Root exudate chemical cues of an invasive plant modulate oviposition behavior and survivorship of a malaria mosquito vector

Experiments using P. hysterophorus root exudate water samples

Insects for oviposition bioassay

Anopheles gambiae mosquitoes (Mbita strain) used in this study were from a colony established in 2018 and maintained at the International Centre of Insect Physiology and Ecology (icipe), Duduville Campus, Nairobi, Kenya. The adults were reared using standard insectary conditions at 28 ± 2 °C, and 72% relative humidity (RH) under a photoperiod of 12 h: 12 h (L:D). For egg development, the mosquitoes were offered human blood through arm feeding, one to two times weekly and had ad libitum access to a 6% glucose solution (wt:vol). The blood fed females were kept together with males for 2 days and used on the third day for the oviposition experiment. Eggs were laid in oviposition cups (7 cm top diameter, 4 cm base diameter and 7 cm depth) lined with filter paper (Whatman 90 mm, GE Healthcare UK Ltd, Buckinghamshire, London, UK) and transferred to larval rearing trays (39 × 28 × 4 cm depth). Upon hatching, the larvae were separated into groups of ~ 500 larvae per rearing tray and fed on commercial Tetramin^® fish food (Tetra, Germany). The rearing room was maintained at 32 ± 2 °C, and 52% RH during the day, and 24 ± 2 °C and 72% RH at night with a photoperiod of L:D 12:12 h¹⁵. The rearing water was changed after every 2 days. Pupae were transferred daily to emergence cups containing 15 mL water and placed in a new cage (15 × 15 × 15 cm). Emerged adults (1 day old) were maintained on 6% glucose solution as described above.

Collection of root exudate water samples from wild growing P. hysterophorus

Root exudate water samples were collected from wild growing P. hysterophorus (~ 30, 60 and 90 cm tall) on the icipe campus and used in various biological assays (i.e., oviposition response, mosquito growth and development assays). Briefly, wild growing P. hysterophorus were transplanted into a plastic pot (23 cm top diameter, 12 cm base diameter and 22 cm depth; batches of ten plants per pot in garden soil from the icipe campus) and watered with rainwater to obtain root exudate water. The root exudate water was collected from the plants after 1 week: to allow the plants to stabilize and to eliminate possible contamination of it with defense compounds released in response to uprooting. Water obtained from potted soil (same volume as used for the potted P. hysterophorus) without P. hysterophorus plants but from the same site, served as control water.

Oviposition response assay with root exudate water

Root exudate water was evaluated for its ability to influence oviposition response of gravid females in a dual-choice assay (Fig. 1A). The gravid females (n = 12 as described in Dieter et al.¹⁸; electronic supplementary material, methods) were presented a choice between the treatment and distilled water contained in similar polypropylene cups and the number of eggs laid counted using a microscope (Leica M127, Switzerland) after every 24 h for four consecutive days⁷. The cups were placed in opposite corners of the experimental cages and their positions interchanged every 24 h to avoid positional bias. Each cup was lined with a filter paper (Whatman 90 mm, GE Healthcare UK Ltd, Buckinghamshire, London, UK) and filled with 30 mL of the test solution (to keep the filter paper moist; as previously described in¹⁹) or an equivalent volume of distilled water as the control. A similar experiment was performed using water obtained from soil without P. hysterophorus plants (Fig. 1A). The bioassays were performed in triplicate as previously described in Ilahi et al.²⁰ and repeated once.

Headspace collection and analysis of root exudate volatiles

To collect headspace volatiles, 500 mL of the root exudate water were placed in air-tight glass jars and activated charcoal-filtered and humidified air passed over it. The volatiles were collected for 24 h on three pre-cleaned (dichloromethane (DCM) and nitrogen-dried) Super Q adsorbent filters (30 mg each, Analytical Research System, Gainesville, Florida, USA) at a flow rate of 170 mL/min. The three Super-Q filters (each treatment), were each eluted with 200 µL GC-grade DCM (Sigma Aldrich, St. Louis, Missouri, USA) into 2 mL clear glass vials, each containing 250 µL conical point glass inserts (Supelco, Bellefonte, PA, USA) and immediately analyzed by coupled gas chromatography/mass spectrometry (GC/MS). Also, natural water from depressions harboring growing P. hysterophorus in the field was sampled during the rainy season (described under parthenin detection) to allow comparison of its volatiles with that of the root exudate water. Volatiles collected from distilled water served as negative controls. Compounds were identified by comparison of their mass spectral data with library data: Adams2, Chemeco and NIST11 and confirmed with those of authentic samples. The relative peak area (%) of each constituent as generated by the NIST11 software, following GC/MS analysis was used to determine the natural ratio of the volatiles in the component blend. The volatiles were stored in glass vials at − 80 °C until used for oviposition assays.

Oviposition bioassay with identified volatiles

Dual choice bioassay was performed to determine the role of root exudate volatiles in egg laying behavior of An. gambiae. Of the volatiles identified, seven compounds: α-pinene, β-pinene, 3-carene, (E)-caryophyllene, camphor, α-phellandrene, β-phellandrene were selected and tested based on results obtained from Random Forest Analysis (RFA) (see electronic supplementary material, methods) and their commercial availability²¹. In dose–response assays, several tests were carried out: (i) a blend of the seven components (7-component blend) mimicking their naturally-occurring ratio in the root exudate volatile extract based on their GC/MS peak areas, (ii) single compounds, and (iii) a blend of the attractive components (5-component blend) guided by the positive attractive responses obtained from (ii). The compounds were dissolved in dimethyl sulfoxide (DMSO) and serially diluted from a stock solution to generate a concentration ranging from 0.25 to 4 µg/µL. Thereafter, for each concentration tested, 50 µL of each compound or blend solution were dispensed into the oviposition cup containing distilled (50 mL) water and monitored for egg laying. Each concentration was tested against a control (distilled water (50 mL) containing 50 µL of DMSO). The bioassays were performed in triplicate.

Mosquito growth and development assays

The root exudate water and the control water (water from soil) were used separately for mosquito rearing. First, mosquitoes were provided with root exudate water to lay eggs in a no-choice oviposition bioassay. Thereafter, a total of 200 eggs were counted and placed into each rearing tray (24 × 34 × 4 cm) and the number of hatched eggs was determined by counting the first instars larvae that emerged in each tray. The larvae were fed daily on a standardized regimen of ground fish food (Tetramin, Tetra, Germany) and water was changed every other day. Daily survival of larvae (from first instar (L1) to pupation) was recorded, and pupae transferred into cups in experimental cages (30 × 30 × 30 cm) until adult emergence. The conditions of the bioassay room were the same as that of the rearing room described above. All experiments were performed in four replicates.

Experiments using parthenin

Detection of parthenin in root exudate water

One liter (1 L) of water was collected from flooded depressions/open puddles in which wild P. hysterophorus was growing on the icipe campus and pooled (the field collection was repeated twice, 1 week apart). The habitat had other non P. hysterophorus plants such as grasses. The water was filtered using a muslin cloth and stored at − 80 °C overnight and then freeze-dried (VirTis SP scientific, Model Advantage EL-85) for 72 h to obtain 38 mg of root exudate. This was extracted with dichloromethane (DCM) and analyzed by GC/MS as described under chemical analysis. A similar analysis was carried out on the root exudate water (four replicates) obtained from potted plants described above.

Preparation of parthenin stock solution

A sample of parthenin obtained from a methanolic extract of P. hysterophorus from a previous study conducted in our laboratory²², was used as a standard. A sample of this isolate (300 mg) was dissolved in 0.3 mL DMSO and then diluted to 300 mL with distilled water to obtain a stock solution of 1000 ppm.

Oviposition response assay with parthenin

Parthenin was evaluated at a concentration of 0.13 µg/µL corresponding to the estimated amount of the root exudate tested (described above). The amount of parthenin in the root exudate was estimated by comparing the relative peak area (%) of parthenin in the root exudate to that recorded for the standard parthenin of known concentration. The oviposition assays were performed as described for root exudate. However, the distilled water which served as a negative control was prepared in 0.1% DMSO. A similar experiment was performed using parthenin water spiked with 50 µL of headspace volatiles collected for 24 h from the plant root exudate. All experiments were performed in triplicates.

Mosquito growth and development assays using parthenin

Parthenin prepared as described for the oviposition bioassay was used for mosquito rearing. Mosquitoes reared on distilled water prepared in 0.1% DMSO served as the control group. The experiment was performed as described for root exudate water.

To determine the effect of exposure to parthenin on mosquito adult survival, the female mosquitoes that emerged from parthenin treated water and controls were monitored separately in different cages. The mosquitoes were maintained on P. hysterophorus potted plant until their natural death (survival). The conditions in the bioassay rooms were the same as those of the rearing room described above.

Chemical analysis

Gas chromatography/mass spectrometry (GC/MS)

To detect parthenin in the root exudate, the sample was freeze-dried and prepared at a concentration of 600 ng/µL in dichloromethane (DCM) and dried over anhydrous Na₂SO₄ (Sigma Aldrich, St Louis, MO USA). The standard parthenin sample was prepared at a concentration of 300 ng/µL in DCM. For GC/MS analysis, 1 µL of each sample (parthenin and root exudate) was analyzed on a 7890B gas chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA) linked to a 5977A mass selective detector under the following conditions: Inlet temperature 270 °C, transfer line temperature 280 °C, and column oven temperature programmed from 35 to 285 °C, with the initial temperature maintained for 5 min then 10 °C/min to 280 °C for 5.5 min and finally 5 °C/min to 285 °C for 34.9 min. The GC was fitted with a HP-5 MS low bleed capillary column (30 m × 0.25 mm i.d., 0.25 µm) (J&W, Folsom, CA USA). Helium at a flow rate of 1.2 mL/min served as the carrier gas. The mass selective detector was maintained at ion source temperature of 230 °C and a quadrupole temperature of 180 °C. Electron impact (EI) mass spectra were obtained at the acceleration energy of 70 eV. Compounds were injected in the splitless mode using an auto-sampler 7693 (Agilent Technologies, Inc., Beijing, China). Fragment ions were analyzed over 38–550 m/z mass range in the full scan mode. The filament delay time was set at 3.0 min. Parthenin was identified based on its general fragmentation pattern and compared also with previously published results²³. The root exudate samples were analyzed in triplicate, with each replicate collected from a different batch of plants.

Statistical analysis

The oviposition activity index (OAI) for dual choice oviposition assay data was calculated according to the formula described in¹⁹;

where Nt is number of eggs laid in the treatment and Nc the number of eggs laid in the control. The OAI ranges from − 1 to + 1, with 0 indicating neutral response, positive value indicating an attraction towards the treatment and a negative value indicating the converse. The oviposition data was analyzed by generalized linear model using negative binomial. The model validity was assessed by inspection of residuals²⁴: Number of eggs deposited served as the response variable while the treatments were used as the predictor variable.

Hatch rate of mosquito eggs exposed to different treatments was calculated as follows:

The % hatchability data was also analyzed using generalized linear model with negative binomial error structure. The variation in survival was analyzed by the Kaplan-Meir method and statistical significance comparisons made using log-ranks test. All statistical analyses were performed using SPSS 23.0 software (IBM SPSS Statistics) and results considered significance at p < 0.05.

Ethics statement

Ethical approval for mosquito blood feeding was obtained from Kenya Medical Research Institute (KEMRI) Scientific and Ethics Review Unit (SERU) (Approval number 593). Plant ethics Additional approval to obtain samples from the plant was received from National Commission for Science, Technology and Innovation (permit number: NACOSTI /P/20/4177). NACOSTI (http://www.nacosti.go.ke) has a mandate to accredit research institutions and approve all scientific research in Kenya. The current study complied with NACOSTI guidelines (National guidelines).

Source: Ecology - nature.com