1. 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 ls
Introduction
Results Cont.
Methods
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 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 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, non-transmitting mosquitoes. Moreover, modeling (Read et al PLoS Biology) predicts that an insecticide that disproportionally kills older mosquitoes will decrease resistance selection because reproduction has already occurred in these mosquitoes.
Mosquito Blood Feeding Timeline
Day Day Day Day Day Day Day Day 15 b
(P =0.0544) b
Record daily mortality for 5 days
2 groups An. gambiae
IVM*
Blood Feed
2 DPE Group a
Record daily mortality for 5 days
2 groups An. gambiae
IVM*
Blood Feed
6 DPE Group
Blood Feed
Record daily mortality for 5 days
2 groups An. gambiae
IVM*
Blood Feed
14 DPE Group
Blood Feed
Blood Feed
Blood Feed a b
*10 mg/mL Ivermectin in DMSO stock solution diluted in PBS and added to defibrinated calf blood for a final concentration of 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.
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. a a a b
Purpose and Hypothesis
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. b b b b b
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.
Figure 2 – Absorbance values for enzymatic assay. Top: α-esterase. Middle: β-esterase. 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)
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.
Ivermectin Structure and Mechanism of Action
Conclusions/Future Directions
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.
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.
Results b c
Acknowledgements a
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 R01AI A1
References:
Sylla, M., Kobylinski, K. C., Gray, M., Chapman, P. L., Sarr, M. D., Rasgon, J. L., and Foy, B. D Mass drug administration of ivermectin in south-eastern Senegal reduces the survivorship of wild-caught, blood fed malaria vectors. Malaria Journal 9: e
Rajatileka, S., Burhani, J., Ranson, H Mosquito age and susceptibility to insecticides. Transactions of the Royal Society of Tropical Medicine Hygiene, doi: /j.trstmh
Lines, J. D., Nassor, N. S., 1991 DDT resistance in Anopheles gambiae declines with mosquito age. Medical and Veterinary Entomology. 5(1):
Read, A. F., Lynch, P. A., Thomas, M. B How to Make Evolution-Proof Insecticides for Malaraia Control. PLoS Biology. 7(4): 1-10
Sigma-Aldrich 2014 (
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.
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.