Energy allocation and fitness cost of cotton aphid, Aphis gossypii Glover (Hem: Aphididae) in counter of neonicotinoids Shadieh Gerami (Corresponding author) Dept. of Plant Pathology, College of Abouraihan, University of Tehran, P.O.Box 33955-159, Pakdasht, Iran; E-mail:
[email protected] The research is financed by university of Tehran Abstract Three populations of Aphis gossypii of distinct susceptibility to neonicotinoids show differences in the accumulation and mobilization of energy reserves, what may allow the production of their defensive tools against two neonicotinoid insecticides without impairing their reproductive performance. Bioassays were also carried out for these aphid populations. According to the results obtained, the Ag-R population showed resistant rate significantly higher than the other two populations. Levels of some biochemical characteristics were determined in the resistant, susceptible and semi susceptible populations of cotton aphid. The results obtained in the assays with biochemical parameters indicated significant differences in activity among the populations, with higher activity in using glycogen in the Ag-R population. The inverse activity trends of T (Generation time) and DT (Doubling time) in both resistant and susceptible populations, one showing fitness disadvantage without insecticide exposure and the other not showing it, may underlay the mitigation of insecticide resistance physiological costs observed in the Ag-R population. Key words: Neonicotinoid resistance, fitness cost, cotton aphid, energy metabolism, biochemical characteristics 1. Introduction Aphids are major polyphagous crop pests and induce very high loss in crop yields notably. They have a very wild feeding host plants. The capability to use a so large range of food sources has to be closely linked to high potential adaptation systems to cope with various defense mechanisms or secondary metabolites in host plants (Blackman and Eastop, 1994, Francis et al., 2006). For coping to the secondary metabolites, biochemical characteristics were involved in the metabolization by several herbivores (Cohen et al., 1992). So the kind of plants change the aphids' biological parameters reveals that aphids imposed selection favoring increased or reduced on energy sources concentration or density. It is clear that the relative contributions of sources of energy are vary widely with tissue and gender (Douglas, 1993), but it is of greater interest, in the present context, that the relative contributions can be perturbed by insect biochemical characteristics as a crucial agents such as materials using for providing energy that exert their activity by inhibition or induction the ability of aphids countered to the plant defensive system. Generalist insect herbivores, such as Aphis gossypii, need more complex adaptive mechanisms since they need to respond to a large array of different plant defensive chemicals (Francis et al., 2006). Until now, plant- insect interactions were only investigated to determine the effect of the insect on the plant adaptation (Francis et al., 2005). The objective of the present study was investigating the effect of plant defense system on the tolerance in A. gossypii and consuming energy pattern in different aphid’s strains using a range of studies of biochemical assays, including the measurement of parameters influenced aphids' biology. The aim of this study was to determine whether changing in recourses of energy in cotton aphid countered with insecticide’s treatment are the signs of adaptation to insecticides or not. The present work attempts to clarify whether energy sources indices are of any diagnostic value in respect to insecticide stress and to provide basis for searching for mechanisms of adaptation to complex insecticide stress. This may reflect severe tolerated of cotton aphid populations in counter of insecticides.
2. Materials and methods 2.1 Rearing Conditions Three cotton aphid colonies were obtained from a colony maintained on the Department of Plant Protection at the Faculty of Agriculture, University of Tehran; one strain which selected from the population neonicotinid population selected from 16 generation neonicotinoid spraying and another from a greenhouse that mostly sprayed by the insecticides of imidacloprid (350 SC Confidor) ® and thiametoxame (500WG Actara) ®. We called after that the mentioned colonies as Ag-R, Ag-S and Ag-M respectively. From these aphids a single apterous partenogenically reproducing female was selected to establish a parent colony. These colonies were used as a source for all aphids used in our laboratory assays. Colonies were maintained environmentally controlled conditions. Aphids were reared on free- pesticide plants at 23 ± 2 °C, under photoperiod 16:8 and 70 % R.H. Cotton aphid populations were reared on the the sqaush from Cucurbiacae family, Cucurbit zucchini. 2.2 Bioassays The bioassay procedures were used by dipping approach. In this method after dipping freshly leaves of three mentioned plants in chosen solutions of insecticides for 30 seconds, let them to dry for 30 min. Then one day old age apterous aphids were transferred onto freshly excised leaves on 6 cm diameter Petri-dishes placed upside down on a layer of moist paper towel. Mortality was assays after 24 hours. LC 50 values and their 95% confidential limits were calculated from probit regressions using the computer program POLOPC (LeOra Software, Berkeley, CA). 2.3 Biochemical analysis We used standard biochemical techniques to assay the amount of protein, lipid and carbohydrate present in aphids’ body. Protein was assayed using Bradford reagent according to Bradford (1976) method, lipids with vanillin in phosphoric acid (Van Handel & Day, 1988) and carbohydrates with anthrone reagent by some modifications according to Yuval et al. (1994) method. The analytical protocol was as follow: Twenty aphids were homogenized individually in 200 µl of 2% Na 2SO4. Lipids and sugar were extracted in 1300 µl of chloroform: methanol (1: 2). Individual tubes were centrifuged for 10 min at 10000g and 500μl were taken from each tube and dried. Samples were then dissolved in 500µl H 2SO4 and incubated for 10 min at 90°C. Samples of 30µl were put into wells on 96- well plates; together with 270 µl of vanillin reagent (600 mg vanillin dissolved in 100 ml distilled water and 400 ml 85% H3PO4).The plate was shaken for 30 min at room temperature, and then optical density was read at 530 nm. Total lipids in each aphid were calculated from standard curve. To determine the amount of sugar in each aphid, 300µl were taken from the chloroform: methanol extract. After adding 200µl water, the sample was reacted for 10 min at 90°C with 1 ml of anthrone reagent (500mg anthrone dissolved in 500ml H2SO4). Samples of 300µl were then put into wells on ELISA plates and optical density was read at 630 nm. Total sugars in each individual aphid were calculated from standard curves. The glycogen content was determined from the pellet that resulted from the centrifugation. After washing it with 400 μl of 80% methanol to remove any traces of sugar, 250 μl water were added and heated for 5 min at 70°C in order to extract the glycogen. 200 μl were taken from each tube and incubated for 10 min at 90 °C with 1 ml anthrone reagent (this time 600 mg anthrone dissolved in 30 ml concentrated H 2SO4). Samples of 300 μl were put into wells on ELIZA plates and optical density was read at 630 nm. Total glycogen was calculated from standard curves.
2.4 Energy assay This assay was done with converted microgram value of energy resources to calories (with multiplying sugars by 0.004 and lipids by 0.009). The ratio of sugar to lipids (using caloric values) is equal to the proportion of used energy to total energy (Warburg and Yuval, 1997). 2.5 Construction of the fecundity life table For each treatment, a life table was established by an insect cohort (100 one-day-old nymphs), and the precise value of the intrinsic rate of increase (rm) is obtained by solving the Euler equation (Anderwartha and Birch; 1954): ∑Yx=0 Lxmxe-rx=1 Where y is the oldest age class, Lx is the survival of a new-born parthenogenesis aphid to the midpoint of an age interval, and x is the age of each female at each age interval. In addition to r m the main reproductive life table parameters including net reproductive rate, generation time, doubling time, and finite rate of increase were computed using the formula; R0=∑Lxmx, T=∑xLxmx/∑Lxmx, DT=In(2)/rm and λ=erm. 2.6 Estimation of uncertainty associated with the parameters The Jacknife technique was used for ease of statistical comparison among life table parameters related to each treatment and for estimating the standard errors (SE) associated with the parameters. First, the precise value of rm was calculated for all of the raw data(r total( . Then, one of the insects was removed and an r m was computed for the remaining insects (n-1). Based on the suggested equation by Meyer et al. (1986) the Jackknife pseudo- value ( ~ ri ) is calculated for this subset of the original data to be:
~ ri n.rtotal (n 1)rˆi
The value of n is the number of insects needed to construct a reproduction life table. This process was repeated until pseudo- values were calculated for all n possible omissions of one insect from the original data set. Finally n number of calculated ~ ri was provided to calculate the mean and its SE. This algorithm was used for estimating the uncertainties associated with the four other parameters. 2.7 Statistical analysis Data were analyzed by means of analysis variance (ANOVA) using the General Linear Model (GLM) model procedure Minitab 18. and for bioassay experiments,. Data were analyzed by probit analysis (Finney 1971) using the software package POLO- PC .Resistance ratios (RR) were calculated by dividing LC50 values computed for the resistant populations by the corresponding LC 50s for the susceptible colony. LC50- values and their 95% confidential limits were calculated from probit regressions using the computer program POLO- PC (LeOra Software, 2007 Berkeley, CA). 3. Results Bioassays with aphids placed on toxic leaves immersed in imidacloprid and thiametoxam solutions revealed a high resistance in strain Ag-R. According to the studies, imidacloprid and thiametoxam also acts as an antifeedant on aphids after oral administration clearly show that imidacloprid and thiametoxam has a strong effect on the feeding of three aphid populations. Resistant strain in the natural condition use glycogen more than sensitive strain while sensitive strain use sugar more, because it is more available .In cotton aphids countered with insecticide stress ,amount of glycogen will increased while lipid, sugar and protein will reduced(Tables 2 , 3). Total lipid in susceptible strain was decreased more than resistant strain whereas total protein increase in resistant strain compared with sensitive strain. Total Glycogen was affected significantly which caused to increase and was the most in resistant strain (Table 2, 3). Total energy consumption was lower in starved aphids than other ones (Table 2 and 3).Sensitive strains use sugar more than other feeding elements while resistance stains mostly use protein and glycogen. It is shown that sensitive strains render to use lipid more than the others. Using energy was more in resistance strains. When aphids treated with distilled water, the generation time was the most in the sensitive strain; however the parameter of the intrinsic rate of increase (r m) was the most in the resistant strain. When aphids' exposure to insecticides, the generation time (T) will be the most in the sensitive strain but the maximum r m
was on the semi-sensitive strain (Tables 4, 5). So the intrinsic rate of increase is the most in the resistant strain rather than sensitive strain. Showing semi-sensitive strain has a hormolygosis effect when exposure to imidacloprid and thiametoxam. Amount of available energy in resistant strain is more than sensitive strain which helps strains to grow and reproduce. Sensitive strain has the less energy for cost for growth and reproduction. 4. Discussion Plants synthesize carbohydrates from atmospheric gases by photosynthesis storing the absorbed energy internally, often in the form of starch or lipids. Plant components are eaten by insects and used as fuel for cellular respiration (Nation 2002). Oxidation of one gram of carbohydrate yields approximately 4 kcal of energy and from lipids about 9 kcal. Energy obtained from metabolism (e.g., oxidation of glucose) is usually stored temporarily within cells in the form of ATP. Organisms capable of aerobic respiration metabolize glucose and oxygen to release energy with carbon dioxide and water as by products (Nation 2002). Energy availability can limit the ability of organisms to survive under stressful conditions (Marron et al., 2003). In cotton aphid laboratory experiments have revealed that energy storage patterns differ between populations separated with insecticide spraying compared with population which not exposure to insecticides, because aphids may use different sources of energy when exposed to insecticide stresses. Energy metabolism may also play a critical role in stress resistance. According to the results, reductions in metabolic rate will increase the amount of time aphids can survive and cause to lose energy sources in exposure to neonicitinoids in stressful conditions. An important link missing from these studies is the knowledge of which energetic substrates are actually consumed when aphids exposure to insecticides. Increased lipid storage will only help aphids to survive starvation if they actually metabolize lipids under these conditions. Thus, we can predict that aphids will regulate their metabolism to use different energy sources depending on the type of stress imposed (Marron et al., 2003). Measuring the rates of disappearance of energetic substrates (lipid, carbohydrate, and protein) is necessary to test the hypothesis that aphids with different neonicotinoid exposure method will contain greater levels of metabolic reserves, particularly carbohydrates. For more reasons, sensitive strain is responsible for the observed loss of fitness and energy and finally starvation hardiness, so it is less tolerated compared with resistant strain and also has less fitness parameters. Experiments with three hardly strains will clarify which of the reasons are responsible for observed loss fitness and energy finally causing starvation death. Biochemical properties are regarded as fast and prognostic indicates of individual reaction to the insecticide stress. Energy sources and fitness ability in residue bioassay where oral ingestion of the insecticide is necessary in order to detect possible tolerance become less than natural condition because of imidacloprids’ ability to act as an antifeedant. According to table (4, 5) resistant strains has more intrinsic rate of increase, but this parameter is less in sensitive strain. However, when aphids did not exposure to the insecticides, life- span in sensitive strain is more than resistant strain. While in the aphid populations stressed by imidacloprid and thiametoxam, rm is increased in semi- resistant strains. This increasing has observable lost in resistant strain. It shows hormolygosis phenomenon in cotton aphids countered with neonicotinoids. Differences from energy metabolism were expected between insecticide- susceptible and resistant populations showing the fitness costs associated with insecticide resistance. Indeed such differences were observed in the present study. Biochemical parameters and energy sources were always higher in the insecticide- resistant populations suggesting higher energy storage and eventual mobilization compared with the susceptible population. The resistant population exhibits significantly higher energy sources than the Ag-S strain. The differences were also particularly high for Glycogen and do not seem to be as important as energy sources in cotton aphid populations regarding the mitigation of insecticide resistance costs, since the resistant population, shows fitness disadvantaged in the absence of insecticide. Adaptative costs associated with insecticide resistance were reported in the Ag-R population of the cotton aphid but other populations of the same species do not exhibit costs of insecticide resistance. According to the results, Energy resources accumulated during developmental time feeding are not used for maintenance (e. g., basal metabolism) (Nation, 2002) can be allocated toward some major life history parameters like as intrinsic rate of increase. In this study, it is shown that resistance phenomenon is a correlation between
susceptibility and fitness cost in aphids which are associated with greater survival and fecundity. It is well known that the kind of plants feeding can alter the biological parameter (Francis et al., 2005) and also growth in cotton aphid. It should be applied as supplementary approaches for demonstrating links between sub- lethal effects of mentioned insecticides on the biochemical characteristics and also its adverse effects on natural populations in this study. Survival insects under stress conditions can be maximized by two physiological mechanisms: increasing the storage or resources (energy or water) that are utilized during stress, or conserving resources by reducing the rates at which they consume (Nation 2002). Because of its high- energy content, lipid is the primary stored nutrient in insects and most of other animals (Nation 2002). In addition to rates of energy consumption, both the amount and form of the energy storage can affect resistance to stress. Selected populations of cotton aphids accumulate high lipid and carbohydrate levels, as predicted from comparative studies. Selection stressed populations, however, use less lipid but much more glycogen than control populations that is similar to the studying of Djawdan et al., (1998). Total lipid decrease probably because of they are being routed to main metabolic pathway and utilized for energy production after treatment. Total lipid in susceptible strain was decreased more than resistant strain whereas total proteins were increasing in resistant strain compared with sensitive strain. Total Glycogen was affected significantly which caused to increase and was the most in resistant strain. Total energy consumption was lower in starved aphids than other ones. In addition to rates of energy consumption, both the amount and form of energy storage can affect stress resistance. Starvation- selected populations of cotton aphids accumulate high lipid and carbohydrate levels (Chippindale et al., 1998), as predicted from comparative studies. Selection stressed populations, however, store less lipid but much more glycogen than control populations (Djawdan et al., 1997). Elucidation of the biochemical mechanisms conferring resistance and therefore, are absolutely necessary for managing resistance. Upon starvation, the absolute content of energy sources is reduced rapidly over the starvation. Only the lower dose of imidacoprid will induce a sustained effect in contrast to thiametoxam which acts at higher doses. Resistant strains use more energy compared with sensitive strains. It may be that resistant strains need more energy to cope with chemical stress. References Andrewartha, H. & Birch, L. (1954). The Distribution and Abundance of Animals. University of Chicago, Illinois, 782p. Blackman, R. L., Eastop, V. F. (1994). Aphids on the World’s Trees, an Identification and Information Guide. CABI Edition, Oxon. UK. 989p. Bradford, M. M. (1976). A Rapid and Sensitive Method for the Quantification of Microgram Quantities of Protein, Utilizing the Principle of Protein- Dye Landing. Analytical Biochemistry, 72, 284- 254. [Online] Available:http://www.ciens.ucv.ve:8080/generador/sites/lab-bioq-gen/archivos/Bradford%201976.pdf Chippindale, A. K., Gibbs, A. G., Sheik, M., Yee, K.J., Djawdan, M., Braley, T. J. & Rose, M. R. (1998). Resource acquisition and the evolution of stress resistance in Drosophila melanogaster. Evolution, 52, 1342-52. [Online] Available:http://faculty.unlv.edu/gibbs/Evolution1998.pdf Cohen, M. B., Schuler, M. A. & Berenbaune, L. X. (1992). A Host Inducible Cytochrome P-450 from a Host Specific Caterpillar: Molecular Cloning and Evolution. Proceedings of the National Academy of Science. USA. 89, 10920- 10924 Djawdan, M., Rose, M. R. & Bradley, T. J. (1997). Does Selection for Stress Resistance Lower Metabolic Rate? Ecology, 78, 828- 837 [Online] Available:http://www.jstor.org/stable/2266062 Douglas, A. E. (1993). The Nutritional Quality of Phloem Sap Utilized by Natural Aphid Populations. Ecological Entomology, 18, 31- 38. DOI: 10.1111/j.1365-2311.1993.tb01076.x Finney, D. J. (1971). Probit Analysis. Cambridge. England. 272p. Francis, F., Gerkens, P., Harmel, N. & Haubruge, E. (2006). Proteomic in Myzus persicae: Effect on Aphid Host Plant Switch. Insect Biochemistry and Molecular Biology, 36, 219–227. DOI: 10.1016/j.ibmb.2006.01.018.
Francis, F., N. Vanhaelen, E. Haubruge. 2005. Glutathion S- Transferase in the Adaptation to Plant Secondary Metabolites in the Myzus persicae Aphid. Archives of Insect Biochemistry and Physiology, 58, 166–174. DOI: 10.1002/arch.20049 Leora Software (2007). Polo- PC: A user Guide to Probit or Logit Analysis. LeOra Software, Barekeley, California. Maraise E. & Chow, S. L. (2003). Repeat ability of Standard Metabolic Rate and Gas Exchange Characteristics in a Highly Variable. The journal of experimental biology, 206, 4565-4574. DOI:10.1242/jeb.00700 Marron, M.T., Markow, T.A., Kain, K.J. & Gibbs, A.G. (2003). Effects of Starvation and Desiccation on Energy Metabolism in Desert and Mesic Drosophila. Journal of Insect Physiology, 49, 261- 270. DOI:10.1016/S0022-1910(02)00287-1 Meyer, J.S., Ingersoll, C.G., McDonald, L.L. & Boyee, M. S. (1986). Estimating Uncertainty in Population Growth Rates: Jacknife vs. Bootstrap Techniques. Ecology, 67, 1156- 1166. DOI: http://dx.doi.org/10.2307/1938671 Nation, J. L. (2002). Insect Physiology and Biochemistry. CRC Press, Boca Raton, Florida, 485 p. Van Handel, E. & Day, J.F. (1988). Assay of lipids, glycogen and sugars in individual mosquitoes: Correlations with wing length in field collected Aedes vexans. Journal of the American Mosquito Control Association, 4, 549- 550. Warburg. M.S. & Yuval, B. (1997). Circadian Patterns of Feeding and Reproductive Activities of Mediterranean Fruit Flies (Diptera: Tephritidae) on Various Hosts. Annals of the Entomological Society of America, 90, 487-495 Yuval. B., Holliday- Hanson, M. & Washino, RK. (1994). Energy Budget of Swarming Male Mosquitoes. Ecological Entomology, 19, 74- 78. DOI: 10.1111/j.1365-2311.1994.tb00392.x
Table 1- LC50 value in different strains of cotton aphid in counter of imidacloprid and thiametoxam Strain/clon e
Plant
LC50(mgL-1)
95%CL *
Slop e
RF*
Imidaclopri d
LC50(mgL-1)
95%C L
Slop e
RF
Thiametoxa m
Ag-R
Squas h
3673
34203880
1.9
183.6
3680
34573871
1.8
92
Ag-S
Squas h
20
17-22
1.4
_
40
32-53
0.2
_
Ag-M
Squas h
127
124130
1.2
28.9
183
165190
1.3
20.1
*95% CL: 95%Confidence Level, *RF: Resistance Factor= LC50 Resistant Strain/LC50 Sensitive Strain
Table 2- Correlation between storage biochemical parameters and total energy in different populations of cotton aphid countered with imidacloprid Lipid
Cont.
Glycogen
Cont.
Sugar
Cont.
Protein
Cont.
Energy
Cont.
Ag-R
Squash
24.6±0.01a
34.6±0.4a
10.3±0.2b
7.4±0.04b
114.1±0.01a
245±0.3
161±1a
315±3a
20.5±0.1b
37.6±0.1a
Ag-S
Squash
10.1±0.3c
33.3±0.1b
4.5±0.1c
6.2±0.2c
45.5±0.05c
204±21.2
111±1.4c
212.2±4c
35.3±0.3a
45.8±0.1b
Ag-M
Squash
13.6±0.05b
30.5±0.3c
11.3±0.02a
8.1±0.4a
72.5±0.2b
230.02±21.2
153±0.7b
310.2±0.2b
21.7±0.1b
31.1±0.1b
Means within a column followed by the same letter are not significantly different (P
