Materials and Methods – part 02

24 martie 2009

Thyrinteina leucocerae and Glyptapanteles sp. were collected from guava (Psidium guajava) and Eucalyptus grandis
trees on the campus of the Federal University of Viosa, Minas Gerais,
Brazil (2045? S, 4251? W). The parasitoid species awaits further
taxonomic description, and voucher specimens are deposited with Prof.
A. Menezes Jr. at the University of Londrina, Brazil. Caterpillars were
reared either in groups on small eucalyptus or guava trees (3090 cm
high) in cages (7070 cm, 100 cm high) outside the laboratory, or
individually in plastic cups (500 ml) in the laboratory at ambient
temperature and light conditions. The cups contained small (510 cm)
twigs of eucalyptus or guava with some 17 leaves, and were closed with
a mesh. The twigs were inserted into moist vermiculite to maintain leaf
turgor. Fresh twigs were added twice per week. Moth pupae were
transferred to cages (as above) outside the laboratory, each containing
a small tree and filter paper moistened with a solution of honey in
water (10% v/v). Moths were allowed to emerge and adults mated and
oviposited inside the cages. Eggs were collected from the cages once a
week, and were left to emerge in cages containing small trees. The host
cultures were frequently supplemented with field-collected individuals.

Recently emerged
adult parasitoids, one female and 12 males, were incubated for 24
hours in a glass tube containing a piece of host plant leaf to allow
them to mate. They were subsequently placed in glass tubes (containing
agar and some honey, closed with foam rubber) and either kept in the
laboratory when caterpillars were available or stored in a climate box
(12C3, L12: D12 ) until there was a supply of caterpillars.
Subsequently, the adult parasitoids were incubated for 24 hours in a
plastic cup (500 ml) containing some leaves and up to 8 first-instar T. leucocerae
caterpillars of the same age. Parasitism is very rapid, occurring as a
female parasitoid apparently walks over a host caterpillar. Immediate
dissection of the caterpillar reveals up to 80 eggs inside (A. Janssen ,
pers. obs.). Parasitoid larvae egress from parasitized caterpillars
through exit holes they make in the host cuticle and pupate after 1116
days (A.H. Grosman, pers. obs). Parasitoid pupae were collected from
the cups and incubated in glass tubes in the laboratory until adult
emergence. As with the host, the parasitoid cultures were frequently
supplemented with field-collected individuals.

For all experiments,
we used caterpillars emerging from the same egg batches, which were
subdivided into groups: one group was exposed to parasitoids to obtain
parasitized caterpillars, whereas the other group was not exposed (i.e.
caterpillars remained unparasitized). Because each group had an equal
probability of containing hosts with aberrant behaviour, this minimized
the possibility that any behavioural changes observed were due to
parasitoids selecting hosts with atypical behaviour, rather than a
consequence of parasitism [4].

Effect of parasitism on host locomotion

First-instar hosts (parasitized and unparasitized) were placed individually on small E. grandis
trees (c. 50 cm high) in cages outside the laboratory. Caterpillars
were prevented from walking off the plant using a ring of insect glue
(Cola Entomolgica, Bio Controle, So Paulo, Brazil) applied to the
stem of the seedlings. Replicates in which the caterpillar disappeared
(<16%) were discarded. Upon egression, half of the twigs with
parasitoid pupae were cut off, while the caterpillar was left
undisturbed on the plant. The twigs with pupae were stapled to a leaf
close by an unparasitized caterpillar, resulting in four treatments:
parasitized and unparasitized caterpillars either with or without
parasitoid pupae close by. We marked the position of the caterpillars
by tying a thin thread on the plant just behind the abdominal prolegs,
taking care not to disturb the caterpillars. Each subsequent day, we
measured the distance moved by the caterpillar from the original thread
(by tying another thread just behind the abdominal prolegs).
Caterpillar locomotion was scored until either five days after
parasitoid egression or five days after the addition of parasitoid
pupae. Caterpillar size was measured in a similar way with another
piece of thread. Locomotion of unparasitized caterpillars without pupae
was scored until 5 days after the average caterpillar age at parasitoid
egression (23 days). Although no parasitoid larvae egressed from
unparasitized caterpillars, for brevity we refer to the movement of
parasitized and unparasitized host before and after egression in all
treatments. The distribution of movement data was non-normal due to
zero inflation, even after transformations; we therefore used the more
conservative non-parametric Kruskal-Wallis test [29] to compare locomotion among treatments before or after parasitoid egression. A Wilcoxon matched pairs test [29] was used to compare caterpillar locomotion before and after egression within treatments [29]
using R statistical software (R, version 2.3.1, 2006. R Development
Core Team 2006, R Foundation for Statistical Computing, Vienna,
Austria). Caterpillar body length was compared using a t-test.

Defensive behaviour in the laboratory

We used third-instar stinkbugs (Supputius cincticeps
(Stl), Heteroptera, Pentatomidae) to quantify the response of
parasitized and unparasitized hosts to predators. Predators of this
genus attack parasitoid pupae as well as T. leucocerae
caterpillars in the field (A.H. Grosman, pers. obs.). Predators were
obtained from a mass culture at the Federal University of Viosa fed
with Tenebrio molitor L. larvae and were individually incubated
for one day in Petri dishes (14 cm diameter) containing a source of
water (a moist piece of cotton wool) and some parasitoid pupae to
familiarize predators with pupae as food. Subsequently, they were
incubated for another day without parasitoid pupae to starve them, thus
increasing their tendency to search for prey.

Twigs with
unparasitized or parasitized caterpillars with their pupae were
inserted into a foam block, so that the twig was positioned vertically.
A starved predator was introduced gently at some 24 cm from the
caterpillar without disturbing the latter, and was allowed to search.
It was reintroduced if it left the twig before encountering the
caterpillar or pupae. Parasitized and unparasitized caterpillars were
tested in an alternate sequence, and each caterpillar and each predator
was tested once. Average observation time was 5.40.87 min
(means.e.m.) for parasitized caterpillars and 6.70.87 min for
unparasitized caterpillars. When the predator encountered the
caterpillar, we scored the number of head-swings the caterpillar
directed towards the predator, as well as the outcome of the
interaction (escape of the predator, predator knocked off by the
head-swings). The number of head-swings by parasitized and
unparasitized caterpillars were compared with a generalized linear
model with quasi-Poisson error distribution to correct for
overdispersion [30],
using R statistical software. The numbers of predators that gave up or
were chased away by the defending caterpillar were compared with a
Fisher’s exact test [29].

Effect of host on parasitoid pupa mortality in the field

Field
experiments were carried out from 1 July to 17 August 2005 in two guava
plantations on the campus of the Federal University of Viosa. The
vegetation covering the soil consisted mainly of grasses; the
plantations were surrounded by more diverse native vegetation. One of
the guava plantations was managed organically; the other plantation was
not managed.

We obtained
parasitized caterpillars as described above. All batches of parasitoid
pupae that emerged on the same day were placed in the same field within
one day of egression and pupation of the parasitoids. The guarding
caterpillar was removed from 43% of the batches. Each batch was
attached to a separate guava tree by stapling the twig (with or without
caterpillar, depending on the treatment) to a leaf, thus exposing it to
predators and parasitoids. The number of pupae in batches with and
without host did not differ significantly between treatments (with
host: 35.51.8, without host: 33.12.0, t-test, P = 0.37). A total of
118 batches of parasitoid pupae were exposed in the two guava
plantations.

To measure
mortality due to causes other than predation and hyperparasitism, we
covered branches, to which twigs with pupae and caterpillars were
attached, with a sleeve cage of fine mesh (below referred to as
unexposed batches). Insect glue applied to the base of each branch
prevented walking predators and parasitoids from accessing these
unexposed batches. Batches were recollected after three days (c. half
of the pupal period), pupae were counted, and the presence or absence
of the caterpillar recorded. Pupae were subsequently incubated for one
month (25C5, L12:D12) to allow emergence of parasitoids and
hyperparasitoids. The proportion of pupae per batch which were eaten by
predators or hyperparasitized was compared among treatments using GLM
with quasi-binomial error distributions to correct for overdispersion [30], using R statistical software.

Results

Effect of parasitism on host locomotion

Before
egression of the parasitoid larvae, parasitized and unparasitized
caterpillars did not differ in body length (parasitized: n = 17,
2.84014 cm (means.e.m.); unparasitized: n = 17, 3.000.08 cm, t-test:
t = 0.995, P = 0.33). All caterpillars moved, and although parasitized
caterpillars moved more than unparasitized caterpillars (7.30.50 and
5.60.45 cm/day respectively), there were no significant differences in
movement among treatments (Fig. 2, Kruskal Wallis test: KW = 7.12, d.f. = 3, P = 0.068).

Fifteen out
of 17 (88%) parasitized caterpillars stopped feeding and moving over
the plant within one day after the parasitoids had egressed (and
pupated), and all remained close to the parasitoid pupae, standing on
their two pairs of abdominal prolegs, often bent over the cluster of
pupae (Fig. 1).
The two parasitized caterpillars that moved following parasitoid
egression (one with pupae and one without pupae) covered a distance of
0.12 and 0.67 cm respectively. There was a highly significant
difference in distances travelled by caterpillars before and after
parasitoid egression (Wilcoxon Matched Pairs test: V = 153,
P<0.001). All parasitized caterpillars died soon after the adult
parasitoids emerged from the pupae, some 67 days after egression of
the larvae. This shows that the behavioural changes described here (and
below) do not benefit the parasitized host.
 
 
 

Figure 2. Effect of parasitism on host locomotion on the plant.

The
distance covered by parasitized and unparasitized caterpillars was
measured daily. Parasitoid pupae were either removed from parasitized
caterpillars (No pupae) or not (With pupae). Unparasitized caterpillars
were supplied with pupae (With pupae) or not (No pupae). Before
parasitoid egression (black bars: mean+s.e.m.), the difference in
displacement of parasitized and unparasitized caterpillars was not
significant (Kruskal-Wallis test, KW = 7.12, d.f. = 3, P = 0.068).
After egression (white bars: mean+s.e.m.), parasitized caterpillars
moved significantly less far than unparasitized caterpillars (KW =
24.0, d.f. = 3, P<0.001). The difference in displacement of
parasitized caterpillars before and after egression was significant
(**: Wilcoxon Matched Pairs test, P<0.01). Numbers of replicates are
given in brackets.

doi:10.1371/journal.pone.0002276.g002

 
10x to this great website

 part 03 soon to come up

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