Increased ladybird predation and metabolism do not counterbalance increased field aphid population growth under experimental warming
Data files
Feb 08, 2024 version files 282.27 KB
-
2024-01-30_Field_GutContent.csv
-
2024-01-30_Functional_response_lab.csv
-
2024-01-30_Gut_DNA_degradation.csv
-
2024-01-30_Population_dynamics.csv
-
2024-01-30_Standard_calibration_curves.csv
-
2024-02-01_Lab_predation_choice_expe.csv
-
README.md
Abstract
- Climate change may have diverse and complex impacts on species interactions, destabilizing food webs and ecosystem services. The effects of warming on the top-down biological control of crop pests have been considerably less studied than bottom-up effects through crop physiological changes.
- We studied the effect of a 2 °C warming in the laboratory and in wheat fields on the predation and metabolism of Harmonia axyridis on wheat aphids using molecular gut content analysis. We also measured the effects of warming on the predation rate and functional response of H. axyridis on each aphid species in the laboratory, as well as on DNA degradation rate.
- Field densities of Sitobion avenae and Rhopalosiphum padi, the two most abundant wheat aphid species, were increased by 2 and 2.5 times, respectively, under experimental warming, but densities of H. axyridis were not. Field predation rate of H. axyridis on these two aphids was found to be about 25% lower under elevated temperature. This could have been due to faster prey digestion, since degradation of the preferred aphid species, Sitobion avenae, was 1.5 times faster under elevated temperature. However, the functional response of H. axyridis larvae on these two species was 1.5 times higher under warming over the range of prey densities tested (50 to 250 over 24 h). The total predation rate of H. axyridis larvae on a mixture of S. avenae, R. padi and Schizaphis graminum aphid prey was also increased by 1.4 times, but consumption of R. padi aphids was increased while that of S. graminum was decreased under warming.
- Overall, our results show that global warming could strongly increase pest outbreaks and destabilize biological pest control, which would likely result in accrued yield losses.
README: Increased ladybird predation and metabolism do not counterbalance increased field aphid population growth under experimental warming
https://doi.org/10.5061/dryad.2fqz612x5
2024-01-30_Population_dynamics.csv
Population dynamics data (M&M section 2.2)
2024-01-30_Standard_calibration_curves.csv
Standard calibration curves data (M&M section 2.3, 2.4, 2.5)
2024-01-30_Field_GutContent.csv
DNA gut content of field specimens of ladybirds (M&M section 2.5)
2024-01-30_Functional_response_lab.csv
Functional response data (M&M section 2.6)
2024-02-01_Lab_predation_choice_expe.csv
Laboratory predation choice experiment data (M&M section 2.7)
2024-01-30_Gut_DNA_degradation.csv
Data on digestion of wheat aphid DNA in ladybirds gut in the laboratory (M&M section 2.7)
Description of the data and file structure
2024-01-30_Population_dynamics.csv
- Treatment: warming treatment (A=ambient 19°C; E=elevated 21°C)
- Plot: Plot ID
- Site: Site (Langfang versus Yuanyuan research station)
- Replicate: Replicate ID
- Date: Sampling date (yyyy-mm-dd)
- Year: Sampling year
- JDay: Julian day (January 1st = 1)
- Haxy: Number of H. axyridis per ten wheat tillers
- Save: Number of S. avenae per ten wheat tillers
- Rpad: Number of R. padi per ten wheat tillers
2024-01-30_Standard_calibration_curves.csv
- Treatment: Target species (RpCOI = R. padi; SgCOI = S. graminum; SaCOI = S. avenae)
- Gene_copy_num: Amplified gene copy number
- log10_gene_copy: log10 of the amplified gene copy number
- CT_value: CT value (from qPCR)
2024-01-30_Field_GutContent.csv
- Treatment: warming treatment (A=ambient 19°C; E=elevated 21°C)
- Species: Species (Rpad = R. padi; Save = S. avenae; Sgra = S. graminum)
- log10_gene_copy: log10 of the amplified gene copy number
2024-01-30_Functional_response_lab.csv
- Ha_stage: H. axyridis developmental stage (larva versus female adult)
- Treatment: warming treatment (A=ambient 19°C; E=elevated 21°C)
- Prey: Species (Rpad = R. padi; Save = S. avenae)
- samples: Sample ID
- Prey_density: Initial prey number
- Live_aphids_24h: Number of live aphid prey after 24h
- Dead_aphid_24h: Number of dead aphid prey after 24h
- Predation_24h: Number of aphids preyed upon after 24h
2024-02-01_Lab_predation_choice_expe.csv
- Treatment: warming treatment (A=ambient 19°C; E=elevated 21°C)
- ID: Sample ID
- Species: Species (Rpad = R. padi; Save = S. avenae; Sgra = S. graminum)
- InitPreyNum: Initial prey number
- Preyed24h: Number of aphids preyed upon after 24h
2024-01-30_Gut_DNA_degradation.csv
- Treatment: warming treatment (A=ambient 19°C; E=elevated 21°C)
- Species: Species (Rpad = R. padi; Save = S. avenae; Sgra = S. graminum)
- ID: Sample ID
- DNA_concentration(nG/µL): DNA concentration in ng per microliter
- CT1: CT value of first replicate
- CT2: CT value of second replicate
- CT3: CT value of third replicate (NA=not available)
- CTM: mean CT value (= mean(CT1;CT2;CT3))
- log_gene_copy: log10 of the amplified gene copy number
Methods
Experimental design
Field experiments were conducted in Langfang, Hebei Province (Langfang Research Station, Institute of Plant Protection, Chinese Academy of Agricultural Sciences; 39°30’N, 116°36’E) and in Yuanyang, Henan Province (Modern Agricultural Science and Technology, Henan Academy of Agricultural Sciences; 34°55’N, 114°15’E). The climate at both stations is continental warm temperate with a monsoon season from April to September and mean annual precipitations of 550 mm; the mean annual temperature is 12.3 °C in Langfang and 14.4 °C in Yuanyang (http://data.cma.cn/). In each station, one field (23 × 16m) was selected and grown with wheat Triticum aestivum L. variety Hengguan 35, which is adapted to dry conditions. In each field, twelve plots of 2 m × 2 m were selected and randomly assigned to control (ambient temperature; six plots) or to treatment (warming; six plots). Plots were separated by a buffer of at least 5 m of bare ground. MSR-2420 infrared heaters (Kalglo Electronics Inc., Bethlehem, Pennsylvania, USA; 165 cm × 15 cm) were placed above treatment plots, setting a radiation output of 2000 W and with a 4 m2 effective area (Han et al., 2019). Air temperature and relative humidity were monitored hourly 80 cm above ground using a data logger (JL-17; Hebei Qingsheng Electricity Company, Hebei, China) placed in the middle of each plot. Wheat was sown in mid-October 2014, 2015 and 2016, and harvested in early June 2015, 2016 and 2017. Two months after wheat planting, infrared heaters were set up until harvesting the wheat. Bare ground borders between plots were regularly manually weeded, and no pesticides were applied.
Field population dynamics of wheat aphids and of H. axyridis
To evaluate the influence of warming on insect population dynamics, aphids and H. axyridis numbers were recorded every seven days from the beginning of March to harvest each year in the twelve experimental plots. In each plot, a ‘Z-shaped’ sampling pattern was used, along which five sampling sites were selected. At each sampling site, ten wheat tillers were randomly selected and carefully inspected, and the number of aphids (adults and nymphs) and of H. axyridis were counted on those tillers.
H. axyridis specimen collection and DNA extraction
Twenty-seven and 33 larvae of H. axyridis were collected in 2018–2021 at the Yuanyang station in ambient versus elevated temperature plots, respectively, and placed individually in 50 mL centrifuge tubes sealed with gauze. Brought back in the laboratory, their guts were immediately dissected under a stereoscope, and placed individually in a DNA Preservation Solution (Phygene Biotech, Fuzhou, China) to protect DNA from degradation and contamination, then stored at -80 ℃ until DNA extraction.
Later, collected guts were placed individually in 1.5 mL microcentrifuge tubes and homogenized in 500 µl of DNA extraction buffer (100 mM NaCl, 50 mM Tris–HCl, 100 mM EDTA, 5% SDS, and 20 mg.mL-1 proteinase K, pH 8.0) using a TGrinder homogenizer (OSE-Y50, Tiangen Biotech, Beijing, China) at 11,000rpm.min-1 for 2 min. The mixture was agitated at 1,000 rpm.min-1 for 30 s using a BS14-Vortex 3000 (Wiggens, Straubenhardt, Baden-Württemberg, Germany) and incubated at 56 °C for 30 min. Then, 300 µl of phenol reagent for DNA extraction (Solarbio biotech, Beijing, China) and 300 µl of chloroform were added, mixed, and centrifuged at 14,000 rpm for 10 min. This last step was repeated another time to remove all impurities and obtain pure DNA. Once the supernatant was transferred into a new 1.5 mL centrifuge tube, 600 µl of chloroform were added, mixed, and centrifuged at 14,000 rpm for 10 min. The aqueous phase was mixed with 0.13 volumes of ammonium acetate (7.5 M) and two volumes of 100% ethanol, and placed for 20 min at -80 °C. The precipitated DNA was centrifuged, dried, resuspended in 25 µl of ultrapure water, quantified using a spectrophotometer (NanoDrop 2, Thermo Fisher Scientific, Massachusetts, USA), and stored at -40 °C. The same DNA extraction procedure was used for both the field and laboratory samples.
Primer design and specificity tests
To analyse H. axyridis gut content and quantify the relative consumption of the three main aphid species, a molecular analysis was conducted. Three pairs of primers specific to the cytochrome C Oxidase subunit I (COI) sequences of each aphid species were designed using the online Eurofins Genomics qPCR Assay Design tool (https://eurofinsgenomics.eu/en/ecom/tools/qpcr-assay-design/), and using GenBank reference sequences for each aphid species. The screening conditions were set as follows: primer length 15-30 bp, GC content 40-80%, Tm > 55 °C, qPCR product 150-400 bp. The specificity of each primer to its target species was verified by PCR amplification of the DNA extract of each species (S. avenae, S. graminum, R. padi and H. axyridis) using each pair of primer (from each aphid species) in separate PCRs. For each PCR, 1 µL of DNA extract was mixed into 12.5 μL of 2×F8 PCR MasterMix (Aidlab, Beijing), 0.5 µL COI-F (10µM), 0.5 µL COI-R (10µM), and ddH2O up to 25 µL. The PCR steps were as such: initial denaturation at 94℃ for 2 min; 30 cycles at 94℃ for 10 s, 57℃ for 10 s, 72℃ for 10 s; and final extension at 72℃ for 5 min. Each PCR was carried out in a DNA engine gradient thermal cycler (Bio-Rad, USA). Sterile water was also included as a negative control in the PCR, for which no CT value was detected after amplification. The COI amplified products were purified using the DNA gel extraction kit (Aidlab, Beijing, China), then inserted and cloned into pTOPO-T vectors (Aibosen biology, Beijing, China) and propagated into DH5α competent cells, resulting in the production of pTOPO- S. avenae, pTOPO- S. graminum and pTOPO- R. padi plasmids. The plasmids were then purified with a High purity plasmid extraction kit (Aidlab, Beijing, China), and run on 1% agarose gels to check fragment purity and amplification success. Positive clones were sequenced in both forward and reverse orientations to verify that the fragment sequences were correct, using the Dnaman (2004) software. The cloned plasmids were then used to generate the standard calibration curves. The three wheat aphid species – S. avenae, R. padi and S. graminum – together account for more than 99% of all aphid individuals found in the field. In addition, the remaining extremely rare species, including Metopolophium dirrhodum (Walker) belong to other aphid genus, and we found that the three primer pairs for each aphid species were very specific to their target species. Therefore, we did not test the specificity of these three primer pairs towards other, locally extremely rare aphid species.
Molecular gut content quantification from field individuals of H. axyridis
To quantify the relative consumption of H. axyridis on the three main aphid species based on molecular gut content (COI copy numbers), standard calibration curves were produced using the three recombinant plasmids pTOPO- R. padi, pTOPO- S. avenae and pTOPO- S. graminum. Each plasmid was first diluted in sterile Millipore water resulting in a stock preparation containing 1010 copies per μL, aliquoted and stored at -80 °C, respectively. From this stock, serial 10-fold dilutions containing from 109 to 100 copies of plasmids per μL were prepared for each plasmid. Then, 1 µL of each dilution was mixed into 10 μL of SYBR qPCR Mix (Aibosen, Beijing, China), 0.6 μL of each of the three primers, 7.8 μL ultra-pure H2O, and 0.01 μL ROX Reference Dye (Aidlab, Beijing, China). A negative control was also prepared by adding 1 µL of ultra-pure water instead of plasmid dilution. qPCR amplifications were then performed for each sample using an Applied Biosystems 7500 (ThermoFisher, Massachusetts, USA). The qPCR steps were as such: pre-denaturation at 95°C for 2min; 40 cycles at 95°C for 15 s, 57°C for 20 s, 72°C for 40 s. After each reaction, a melting curve analysis was performed to verify amplicon purity. Primers’ amplification efficiency was calculated based on standard curves’ slopes. The copy number of plasmids per µL in each sample was calculated as the DNA concentration (ng.µL-1) divided by the plasmid + insert DNA molecular weight (Da; calculated from the website tool https://www.novopro.cn/tools/dna_mw.html) × 6.023 × 1023. The number of COI copies of each aphid species in gut DNA extracts from H. axyridis individuals was quantified via qPCR using the same methodology, but using 1 µL of undiluted gut DNA extract (section 2.3).
Functional response of H. axyridis to wheat aphids under elevated temperature in the laboratory
To better understand the effects of increased temperature on H. axyridis predation in conditions of resource limitation, we assessed how experimental warming in the laboratory affected the functional response of H. axyridis to the two most abundant aphid species, R. padi and S. avenae. Wheat aphids were reared on wheat variety Hengguan 35 in separate cages in an insectary under 19 °C, 70 ± 5% RH, and a 16 L:8 D photoperiod, which are the optimal conditions for aphid rearing in the insectary (Park et al., 2017; Wang et al., 2019; Sun et al., 2022). Individuals of H. axyridis were provided by the Institute of Plant Protection, Beijing Academy of Agricultural and Forestry Sciences and reared in the laboratory on a M. japonica aphid diet on broad bean, under 25 °C, 70 ± 5% RH, and a 16 L:8 D photoperiod, which are the optimal conditions for H. axyridis rearing in the insectary (Wang et al., 2022). This alternative aphid species was selected to not affect the results of H. axyridis predation on wheat aphids. Fourth instar larvae and adult females of H. axyridis were used in the experiment after being starved for 24 h in individual Petri dishes (9 cm diameter × 1.8 cm height) to standardize their nutritional state. Third to fourth instar aphid nymphs of either R. padi or S. avenae were selected and deposited on wheat leaves at densities 50, 100, 150, 200, or 250 in hermetic Petri dishes (diameter 12.5 cm) with a hole for ventilation covered by a fine mesh to prevent insect escape. The wheat leaves were cut from roughly three-week-old plants at a length of about 10 cm, and moisturised with absorbent cotton. Five replicates were prepared for each aphid species, density, H. axyridis stage, and temperature treatments. The experiments were conducted in climate chambers at 19 °C (control, ambient temperature) and 21 °C (treatment; elevated temperature), 70 ± 5% RH, and a 16 L:8 D photoperiod. Starved predators were placed in the Petri dishes containing wheat leaves and aphids for 24 h, after which the number of remaining aphids was counted.
Laboratory predation choice test among the three aphid species under elevated temperature
The larvae of H. axyridis were reared as described above. All fourth-instar larvae were also starved for 24 h to standardize nutritional states in Petri dishes before trials. 50 3rd-4th instar nymphs of each wheat aphid species (total 150 aphids per replicate) were placed on 10 cm-long wheat leaves moisturised with absorbent cotton in a hermetic transparent plastic box (21 × 16 × 5.5 cm) at either 19 or 21 °C. After aphids had colonized the leaves (two hours later), one fourth-instar H. axyridis larva was introduced inside each box and allowed to prey upon aphids for 24 h under its respective temperature treatment (either 19 or 21 °C). The number of aphids of each species was counted after 24 h. Seven replicates (seven H. axyridis larvae) were prepared for each temperature treatment.
Digestion of wheat aphids by H. axyridis under elevated temperature in the laboratory
To measure the digestion and DNA degradation rate of the three aphid species in H. axyridis in the laboratory, two rearing conditions for H. axyridis were established: egg batches were placed in two climate chambers at 19 °C and 21 °C, respectively. After the eggs hatched, H. axyridis larvae were reared in their respective temperature environment until the fourth instar stage, on a mixed diet of R. padi, S. avenae, and S. graminum provided ad libitum in the approximative ratios 2:2:1 matching relative abundances measured in the field. 25 fourth instar H. axyridis larvae from each temperature treatment were used in the experiment, after being starved for 24 h in individual Petri dishes. A set of about 20 second-instar nymphs of each aphid species weighing precisely 4.0 mg in total (range 4.00–4.06 mg) was placed in a 2 mL centrifuge tube. A small hole was made in the tube cap for ventilation. A single fourth-instar H. axyridis larva was placed in the tube at room temperature (25 ± 1 °C). Each H. axyridis larva was allowed to feed on aphid nymphs for 40 min; this duration was enough for them to always consume all aphids. Each larva was then transferred into a clean Petri dish and placed in its original climate chamber – with room temperature of either 19 °C or 21 °C – during the digestion time. At 0, 2, 4, 6 and 8 h post-feeding, five H. axyridis larvae were collected from each climate chamber and immediately frozen individually in liquid nitrogen and stored at -80 °C until DNA extraction, which was performed as above.