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The multiple effects of the wasp Cotesia
congregata, a parasitic manipulator, on the brain of
its host, the caterpillar Manduca sexta.
LEM McMillan
Dalhousie University
RH Herbison
Dalhousie University
DG Biron
Clermont Université
A Barkhouse
Dalhousie University
DM Miller
Dalhousie University
N Raun
Dalhousie University
SA Adamo
Dalhousie University
Article
Keywords: neuroinammation, polydnavirus, neural activity, parasitic manipulation, feeding,
neuroimmunology, cytoskeleton, extracellular matrix
Posted Date: July 31st, 2024
DOI: https://doi.org/10.21203/rs.3.rs-4680763/v1
License: This work is licensed under a Creative Commons Attribution 4.0 International License.
Read Full License
Additional Declarations: No competing interests reported.
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Abstract
The parasitic wasp,
Cotesia congregata
, manipulates the behaviour of its host, the caterpillar
Manduca
sexta
. The female wasp injects her eggs and a symbiotic virus (i.e. bracovirus, CcBV) into the body of its
host. The host’s behaviour remains unchanged until the wasps exit the caterpillar, and then the caterpillar
becomes a non-feeding bodyguard for the wasp cocoons. Using proteomic, transcriptomic and qPCR
studies, we discovered an increase in antimicrobial peptide gene expression and protein abundance in
the host central nervous system at the time of wasp emergence, correlating with the change in host
behaviour. These results support the hypothesis that the wasps hyperactivate an immune-neural
connection to help create the bodyguard behaviour. At the time of wasp emergence, there was also an
increase in bracoviral gene expression and proteins in the host brain, suggesting that the bracovirus may
also be involved in altering host behaviour. Other changes in gene expression and protein abundance
suggest that synaptic transmission is altered after wasp emergence, and this was supported by a
reduction in descending neural activity from the host’s brain. We discuss how a reduction in synaptic
transmission could produce bodyguard behaviour.
Introduction
Parasitic manipulators are parasites that enhance their tness by altering their hosts’ behaviour1. Our
understanding of how parasitic manipulators alter host behaviour has progressed rapidly in the last few
years2–4. Parasitic manipulators can exploit existing neuroregulatory networks in their host5–7. They can
remodel these networks by secreting chemicals, e.g. venoms8,9 or toxins3,10 and/or by inducing changes
in gene transcription within the host’s neurons and/or glia6,11. Immune-neural connections may be
especially prone to exploitation because parasites are pre-adapted to manipulate immune signaling to
survive within the host5. Parasitic manipulators typically impact multiple neuromodulatory systems
simultaneously8,12 across a range of neural networks1. Using this multi-targeted approach, parasitic
manipulators produce reliable changes in host behaviour13, although the details remain unclear.
The interactions between the parasitic wasp
Cotesia congregata
and its caterpillar host,
Manduca sexta
,
have been studied for decades14,15. However, the effect of the wasp on the host’s central nervous system
(CNS) is still poorly understood, despite the pronounced changes in host behaviour16. Given the
extensive background information available for this host-parasite system, as well as on the neurobiology
of the host17, a more detailed examination of the effects of wasp parasitism on the host’s CNS promises
to provide important insights into how the wasp exerts control over its host’s neural function.
Female
C. congregata
wasps co-inject venom, a polydnavirus, and wasp eggs into the body of
M.
sexta
14. The venom is not necessary for wasp development18 or altering host behaviour14. The
polydnavirus of
C. congregata
(
C. congregata
Bracovirus, CcBV) is a domesticated virus that has
become incorporated into the wasp’s genome19. This non-replicating virus is made in the wasp’s
ovaries20 and acts as a gene delivery agent, inserting its genes into the host’s genome19. CcBV attacks
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host tissues such as the fat body21, allowing the wasp to alter host physiology, optimizing the caterpillar
host for wasp larval development14. CcBV gene expression can also be found within the host CNS soon
after wasp oviposition22, and at least some of these genes are translated into CcBV proteins23. The
genes of other polydnaviruses (e.g.
Microplitis demolitor
bracovirus) also show expression in the brains
of their host (e.g.
Pseudoplusia includens
(Lepidoptera)24). However, whether polydnaviruses promote
parasitic manipulation of behaviour is unknown.
M. sexta
caterpillars show normal feeding and
locomotion behaviour during wasp larval development, despite CcBV activity and the numerous
physiological and endocrinological changes that occur within the host14. However, approximately 1 day
prior to the wasps’ exit from the host, host feeding and spontaneous locomotion decline dramatically,
never to recover16. Once the 50–80 wasp larvae scrape their way through the host’s body wall, the wasps
spin cocoons that remain attached to the cuticle of the caterpillar, and eclose as adult wasps 4 to 5 days
later14. The caterpillar loses all self-generated behaviours after the wasps emerge; however, it retains its
defensive behaviours16,25. The caterpillar becomes a non-feeding (i.e. anorexic) bodyguard for the
wasps, and defends the cocoons from arthropod predators26. This defense is crucial for wasp survival26,
as is preventing the host from eating the wasp cocoons by inducing axorexia16. The host eventually dies
of starvation several days after the wasps emerge from its body16.
Transforming an actively feeding caterpillar into an anorexic bodyguard requires changes in multiple
behaviours, while at the same time ensuring that the mechanisms needed for survival and defensive
behaviour remain functional. As expected, during the bodyguard phase the host’s sensory and motor
systems remain operational16,27–30. However, the ability of the host to initiate feeding and spontaneous
locomotion is greatly reduced30. One of the most parsimonious ways for the wasp to induce the
bodyguard phenotype is to exploit an existing host network that naturally produces a similar behavioural
phenotype. For example, reduced feeding, decreased locomotion and heightened defensive behaviours
are observed in
M. sexta
during an immune response, and these changes are thought to benet the
caterpillar16,31–33. The details of how sickness behaviours are activated and maintained in insects is
poorly understood, however immune-neural connections appear to be involved34. During an immune
response, insects release immunomodulators (e.g. octopamine35, cytokines34). These
immunomodulators can activate receptors in the brain36, and this activation is thought to induce
sickness behaviours such as illness-induced anorexia37, a phylogenetically conserved host behaviour38
that promotes recovery in
M. sexta39,40
. The wasp larvae activate a massive systemic immune response
as they scrape their way through the host’s bodywall32. Therefore, wasps could exploit an immune-neural
connection to create an anorexic bodyguard16,25. However, there is no direct evidence that systemic
immune activation in the body of the
M. sexta
caterpillar has an impact on the caterpillar brain, although
nematode infections can increase the expression of antimicrobial peptide (AMP) genes in the brain of
another lepidopteran,
Galleria mellonella41
. We use qPCR to test whether an immune response in the
body of
M. sexta
also produces an increase in immune gene expression in its brain. Such an activation
would help explain how the wasp larvae could alter the neural function of the caterpillar without
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physically contacting its CNS. We also perform proteomics and transcriptomics analyses of the
M. sexta
CNS to help determine whether neuroinammation (i.e. excessive immune activation within the brain42)
occurs in the host concomitant with the change in host behaviour. Neuroinammation is known to alter
synaptic transmission43. Neuroinammation is common in the hosts of parasitic manipulators and may
be critical for host manipulation in some systems44–47.
CcBV activity within the CNS could also activate neuroimmunological responses and potentially cause
neuroinammation. If viral genes and proteins increase in the brain at the time of wasp emergence from
the host, CcBV could also play a role in altering host behaviour. We examine whether there is a burst of
CcBV gene expression and/or protein abundance at the time of host behavioural change. We further
examine whether the expression of specic CcBV genes, and/or the presence of certain CcBV proteins,
within the caterpillar CNS correlates with the host’s change in behaviour. Such a temporal correlation
would suggest that specic CcBV genes and/or proteins play a role in mediating host behavioural
change48. CcBV gene expression remains measurable within the CNS for at least six days after wasp
emergence49, and, therefore, appears to persist for the duration of bodyguard behaviour.
Finally, changes in neural activity are required to produce changes in behaviour. Transcriptomics and
proteomics alone cannot demonstrate that neural activity has been altered. Nervous systems have
powerful homeostatic mechanisms to maintain neural circuit function despite perturbations in ion
channel performance or neurotransmitter abundance50,51. Therefore, we assessed neural activity
descending from the brain during different stages of parasitism, allowing us to correlate transcriptomic,
proteomic and electrophysiological changes within the CNS of the host with the expression of the
bodyguard phenotype. We predict that: 1) systemic immune activity results in increased immune activity
in the brain of
M. sexta
, 2) immune activity within the brain increases dramatically with the change in
host behaviour; 3) CcBV gene expression and protein abundance correlate with changes in host
behaviour, and 4) the change in host behaviour correlates with changes in descending neural activity
from the brain. Examination of the correlations across the different measures will provide insight into
how the wasps alter the host’s brain to create the bodyguard phenotype.
Results and Discussion
Overview
Changes in protein abundance (Table S1, Table S4) and gene expression (Fig. 1) within the CNS of
parasitized
M. sexta
correlated with the stage of wasp development. Using the brains of parasitized
caterpillars prior to the change in host behaviour (i.e. pre-emergent brains) as a baseline to remove the
effects of parasitism without behavioural change, we found over 200 genes changed in expression as
hosts adopted the bodyguard phenotype (Fig. 1), although the number of changes declined by three days
after the wasps had emerged from the caterpillar (i.e. 3-Days Post emergence, Fig. 1). Interestingly, most
of the changes in gene expression were an upregulation (Fig. 1), even though the host suffers a dramatic
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decline in its resting metabolic rate during the bodyguard phase (approximately 40%52). The change in
host behaviour also correlated with the change in abundance of almost 100 proteins within the CNS
(Table S4). However, it should be noted that given the total number of identied genes and proteins (Fig.
1, Table S1), these products represent less than 5% of
M. sexta
genes and proteins.
The burst in gene expression and change in protein abundance that occurs during wasp emergence
probably requires a trigger from the exiting larvae. This trigger could be the massive immune response
that occurs during wasp emergence (i.e. an immune-neural signal29,32), and/or the ecdysteroid pulse that
occurs 1 day prior to host emergence14, and/or secretions from the wasp larvae29 themselves. There are
receptors for immune signaling molecules33 and ecdysteroids53,54in the
M. sexta
brain.
Parasitism resulted in changes in the expression of genes and in the abundance of proteins related to
gene transcription, cytoskeleton architecture, intracellular transport, the extracellular matrix and immune
defense (Table S3, Table S4, Figs. S1 – S23). These changes began prior to the change in host behaviour.
However, there is a deepening in many of these changes as the caterpillar transitions into a bodyguard
(e.g. Tables 1, 2, 5, 6). We focus on the changes in three physiological networks that we believe are most
likely to be involved in creating the bodyguard phenotype.
1. Immune Activity in the Brain
Our results support our hypothesis that the bodyguard phenotype is produced, at least in part, by
activating immune-neural connections. We conrmed that a systemic immune challenge in unparasitized
M. sexta
induced an increase in gene expression for 2 different antimicrobial peptides (
gloverin and
attacin-1
) and a pattern recognition molecule (
hemolin
) in the brain (Fig. 2), correlating with the
expression of illness-induced anorexia32. This result demonstrates that immune activity in the brain
correlates with the expression of sickness behaviour. AMPs themselves may act as neural signals,
although the details remain unclear55,56.
The caterpillar exhibits a massive systemic immune response at wasp emergence29,32 and, as predicted,
parasitized caterpillar brains showed proteomic (Table 1), transcriptomic (Table 2) and qPCR (Fig. 2)
evidence of increased immune activity at this time, compared with parasitized caterpillars prior to wasp
emergence. Moreover, expression of CcBV genes (Table 4) and the abundance of CcBV proteins (Table
3) increased after the wasps emerge, and this increase could also initiate immune activity in the brain as
a host response. Although there was also an increase in immune activity in the brain before the wasps
emerge, compared with unparasitized controls (Fig. 2, Tables 1 and 2), caterpillar behaviour remained
normal29 during this smaller increase in immune activity within the CNS.
The large and long-lasting increase in immune activity that occurs in the brain of
M. sexta
after the
wasps emerge (Tables 1, 2) could be producing neuroinammation. In mammals, increased immune-
neural activity causes neuroinammation57, resulting in altered neural function58. Such hyperactivation
of immune-neural connections could help produce the bodyguard phenotype by heightening illness-
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induced anorexia and other sickness behaviours (e.g. lack of locomotion and enhanced defensive
behaviours)33. In
D. melanogaster
, increased AMP production in the brain59 and hyperactivation of
immune pathways60 also leads to neural damage. Neuroinammatory damage is key to creating a
bodyguard host in the
Dinocampus coccinellae-Coleomegilla maculata
wasp-ladybug system45. The
exiting wasp larva induces an increase in the production of the wasp’s symbiotic RNA virus within the
host’s brain45 Electron microscopy of the
C. maculata
brain suggest that the increase in viral production
damages the host brain, leading to a partially paralyzed, trembling host that sits on top of the wasp
cocoon45. This behaviour is sucient to repel arthropod predators, increasing wasp reproductive
success61. However, such uncoordinated behaviour would not produce the bodyguard phenotype
observed in
M. sexta
.
M. sexta
defends the cocoons using behaviours such as the defensive strike, that
requires a coordinated motor response62. Moreover, the polydnavirus in
C. congregata
(CcBV) is non-
replicating, and, therefore, cannot damage the brain in the same way as
D. coccinellae
’s RNA virus.
Further studies are needed to determine the extent (if any) of neuroinammatory damage and its
location within the
M. sexta
CNS.
2. Cytoskeleton and Extracellular Matrix (ECM)
Although not part of our original hypothesis, both the proteomic (Table 5) and transcriptomic analyses
(Table 6) showed strong changes in the protein abundance and gene expression of molecules related to
the cytoskeleton (e.g. actin, tropomyosin, troponin-1) and extracellular matrix (e.g. hemicentin, nidogen,
integrin beta 6, teneurin), including those thought to be directly involved in neural function (e.g. lamin
A63, tropomyosin64 and teneurin65). Changes began before the wasps emerged, when the caterpillar still
has normal behaviour. There was a pronounced upregulation of gene expression, and an increase in
protein abundance, of molecules that are key for intracellular transport (e.g. actin, paramyosin, troponin
1, Tables 5,6). However, at the time of wasp emergence, these proteins declined in abundance, compared
with their pre-emergence amounts. Once the wasps had emerged from the host, these proteins then
surged in abundance to levels higher than that observed prior to wasp emergence (Table 5).
Furthermore, after wasp emergence, additional proteins involved in axonal transport (e.g. Atlastin,
Dynactin subunit 6, Vesicle transport protein SEC20) changed in abundance, and additional genes
(
atlastin, dynein heavy chain 1
) changed in expression (Table 6). The increasingly large changes in genes
and proteins related to intracellular transport suggest that axonal transport is probably disrupted after
the wasps emerge. Disruption in axonal transport leads to a decline in synaptic transmission in
D.
melanogaster66
Also correlating with the change in host behaviour, were changes in gene expression
(
hemicentin-1
and
2
,
nidogen
and
integrin beta 6
) and protein abundance (Hemicentin-2-like and
Teneurin-A) of molecules that are important in the ECM (Tables 5 and 6). These changes are also likely
to depress synaptic transmission67.
Viruses commonly alter cellular architectural proteins, and, therefore, some change in cytoskeleton
proteins are expected in virally infected insect cells68. However, the CcBV virus appears to target the
cytoskeleton, at least in
M. sexta
hemocytes
69
. CcBV-induced changes in hemocyte cytoarchitecture
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contribute to the survival of the wasp larvae by reducing host hemocyte activity70. If the virus produces
similar changes in the microglia of the
M. sexta
brain, it could alter the functioning of these cells66. Such
changes could also impact immune-neural signaling.
3. Neural Function
Even before the wasps emerge from the host, both the transcriptomics and proteomics results showed
changes in the gene expression and protein abundance of molecules important for neuronal function
(e.g. potassium channels, Table 8). Although these changes were insucient to produce a change in
host behaviour, they could facilitate the production of the bodyguard phenotype, when combined with the
additional changes that occur after wasp emergence (Tables 7, 8). However, the dynamic nature of
neural networks51 makes interpreting these changes challenging. Moderate changes in neurotransmitter
synthesis, or the number of receptors, can be compensated for by the brain’s homeostatic
mechanisms50. Similarly, different ion channel subtypes can often compensate for each other51,71.
Additionally, synaptic mechanisms tend to keep ring rates within a set-point range, despite
perturbations72. Therefore, changes in proteomics and transcriptomics of the host brain could be a
direct effect of parasitism, but could also be a sign of the activation of host homeostatic mechanisms
(also see discussion in51,73).
CcBV
CcBV is known to rapidly enter host cells and is transcribed within an hour of wasp oviposition in fat
body and hemocytes21. We found that bracovirus gene expression occurs in every region of the CNS
within 24 hours (Fig S24). How CcBV enters the brain this quickly remains unknown, and whether it
enters all brain areas simultaneously is also unknown. Once in the cell, CcBV genes appear to make a
number of proteins that could interfere with intracellular signaling pathways74, and have been shown to
be immunosuppressive when expressed in immune tissues75. Unfortunately, nothing is known about
their effects in the CNS.
Some CcBV proteins could be found in all regions of the CNS at all time points (e.g. CcV1, Table 3),
however two proteins occurred in the brain only during and after wasp emergence (BV7-1 and
CcPL4.001) or in the ventral nerve cord at emergence (Table 3). The timing of the appearance of these
two proteins makes them good candidates for further studies on the possible involvement of CcBV
proteins in changing host behaviour.
Transcripts for CcBV genes could be detected at all late-stage parasitism timepoints measured (Pre-
emergent, Emergent, 1-Day Post, and 3-Days Post) (Table 4).
Likely Overall Effect of Neuroparasitic Changes
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The cytoskeleton and ECM results suggest that synaptic transmission is reduced in parasitized
caterpillars after the wasps emerge. This is consistent with previous research that showed that
immunohistochemical staining for multiple neuropeptides increases after wasp emergence, likely
caused by a build-up of product12. Similarly, at least one biogenic amine (octopamine) increases in
abundance in the brain and CNS after wasp emergence76 as would be expected if release was reduced.
We also found a decline in neural activity descending from the brain (both spontaneous and evoked
activity) in caterpillars after wasp emergence (n = 7) compared to controls (n = 8) or to parasitized
caterpillars prior to wasp emergence (Fig. 3, spontaneous: F(2, 16) = 6.8, p = 0.007. Tukey’s multiple
comparison test, Control vs Pre-emergent caterpillars (n = 4), p = 0.72; Control vs Post-emergent
caterpillars, p = 0.006; Evoked: F(2, 18) = 5.89, p = 0.01; Control (n = 8) vs Pre-Emergent caterpillars (n =
6), p = 0.91; Control vs Post-Emergent caterpillars (n = 8), p = 0.01, Pre-Emergent vs Post-emergent, p =
0.04). These results are consistent with a reduction in synaptic release in post-emergent caterpillars (i.e.
during the bodyguard phase). Further studies are required to determine whether neuroinammation,
bracoviral effects, and/or other factors are causally linked to this decline.
A decline in synaptic transmission throughout the CNS could produce the bodyguard phenotype. Neural
circuits mediating defensive behaviours tend to have few synaptic connections (e.g.
M. sexta77,78
) and
would probably be minimally affected by a small decline in synaptic transmission. However, motivated
behaviours, that rely on more complex multisynaptic circuits79, would be disproportionately reduced.
This differential impact on neural circuits could produce a caterpillar with robust defensive reexes, but
one that lacks motivated behaviours – i.e. a bodyguard.
Limitations
Proteomics and transcriptomics are complementary techniques. They typically do not show a high level
of correlation because of phenomena such as mRNA post-transcriptional processing80. Nevertheless, we
found considerable overlap in the proteomic and transcriptomic results for immune, cytoskeleton and
ECM molecules, giving us condence in these results (Table S2). However, our data likely underestimate
the number of molecular changes occurring during host behavioural change.
Changes in host behaviour can reect parasitic manipulation or host responses (e.g. see discussion
Bernardo and Singer81). The large number of changes that occur within the host during parasitism make
it dicult to determine which of our reported changes are directly caused by the parasite (e.g. via CcBV)
and whether any of these changes are directly responsible for producing the bodyguard phenotype.
Future studies (e.g. suppressing the production of CcBV proteins BV7-1 and CcPL4.001 at the time of
wasp emergence) are needed.
Conclusions
As predicted, we found that: 1) systemic immune activity increased immune activity in the brain of
M.
sexta
, and 2) immune activity within the brain increased dramatically with the change in host behaviour.
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These results support the hypothesis that enhanced immune activity in the caterpillar brain is playing a
role in the change in host behaviour. 3) Changes in CcBV gene expression and protein abundance
correlated with the appearance of the bodyguard phenotype. This correlation suggests that
polydnaviruses may play a role in parasitic manipulation of host behaviour. 4) Changes in genes and
proteins important for intracellular transport and the ECM suggested that synaptic transmission is
reduced in bodyguards. This hypothesis was supported by our nding of a reduction in descending
neural activity from the brain after wasp emergence.
Controlling the ECM and cytoskeleton may be important for parasitic manipulators. The ECM82 and
cytoskeleton83 contribute to neuronal homeostasis. Manipulating the ECM and neuronal cytoskeleton
may be a novel method of circumventing neuronal homeostasis, leading to long term changes of neural
circuits. Many manipulated hosts from different parasite-host systems exhibit altered cytoskeleton and
ECM dynamics (e.g. jewel wasp/cockroach9; trematode/crustacean84; gordian worm/crustacean85;
Hairworm/grasshopper86). The need to overcome homeostatic mechanisms will be especially important
for parasitic manipulators that require their hosts to survive for several days in the altered state. Multi-
targeted interventions appear to allow parasitic manipulators to achieve effective, predictable and long-
lasting control of the host’s brain.
Methods
Animals
All studies were performed on
Manduca sexta
larvae obtained from our in-house colony, and were
maintained as described previously.29
Cotesia congregata
were obtained from an in-house colony. Mated adults were given 3rd instar
M.sexta
(Proteomics, transcriptomics, late-stage qPCR, and electrophysiological studies) or 4th instar (early-
stage qPCR) in which to lay their eggs. Parasitized
M. sexta
were reared on lab-made high nutrition diet
(Frontier Agricultural Sciences, (#F9783B Neward, DE) until tissue extraction.
All studies were approved by the University Committee on Laboratory Animals (Dalhousie University, I-11-
025) and were in accordance with the Canadian Council on Animal Care.
Sample sizes followed minimum guidelines for proteomics and transcriptomics67.
A Benjamini-Hochberg correction was applied when multiple tests were performed on the same data
set87.
Tissue Extraction
Caterpillars were cooled to induce a chill coma88and dissected over ice.
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Methods for Proteomics, Transcriptomics, Immune and
Bracovirus qPCR
Final instar (5th )
M. sexta
caterpillars were sorted into ve groups based on stage of parasitism:
Unparasitized (Control), Pre-emergent (3 days prior to emergence of wasp larvae), Emergent (tissue
collected during the emergence of wasp larvae), 1 day post emergence (1 day after emergence of wasp
larvae), and 3 days post emergence (3 days after emergence of wasp larvae).
The supraesophageal ganglion or subesophageal ganglion or ventral nerve cord (thoracic ganglia +
abdominal ganglia) were extracted, washed in PBS and placed into an empty tube (for transcriptomics);
a tube containing protease inhibitor (for proteomics) (cOmplete, Mini, EDTA-free Protease Inhibitor
Cocktail, Roche, Switzerland); or a tube containing RNA
later
(qPCR) (Invitrogen, MA, USA) and ash
frozen in liquid nitrogen. All tissues were kept at -80°C until use.
Methods for Early-stage RT-qPCR
Fourth instar
M. sexta
caterpillars were sorted into two groups: Unparasitized (control), or 24 hours post-
parasitism (i.e. after wasp oviposition).
Tissues were collected as described above.
PROTEOMICS
Extraction and Quantication
Due to protein concentration required, 10 individuals per group were pooled to create 3 replicates per
group (i.e. CNS samples from 30 individuals in total). The tissues (supraesophageal ganglia,
subesophgeal ganglia, or ventral nerve cords) were suspended in 30 µl of extraction buffer containing
Urea 7M, Thiourea 2M, TrisHCl 40 mM, CHAPS 4%, DTT 1% and protease inhibitor. Samples were then
mechanically homogenized on ice using a micro pestle. Samples were further disrupted using a micro-
sonicator on maximum for 3 cycles of 10 seconds, followed by 20 seconds on ice. Following
homogenization, samples were centrifuged at 15 800 g at 4°C for 12 minutes, after which the
supernatant was collected for protein quantication.
A Bradford test for total protein was conducted on the samples followed by a 1D SDS PAGE on a 10%
pre-cast acrylamide gel (Biorad). Gels were stained overnight using Sypro ruby protein stain (Invitrogen,
Massachusetts, USA) before being visualized under UV light.
Protein Identication
Extracted protein samples were denatured and digested in a trypsin solution. The resulting peptide
solution was suspended in 10 µl of a 0.1% formic acid solution and injected into an HPLC nano debit
(RSLC U3000, Thermo Fisher Scientic) coupled with a nanoelectrospray mass spectrometer (Q Exactive
HF, Thermo Fisher Scientic). Peptides were separated on a C18 reverse-phase capillary column (0.075
Page 11/26
mm x 500 mm, Acclaim Pepmap 100, NanoViper, Thermo Fisher Scientic), using a gradient of 0.1%
formic acid:acetonitrile, 2–40%:98 − 60%, at a ow rate of 300 nL/min.
Resulting mass spectrographs were recorded using Xcalibur 4 software (Thermo Fisher Scientic).
These spectrographs were then analyzed using MaxQuant v1650 and Perseus v1.6.10.43 programs
using the script leading FPP v3.2. As a template for protein comparison, we created four protein
databases that encompassed known proteins from the family braconidae (NCBI txid 7402), the
polydnavirus genus bracoviriform (NCBI txid 2946836), the species
C. congregata
(NCBI txid51543) and
the species
M. sexta
(NCBI txid 7130). These datasets have been made publicly available89. A MaxQuant
database was also used to reduce proteins being identied due to contamination
(contaminants_fpp_180320.FASTA)90. Protein validation was conducted with a 1% false discovery rate
lter at both the peptide and the protein level.
Ratio Analysis
Normalization of protein intensity signals was required to analyze the data. Individual proteins had to
have been detected in all samples (all three replicates of parasitized caterpillars per group, and all three
replicates of control caterpillars) to be included in the analysis. Preliminary examination of the data
found that the raw intensity values were not normally distributed, therefore these values were log2
transformed prior to statistical analysis. Furthermore, the intensity values per replicate were centered
using the following procedure. For each sample, the median intensity value was established taking all
detectable protein intensities into account. This overall median was then subtracted from each individual
protein intensity value within that sample. This process was repeated for each of the three replicates.
Once the data was centered, a ratio was calculated for each protein intensity by subtracting the control
group intensities from the treatment group intensities for each protein. The median ratio of the three
replicates was then Z-Scored. These Z-scores were converted to p-values, and a Benjamini-Hochberg
procedure was used to determine statistical signicance87.
qPCR
RNA Extraction
RNA extraction was performed using a RNeasy lipid tissue mini kit (Qiagen, Hilden, Germany). All steps
adhered to the manufacturer’s instructions and included a DNase 1 treatment (RNase-free DNaset,
Qiagen) step to remove genomic DNA. The integrity of total RNA samples was assessed using a
Bioanalyzer (Agilent, California, USA). The purity of extracted RNA was determined using an EPOCH
spectrophotometer (BioTek, Vermont, USA) using the 260/280 ratio. Only samples with a 260/280
between 1.8 and 2.4 were used in accordance with the MIQE guidelines91. The concentration of total
RNA was determined using a Qubit Fluorometer (Q32857, Invitrogen, California, USA) using a HS RNA
quantication kit. cDNA was synthesized using iScript Reverse Transcription Supermix for RT-qPCR (Bio-
Rad, California, USA) and samples were stored in -80°C until use. Samples sizes are given in Table9.
Primers
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Primer eciency (E) and correlation coecient (R2) were estimated from a standard curve generated
with 10-fold dilutions of mixed cDNA samples (Table 10).
Primer specicity was checked by running endpoint PCR products for each primer on a 1.5% acrylamide
gel. The resulting bands were excited and sent out for Sanger sequencing (Genewiz, NJ, USA). The
resulting sequences were put through a BLAST search on NCBI to conrm fragment identity.
Reference gene selection
Six candidate reference genes were investigated to select two of the most stable reference genes in our
tissues of interest. These genes were used in previous studies in
M. sexta caterpillars: RpL17a93
(
Rewitz
et al., 2006),
actin96
,
glycerol-3-phosphate dehydrogenase (G3PDH)97
,
beta-FTZ-F198
,
ubiquitin92
, and
ribosomal protein S399
. We used NormFinder for R (http://moma.dk/normnder-software) to determine
the stability of pairs of reference genes (Andersen et al., 2004), using Cq values of ve biological
samples per treatment for each candidate reference gene.
RpL17a
and
ubiquitin
were found to be the
most stable across treatment groups and were used as reference genes in further qPCR analysis.
RT-qPCR
RT-qPCR was run using SsoAdvanced Universal SYBR Green Supermix (Bio-Rad) on a CFX96 real-time
system (Bio-rad) with the following parameters: 40 cycles of (95℃ for 10 seconds; 55℃ for 30 seconds;
60℃ for 45 seconds) followed by a nal extension of 60℃ for 10 minutes. After the qPCR a melt curve
analysis was run to assess the specicity of the qPCR product. For each biological sample, qPCR
reactions were performed in duplicate, and for each gene no-template controls were run. Cq values for
each sample and gene target were calculated in CFX Maestro (BioRad).
The qPCR data were analysed using the REST program100 (http://rest.genequantication.info).
TRANSCRIPTOMICS
Sample Group Information
For the transcriptomic analysis there were ve groups of interest: Unparasitized caterpillars, pre-
emergent caterpillars, emergent caterpillars, 1-day post emergence, and 3-days post emergence (see
Tissue Extraction for more details). There were 6 biological replicates per group.
RNA Extraction
RNA extraction was performed using a RNeasy lipid tissue mini kit (Qiagen, Hilden, Germany). All steps
adhered to the manufacturer’s instructions and included a DNase 1 treatment (RNase-free DNaset,
Qiagen) step to remove genomic DNA. The integrity of total RNA samples was assessed using a
Bioanalyzer (Agilent, California, USA). The purity of extracted RNA was determined using an EPOCH
spectrophotometer (BioTek, Vermont, USA) using the 260/280 ratio. Only samples with a 260/280
Page 13/26
between 1.8 and 2.4 were used in accordance with the MIQE guidelines91. The concentration of total
RNA was determined using a Qubit Fluorometer (Q32857, Invitrogen, California, USA) using a HS RNA
quantication kit. All RNA samples were stored at -80°C until library preparation.
Library Preparation and Transcriptome Sequencing
Library construction and sequencing were performed at Farncombe Metagenomics Facility (McMaster
University, ON, Canada). The RNA-Seq libraries were prepared using Illumina TruSeq RNA sample
preparation kit (Illumina, CA, USA). RNA-Seq libraries were sequenced on an Illumina NextSeq for 2 x 50
bp paired-end reads.
Trimming, Mapping of Sequences, and Differential Gene
Analysis
Raw sequencing reads were trimmed using Trimmomatic software101 (v0.39), removing low quality
leading and tailing bases (Q < 3) and reads below 30 bases long. Trimmed reads were aligned using
STAR (v.2.7.5a)102, to either the
M. sexta
genome103 (JHU_Msex_v1.0), the
C. congregata
genome104, or
the
Bracoviriform congregatae
genome105 (ViralMultiSegProj14556. Uniquely mapped reads with a
maximum of four mismatches were counted to genes using FeatureCounts from the Rsubread R
package106 (v2.4.2). First, reads were counted to features in the
M. sexta
genome, which resulted in an
average 32million counted reads per sample. Next, reads were counted to features in the
C. congregata
genome. For the
C. congregata
analysis, some
M. sexta
reads mapped to the
C. congregata
genome,
aligning to a handful of genes in controls (e.g. LOCUS4576). To exclude these reads,
C. congregata
was
aligned simultaneously with
M. sexta
and then separated during counting. We effectively found no
C.
congregata
reads in control samples (an average of 66 counted reads), and in parasitized samples there
was an average of 420,000 reads. Finally, we counted reads to features in the
Bracovirus
genomes,
where control samples had on average 0 counted reads, and parasitized samples had on average 9000
counted reads. Genes were pre-ltered before differential expression analysis by removing genes with <
10 counts across all samples. Differential expression analysis was performed using the R package
DESeq2 (v1.40.2)107, in 4 pairwise comparisons, either using the pre-emergent condition as the reference
level (Pre-emergent vs Emergent, Pre-Emergent vs 1-Day Post, and Pre-Emergent vs 3-Days Post), or the
control (Pre-emergent vs Control). Statistical signicance was determined using a Wald test and
corrected using a Benjamini–Hochberg correction for multiple comparisons87. Differentially expressed
genes were considered for further analysis with a cut-off of an FDR adjusted p-value < 0.05 and a fold
change in expression > 2.0108.
Validation of Gene Expression Proles using RT-qPCR of Select Immune Genes
RT-qPCR was used to validate the expression proles of select immune genes from RNA-seq results. For
more information, please see RT-qPCR section. An additional group was added “Immune Challenge”.
This group allowed us to test whether a systemic infection also increased gene expression for
antimicrobial peptides (AMPs) in the brain. This group was reared on high nutrition diet and were
Page 14/26
unparasitized. On day 1 of their fth instar this group was given a 40 μlinjection of heat-killed mix of
Serratia marcescens
(Gram-negative bacterium, Microkwik culture, Carolina Biological, 1/10 LD50),
Bacillus cereus
(Gram-positive bacterium, Microkwik culture, Carolina Biological, 1/10 LD50),
Beauveria
bassiana
(strain GHA, fungus, 1/10 LD50, BotaniGard 22WP; Laverlam, Butte, MT, USA) into the
hemocoel. Post injection, these caterpillars had their food removed for 24 hours to mimic the large
systemic immune response and lack of feeding observed in Post 1 caterpillars29,32. The
supraesophageal ganglia was collected 24 hours after the injection using the same methods listed
above.
The Effect of Parasitism on the Descending Neural Activity from the Supraesophageal Ganglion.
Caterpillars were cooled for approximately 10 min to induce chill coma and then the gut and a section of
the body wall (between the second and fth abdominal ganglia) was removed without disturbing the
ventral nerve cord. The physiological saline for
M. sexta
was modied from Miyzaki109 by Trimmer and
Weeks77 and contained 140 mM NaCl, 5 mM KCl, 4 mM CaCl2, 29 mM glucose and 5 mM HEPES
adjusted to pH 7.4 using NaOH (all chemicals were from Sigma-Aldrich (St Louis, MO). The posterior
and anterior ends of the caterpillar were pinned to an elastomer-covered dish. A suction electrode
(Bipolar Suction Electrode, A-M Systems, Carlsberg, WA) was connected to a differential amplier (A-M
Systems, Model 3000), and digitized using a PowerLab SP4 (ADInstruments, Colorado Springs, CO). The
output was collected and analyzed using Lab Chart (ver. 7.3.8, ADInstruments). The suction electrode
was used to make en passant recordings of the connective between the subesophageal ganglion and
the supraesophageal ganglion. Spontaneous activity was collected for 3 minutes and then the posterior
end of the animal was electrically stimulated by a Grass S9 stimulator. Low voltage was applied to the
dorsal nerve root of the sixth abdominal ganglion that was ipsilateral to the suction electrode. The
voltage was increased until evoked potentials were visible in the en passant recordings. The suction
electrode was then moved to the opposite supraesophageal – subesophageal connective. The
connective was cut near the subesophageal ganglion, and recordings of the descending activity in the
contralateral connective recorded for 3 min using the suction electrode. The stimulus to the dorsal root
of the contralateral sixth abdominal ganglion was re-applied as described above. If no evoked potentials
were observed, the voltage was increased until evoked potentials were visible. Recordings were made
from control caterpillars (5th instar day 2 to day 3, n=8), pre-emergent caterpillars (estimated as 3 days
prior to emergence, n=4-6, see Results and Discussion section); and post emergent caterpillars (1 to 3
days after wasp emergence, n=7)). The number of potentials at least two times above noise were
counted during spontaneous recordings, as well as after the stimulus artifact. Using this threshold
meant that only the largest spikes were counted (i.e. greater than 30 µV in amplitude and 2 ms in
duration). We used the response of control caterpillars to determine the time frame for counting the
evoked potentials (i.e. for 30 s after the stimulus artifact).
Declarations
Data availability
Page 15/26
Data are available in the supplementary le. Complete transcriptomic and proteomic datasets are
available for download at: McMillan, L., Herbison, R., Raun, N. & Adamo, S. Datasets for "The multiple
effects of the wasp Cotesia congregata, a parasitic manipulator, on the brain of its host, the caterpillar
Manduca sexta" https://doi.org/10.5683/SP3/FRJDPT, Borealis, V1
Ackowledgements
This research was funded by the Natural Sciences and Engineering Research Council of Canada (NSERC)
Discovery Grant to SAA and the France-Canada Research Fund program (SAA, DB and LEM). We thank
Christine Mader at McMaster University Genomics Facility for helpful advice regarding the RNAseq, and
Serge Urbach for help with the proteomics data analysis.
Author contribution statement
L.M. Study design, Data collection, Data curation, Analysis, Writing of manuscript; R.H. Analysis; Data
collection, Manuscript review; D.B. Conceptualization of the study, Study Design, Analysis; A.B. Data
collection, Manuscript review; D.M. Analysis, Manuscript review; NR: Study design, Analysis, Manuscript
Review; S.A.: Conceptualization of the study, Analysis, Interpretation of Results, Writing of manuscript
Competing interests
The authors declare no competing interests.
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Tables
Tables 1 to 10 are available in the Supplementary Files section.
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Figures
Figure 1
Overall change in gene expression in
M. sexta
caterpillars. (A) Gene expression levels of Pre-emergence
caterpillars compared to Unparasitized caterpillars. (B) Gene expression pattern of Emergent caterpillars
compared to Pre-emergent caterpillars. (C) Gene expression pattern in 1-Day Post-emergent caterpillars
compared to Pre-emergent caterpillars. (D) Gene expression pattern of 3-Days Post-emergent
caterpillars compared to Pre-emergent. In all graphs the number of downregulated genes is indicated in
blue on the top left of the graph, the number of upregulated genes is indicated by the red number in top
right of each graph, and the number of unchanged genes is indicated in black in the top center. A grey
horizontal dashed line indicates the signicance cut-off for the false discovery rate of 0.05. Two grey
vertical dashed lines indicate a 2-fold change, which was the chosen cut-off for signicance.
Page 24/26
Figure 2
Targeted qPCR for immune gene expression in the brains of unparasitized, Post-emergence parasitized,
and Immune challenged
M. sexta
compared to Pre-emergence parasitized caterpillars. (A) Relative
expression of
attacin-1
(B) Relative expression of
gloverin
(C) Relative expression of
hemolin.
All groups
compared to the pre-emergence group whose expression has been normalized to 1 (Indicated by dashed
line). * Denotes a signicant change p<0.001.
Page 25/26
Figure 3
Descending activity in the supraesophageal connective contralateral to the stimulus. After the wasps
emerge (Post, n=7) there is a decline in evoked neural activity compared with that of controls (Control,
n=8). A signicant decline was not observed prior to wasp emergence (PreEm, n=6). The horizontal lines
denote the median, and each circle represents an individual data point. The asterisk denotes statistically
signicant differences (p<0.05).
Supplementary Files
