Transcript The Effect of Senescence on Ivermectin

The Effect of Senescence on Ivermectin Susceptibility in Malaria Transmitting Mosquitoes
Jonathan A. Seaman, Haoues Alout, Timothy A. Burton, Wojtek S. Kuklinski, Jacob I. Meyers,
Karla Saavedra-Rodriguez, Brian D. Foy
Arthropod-borne and Infectious Diseases Laboratory, Department of Microbiology, Immunology and Pathology
Colorado State University, Fort Collins, CO
Introduction
Results Cont.
Methods
Mosquito Blood Feeding Timeline
Day 0
In sub-Saharan Africa, malaria has caused disease and death for hundreds of thousands of
years. Plasmodium, the pathogen responsible for this disease, is transmitted between humans
by the bites of infected Anopheles spp. mosquitoes. Vector control is the most effective tool for
reducing malaria transmission, but current methods have various shortcomings including
insecticide resistance and inability to target exophagic vectors, creating the need for novel
ways to target disease vectors. Our laboratory is investigating the compound ivermectin, a
semi-synthetic 16-membered macrocyclic lactone derived endectocide, as a novel malaria
transmission control tool. It has been previously shown that this drug, when administered to
people by mass drug administration (MDA) for the treatment of onchocerciasis and lymphatic
filariasis in Africa, is lethal to wild Anopheles gambiae that bite villagers (Sylla et al. 2010 MJ).
Additionally, multiple studies have shown a relationship between insecticide susceptibility and
mosquito age, with the general trend being higher susceptibility in older mosquitoes (Rajatileka
et al. 2010 TRSTMH, Lines 1991 MVE). Due to the approximately 2-week long Extrinisic
Incubation Period (EIP), referring to the incubation period within the mosquito vector rather
than in the final human host, older mosquitoes are a more favorable target due to their
increased likelihood to be active transmitters of infectious parasites. This suggests that vector
control will shift the age-structure of the mosquito population towards younger, nontransmitting mosquitoes. Moreover, modeling (Read et al. 2009 PLoS Biology) predicts that an
insecticide that disproportionally kills older mosquitoes will decrease resistance selection
because reproduction has already occurred in these mosquitoes.
Purpose and Hypothesis
• To investigate the possible relationship between ivermectin susceptibility and mosquito
senescence in the malaria vector Anopheles gambiae in an effort to discover how
ivermectin by MDA may alter mosquito population structure in sub-Saharan Africa.
• We hypothesize that older females would be more susceptible than younger ones and in
this way decrease the chance of resistance development while specifically targeting
those mosquitoes more likely to transmit Plasmodium.
2 groups
2 DPE
An.
Group gambiae
6 DPE 2 groups
An.
Group
gambiae
2 groups
14 DPE
An.
Group gambiae
Day 2
IVM*
Blood
Feed
Blood
Feed
Blood
Feed
Day 3
Day 6
Day 7
Day 10
Day 14
Day 15
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(P =0.0544)
Record
daily
mortality
for 5 days
b
a
IVM*
Blood
Feed
Record
daily
mortality
for 5 days
Blood
Feed
Blood
Feed
IVM*
Blood
Feed
Record
daily
mortality
for 5 days
a
b
*10 mg/mL Ivermectin in DMSO stock solution diluted in PBS and added to defibrinated calf blood for a final concentration of
11.75 ng/mL. Control blood contained same dilution starting with 100% DMSO. DPE = Days Post Emergence
b
Mortality Statistics
•Due to the shape of the survivorship curve, it was determined that the IVM associated
mortality is maximal by day 3 post-IVM blood feed.
ls
•Mortality values compared using a 2-Way ANOVA with Tukey’s post-hoc multiple
comparisons test (graph not shown).
•Mantel-Haenszel Hazard Ratios were computed from the survivorship curves.
•The 95% confidence intervals of the Mantel-Haenszel Hazard Ratios were compared
between age groups for overlap.
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a
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b
Enzymatic Activity Assay
•Pools of 5 mosquitoes from each age group and blood feed status (Non-blood fed, Ctrl BF,
and IVM BF) homogenized in PBS.
•Activity of α-esterase, β-esterase, PNPA-esterase, Mixed Function Oxidases (MFO), and
Glutathione-S-transferase were quantitated spectrophotometrically according to the
“Quantification Methodology for Enzyme Activity Related to Insecticide Resistance in Aedes
aegypti” by the Brazil Ministry of Health, 2006.
•Absorbance values compared using a One-Way ANOVA with Tukey’s post-hoc multiple
comparisons test.
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b
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Figure 2 – Absorbance values for enzymatic assay. Top: α-esterase. Middle: βesterase. Bottom: Glutathione-S-transferase. PNPA-esterase and MFO not shown
due to no significant differences that correlate with survivorship. Values compared
using a One-Way ANOVA and Tukey’s post-hoc multiple comparisons test (α =
0.05). Lower case letters indicate significance.
Next-Generation Sequencing – Whole Transcriptome Analysis (data analysis in process)
Ivermectin Structure and Mechanism of Action
Ivermectin acts as an agonist of glutamate-gated chloride channels (GluCl) found in the
nervous system of mosquitoes. By binding to the transmembrane portion of the channel,
ivermectin causes artificial channel opening and subsequent influx of chloride ions, leading
to flaccid muscle paralysis. Due to the channel’s location throughout the nervous system, this
causes instability in flight, inability to feed, and eventual mosquito death.
Four different isoforms of the AgGluCl channel are present in An. gambiae and differential
sensitivity to ivermectin among the isoforms seems to exist. Ongoing studies in our
laboratory aim to investigate the isoform sensitivities as well as expression changes with
mosquito age, gender, and physiological state.
•Total RNA extracted from 10 individual females from the 2DPE and 6DPE age groups and
each blood feed state
•12 libraries prepared (each age group and feeding state in duplicate) using Illumina’s TruSeq
Stranded mRNA Sample Prep Kit.
•Sequenced on a HiSeq 2500 platform with 338 million 2x100bp paired end reads.
Results
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Conclusions/Future Directions
Survivorship data shows a relationship between ivermectin susceptibility and An. gambiae
senescence with 6DPE mosquitoes being more susceptible than both 2DPE and 14 DPE
mosquitoes. Hazard ratio comparisons show that 2DPE mosquitoes have the highest odds of
survival followed by 14DPE and with 6DPE having the lowest odds of survival. These findings
suggest that ivermectin may exhibit selective targeting of older, malaria transmitting
mosquitoes.
Enzymatic analyses indicate GST may be involved in ivermectin detoxification, because GST
levels across age is negatively correlated to age-group survival from ivermecin. However,
additional studies need to be conducted to validate if GST activity can reduce ivermectin’s
toxic effect in vitro and in vivo. Alpha esterases may be reduced in 6DPE mosquitoes after
IVM injestion, leading to increased susceptibility. Beta esterases, PNPA esterase, and MFO do
not appear to be related to ivermectin susceptibility in An. gambiae.
Next-generation whole transcriptome sequencing may reveal further mechanistic explanations
to the observed mortality differences. Sequencing has been completed and data analysis is in
progress but not yet completed.
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Acknowledgements
Benjamin Krajacich, Meg Gray, Dr. William Black IV, Dr. Doug Brackney, Dr. Corey Rosenberg,
and Abhishek Prasad for assistance with data analysis, Next-Generation Sequencing library
preparation, support and advice. Funding: NIH R01AI094349-01A1
Sigma-Aldrich 2014 (http://www.sigmaaldrich.com/catalog/product/sigma/i8898?lang=en&region=US)
References:
Figure 1 – Left: Ivermectin molecular structure. Right: Modeled crystal structure of the An.
gambiae glutamate-gated chloride (AgGluCl) channel, depicting glutamate and ivermectin
binding sites. The highlighted beta-sheet is where splice variants in channel isoforms occur.
Figure 1 – Left: Survivorship curves of ivermectin-fed An. gambiae in three different
age groups. Right: Hazard ratio of survivorship curves and 95% CI. Lower case
letters indicate significant differences as determined by no overlap of 95%
confidence intervals.
Sylla, M., Kobylinski, K. C., Gray, M., Chapman, P. L., Sarr, M. D., Rasgon, J. L., and Foy, B. D. 2010. Mass drug administration of ivermectin in
south-eastern Senegal reduces the survivorship of wild-caught, blood fed malaria vectors. Malaria Journal 9: e365.204
Rajatileka, S., Burhani, J., Ranson, H. 2011 Mosquito age and susceptibility to insecticides. Transactions of the Royal Society of Tropical Medicine
Hygiene, doi:10.1016/j.trstmh.2011.01.009
Lines, J. D., Nassor, N. S., 1991 DDT resistance in Anopheles gambiae declines with mosquito age. Medical and Veterinary Entomology. 5(1): 261265
Read, A. F., Lynch, P. A., Thomas, M. B. 2009 How to Make Evolution-Proof Insecticides for Malaraia Control. PLoS Biology. 7(4): 1-10