the sto-
modaeum, and the fact that it is closed from the dorsal
side. The stomodaeum was the most prominent and
important structure involved in the ingestion process.
The T. brassicae larvae studied here underwent an
impressive 40-fold increase in volume during the ca.
9 h feeding period. This remarkably fast increase in
size implies a very rapid and efficient nutrient inges-
tion mechanism. Considering the fact that the gut of
Trichogramma larvae is a blind sack, it is most likely
that these larvae must ingest strictly or predominantly
‘solid’ food, because it is impossible for necessary
nutrients to be sufficiently concentrated in liquids (Co-
hen, 1998). The contention of predominantly ‘solid’
feeding is further supported by continuous observation
of the feeding/swallowing process, where only ‘solid’

particles were observed to be ingested. We consider
the materials as "solids" because they have a definite
and consistent shape and a particulate appearance.
The most important discovery in this study is that
T. brassicae feed extensively on such solid materials.
Jarjees et al. (1998) described ingestion of particulate
materials in T. australicum ranging from 3.5–14 µm
in diameter. The findings that T. brassicae larvae
use ‘solid’ food is directly applicable to development
of artificial diets for Trichogramma spp. For exam-
ple, it now seems important that artificial diets for
Trichogramma spp. should contain high concentra-
tions of particles, forming a nutritional suspension.
Zhong (1992) reported that ‘dregs’ (apparently solid
particles) enhanced development of Trichogramma on
artificial diet. Chicken egg yolk is rich in proteins
and lipoproteins, which appear as spheres and gran-
ules. Their diameter ranged from 25 nm to 40 µm
(Juneja & Kim, 1997), smaller than H. zea egg yolk
particles. Our measurement for H. zea yolk particle
size ranged from 0.7 µm to 65 µm. These findings
may help explain the frequently observed phenom-
enon of abnormal pupae and adult development of
Trichogramma spp. fed fairly dilute liquid artificial
diet (Grenier et al., 1995; Nordlund et al., 1997). A
difficulty inherent in using high concentrations of nu-
trients in diets for rearing Trichogramma spp. larvae
is that of high osmolality or that the emulsifier re-
quired for the high concentration of lipids may be toxic
to larvae. However, our findings indicate that whole
lipoprotein complexes may be useful in providing the
concentrations of nutrients required while maintaining
the medium at physiological osmolality, without re-
course to emulsifiers. This could prove to be a fruitful
line of artificial diet research.
Jarjees et al. (1998) also described large secretory
cells near the mandibular bases, which they suggest
as being involved in extra-oral digestion. We observed
one common opening of the salivary glands ventral to
the oral opening (Figure 2). Its position is the same as
that described by Volkoff et al. (1995) and by Jarjees
et al. (1998). We have shown direct evidence of extra-
oral digestion with the demonstration of hydrolysis of
fluorescently labeled casein. It is not clear from the
present study or from Jarjees et al. (1998) whether the
source of digestive enzymes is the salivary gland or the
mandibular secretory glands.
The types of food that larvae can ingest depend ul-
timately on the structure of their mouthparts and their
feeding mechanisms (Smith, 1985). The feeding be-
havior of the Trichogramma larvae in hosts or artificial
7
diet and morphology and histology of their feeding
apparatus deserves further attention.
Acknowledgements
We thank Drs. C. Collison, P. D. Greany, M. R. Strand,
S. N. Thompson, and S. B. Vinson for reviewing an
initial draft of this manuscript. We also appreciate the
assistance of Ms. Ethel Griffin for maintaining the
Trichogramma colony, and Ms. Brenda Woods, for
providing H. zea eggs. Mr. Joe MacGown (Depart-
ment of Entomology and Plant Pathology, Mississippi
State University) prepared Figure 2. Approved for
publication as Journal Article No. J-9543 of the Mis-
sissippi Agricultural and Forestry Experiment Station,
Mississippi State University.
References
Clausen, C. P., 1940. Entomophagous insects. McGraw-Hill, New
York.
Cohen, A. C., 1995. Extra-oral digestion in predatory arthropods.
Annual Review of Entomology 40: 85–103.
Cohen, A. C., 1998. Solid-to-liquid feeding: the inside(s) story
of extra-oral digestion in predaceous Arthropoda. American
Entomologist 44: 103–116.
Dahlan, A. N. & G. Gordh, 1996. Development of Trichogramma
australicum Girault (Hymenoptera: Trichogrammatidae) on He-
licoverpa armigera (Hübner) eggs (Lepidoptera: Noctuidae).
Australian Journal of Entomology 35: 337–344.
Flanders, S. E., 1937. Notes on life history and anatomy of Tri-
chogramma. Annals of the Entomological Society of America
30: 304–308.
Grenier, S., H. Yang, J. Guillaud & L. Chapelle, 1995. Comparative
development and biochemical analyses of Trichogramma (Hy-
menoptera: Trichogrammatidae) grown in artificial media with
hemolymph or devoid of insect components. Comparative Bio-
chemistry and Physiology. Part B, Biochemistry & Molecular
Biology 111B: 83–90.
Hagen, K. S., 1964. Developmental stages of parasites. In: P.
Debach (ed.), Biological Control of Insect Pests and Weeds.
Chapman & Hall, London, pp. 168–246.
Jarjees, E., D. J. Merritt & G. Gordh, 1998. Anatomy of the
mouthparts and digestive tract during feeding in larvae of the par-
asitoid wasp Trichogramma australicum Girault (Hymenoptera:
Trichogrammatidae). International Journal of Insect Morphology
& Embryology 27: 103–110.
Juneja, L. R. & M. Kim, 1997. Egg yolk proteins. In: T. Yamamoto,
L. R. Juneja, H. Hatta & M. Kim (eds), Hen Eggs, Their Basic
and Applied Science. CRC Press, New York, pp. 57–71.
Nettles, W. C., Jr., 1990. In vitro rearing of parasitoids: role of
host factors in nutrition. Archives of Insect Biochemistry and
Physiology 13: 167–175.
Nordlund, D. N., Z. X. Wu, & S. M. Greenberg, 1997. In vitro
rearing of Trichogramma minutum Riley (Hymenoptera: Tri-
chogrammatidae) for ten generations, with quality assessment
comparisons of in vitro and in vivo reared adults. Biological
Control 9: 201–207.
8

Smith, J. J. B., 1985. Feeding mechanisms. In: G. A. Kerkut & L.I.
Gilbert (eds), Comprehensive Insect Physiology, Biochemistry
and Pharmacology, Vol. 4. Pergamon, Oxford, pp. 233–286.
Snodgrass, R. E., 1935. Principles of Insect Morphology. McGraw-
Hill Book Co. New York.
Volkoff, A., J. Daumal, P. Barry, M. Fancois, N. Harlitzky & M. M.
Ross, 1995. Development of Trichogramma cacoeciae Marchal
(Hymenoptea: Trichogrammatidae): Time table and evidence for
a single larva instar. International Journal of Insect Morphology
& Embryology 24: 459–466.
Wu, Z. X., J. Qin, T. X. Li, Z. P. Chang & T. M. Liu, 1982. Culturing
Trichogramma dendrolimi in vitro with artificial media devoid of
insect materials. Acta Entomologica Sinica 25: 128–135.
Xie, Z. N., Z. X. Wu, W. C. Nettles, Jr., G. Saldana & D. A. Nord-
lund, 1997. In vitro culture of Trichogramma spp. on artificial
diets containing yeast extract and uncentrifuged chicken egg yolk
but devoid of insect components. Biological Control 8: 107–110.
Zhong, L. 1992. The effect of food dregs in pupa tissue of Anther
pernyi on the development of Trichogramma and the treatment
of the pupa tissue. Abstracts XIX International Congress of
Entomology, 344 pp.