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Morphological, ecological and biological variations in the mustard aphid, Lipaphis pseudobrassicae (Kaltenbach) (Hemiptera: Aphididae) from different host plants
Journal of Asia-Pacific Entomology 12 (2009) 169–173
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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j a p e
Morphological, ecological and biological variations in the mustard aphid, Lipaphis pseudobrassicae (Kaltenbach) (Hemiptera: Aphididae) from different host plants B.K. Agarwala ⁎, Kalpana Das, Parichita Raychoudhury Ecology and Biosystematics Laboratories, Department of Zoology, Tripura University, Suryamaninagar 799 130, Tripura, India
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Article history: Received 9 January 2009 Revised 17 March 2009 Accepted 20 March 2009 Keywords: Aphids Lipaphis pseudobrassicae Host plant specialization
a b s t r a c t Genetic and morphological differentiation of insect populations in relation to the use of different host plants is an important phenomenon that leads to ecological specialization. In this study, we describe variations in morphology, and in ecological and biological parameters of Lipaphis pseudobrassicae (Kaltenbach) clones associated with three host species of Cruciferae, Brassica juncea (L.) var. rai sarson Czern and Cross (brown mustard), Brassica campestris L. var. sarson Prain (yellow mustard), and Rorippa indica (L.) Hiern (wild herb). This study was aimed at obtaining evidence regarding phenotypic differentiation induced by, or associated with, the use of distinct host species. Ten morphological characters, 4 growth parameters and 8 biological functions were investigated in wingless aphids collected from plants of the three host species. Aphids from B. campestris and B. juncea clones were bigger in size, heavier in weight and showed higher growth rates and fecundity than the clones from R. indica. Between the two crop plants, clones from B. juncea showed significantly higher growth rates than the clones from B. campestris. Transfer of L. pseudobrassicae populations from B. campestris to B. juncea and R. indica and vice versa resulted in poor performance. Results indicate that the average phenotype of L. pseudobrassicae individuals inhabiting different host plant species differs as a consequence of the contrasting feeding environments the host species provide. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2009 Published by Elsevier B.V. All rights reserved.
Introduction Recent morphological, biochemical and host plant preference studies have shown that several polyphagous and oligophagous aphid species consist of genetically different forms, i.e. host races, or even appear to represent complexes of several separate species. In Schizaphis graminum (Rondani), several biotypes were distinguished on the basis of their performance on different cereals, a difference that is supported by studies of DNA (Beregovoy et al., 1988; Powers et al., 1989; Wilhoit and Mittler, 1991). Similarly, host races and sibling species were distinguished in Cryptomyzus galeopsidis (Kaltenbach) and Aphis gossypii Glover (Guldemond, 1990, Guldemond et al., 1994). More recently, host plant-based clonal diversity was recorded in the polyphagous species, Myzus persicae (Sulzer) (Peppe and Lomonaco, 2003) and Aphis gossypii (Agarwala and Das, 2007), and in the oligophagous species, Brevicoryne brassicae (RuizMontoya et al., 2005). Gorur et al. (2005, 2007) reported high levels of phenotypic plasticity in Aphis fabae Scopoli when reared on broad bean and nasturtium host plants. They suggested that the presence of genotypic and phenotypic variations in natural populations
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facilitate host plant specialization, host race formation and sympatric speciation in herbivore insects (Gorur, 2005). These results clearly indicate that obligate species of herbivores like aphids have the potential to adapt to new host plants and to develop phenotypic and genotypic changes in new food environments. Often the genetic and morphological differences and variations in fitness characters, such as intrinsic rate of natural increase, relative growth rate, fecundity and development time of insect populations, in relation to the use of different host plants cause ecological specialization. Host plant specialized populations of a species can lead to speciation in sympatric conditions (RuizMontoya et al. 2005; Gorur et al. 2005, 2007). Aphid – host plant relationship was examined in Lipaphis pseudobrassicae (Kaltenbach), an oligophagous species of cruciferous plants, in Brassica campestris L. var. sarson Prain (yellow mustard), B. juncea (L.) var. rai sarson Czern and Coss (brown mustard) and a wild herb, Rorippa indica (L.) Hiern. Host transfer experiments, involving reciprocal transfer of aphid clones from field host to laboratory hosts, were conducted to test the host specialization hypothesis. Earlier Agarwala and Das (1998) showed that L. pseudobrassicae aphids [=L. erysimi (Kaltenbach)] collected from radish and aphids collected from Indian mustard differed in length of siphunculi, developmental time and intrinsic rate of increase. These results were not supported by host transfer experiments.
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2009.03.002
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Materials and methods Insect materials Wingless aphids of L. pseudobrassicae were collected in the winter of 2006–2007 from three plant species, Brassica campestris var. sarson, B. juncea var. rai sarson and Rorippa indica. Aphids were collected from the university farm and refuse area at Suryamaninagar, West Tripura, north-east India (23.50°N, 91.25°E). Fieldcollected wingless (apterae) parthenogenetic viviparous aphid females representing three clones, one from each plant species, were used as the source of stock culture in the greenhouse conditions (20° ± 2 °C temperature and b16 h photoperiod) on their respective host plants species. Each culture was comprised of a sufficient number of potted plants of a species infested with aphids. All pots were held in water trays on benches illuminated with photo-simulator lamps. Aphids from the stock culture were used to raise the host plantspecific clones in a rearing cabinet (20° ± 2 °C temperature, 65–70% relative humidity and 16:8 h photoperiod). For cloning, fourth stadium nymphs from different mothers reared on different plants of the same species were used to obtain several sister clones. An individual nymph was placed on the apical part of the pot-grown 3–4 leaf stage plants of similar age. Each potted plant was covered with nylon gauze cage to prevent contamination. Aphids were allowed to settle on each potted plant and then to increase in number. Observations were made at frequent intervals till the clonal population peaked and then started to decline. This practice was repeated at least 20 times for all host plant species. Clones were monitored individually on a daily basis and any winged aphid produced was removed to avoid contamination of clones. Fresh plants were periodically substituted for any that had deteriorated. This practice allowed an uninterrupted supply of aphids from the three host plant species during this study. Ecological variations Variations in population growth rate and carrying capacity were determined for L. pseudobrassicae clones from the three plant species. Twenty replicates were used in each study. Population growth rate (GR), the change in the number of individuals of a population per unit time, was recorded in the rising phase and was calculated using the equation (Odum, 1971): GR = (Nt − N0) / Δt, where N0 is the number of aphids initially released on a potted plant, Nt is the number of aphids recorded at the maximum count of the plant, and Δt is the difference of time between N0 and Nt. Carrying capacity (K), the upper limit of population size of an organism that is acceptable to a given environmental condition, was determined using the equation: K = Σ (Nmax − Nmin) / Nmax, where K is the carrying capacity of the individual host plant, and Nmax and Nmin are the maximum and minimum number of aphids, respectively, present in the population at the beginning and at the peak of growth (Stiling, 2006). Time taken to reach the carrying capacity (Tk) was calculated by the equation: Tk = Σ no. of days to K / n, where n is the number of observations (Agarwala and Das, 2007). Biological variations The following biological attributes of L. pseudobrassicae from the three host plant species were determined: For determining live weight, developmental time, generation time, reproductive time and fecundity, new born aphids were individually weighed and then placed on detached leaves in leaf cages (Blackman, 1987). One nymph was placed per leaf per cage, in a low temperature incubator at 22° ± 1 °C and 16:8 h photo-
period. All nymphs were allowed to become adults and reproduce. Nymphs at final molt (b12 h old) were individually weighed and observed for the duration of pre-reproduction, reproduction and post-reproduction periods. The number of nymphs born to individual adult apterous aphids were counted, and all but one were removed. The remaining aphid was allowed to develop into the next generation. Leaves were changed every 24 h to maintain the vigor of experimental culture. Each aphid was observed for three succeeding generations. As a result of this procedure, birth weight (BW), adult weight at the final molt (AW), developmental time from the birth of a nymph to its final molt (DT), generation time from the birth of a nymph to the time of onset of reproduction by this nymph (GT), reproductive duration from the birth of first nymph to the last nymph by an apterous female (RD), and fecundity (F) were recorded. Twenty replicates were used from each host plant species. The intrinsic rate of increase (Rmax), a measure of rate of increase of a population under controlled conditions, was calculated using the method of Krebs (1985): Rmax = logе(R0) / G, where, R0 is the net reproductive rate and G is the mean length of a generation. G is determined by the equation: G = Σlx bx / R0, where lx is the proportion of females surviving, bx is the number of female offspring born per female during its reproductive time and x is the age of the female adult. The net reproductive rate (R0), the multiplication rate of an organism per generation in terms of number of female offspring produced by a cohort of females, i.e. adult wingless aphids of a colony, was calculated using the following equation (Krebs, 1985): R0 = Σlx bx. Morphological variations Twenty adults of similar age were individually collected from the clones of three host plant species. These were prepared as whole mounted specimens for microscopic examination following the method of Eastop and van Emden (1972). Ten different characters of taxonomic importance were measured with an eyepiece micrometer at 400× magnification using a light microscope: (1) length of body (BL), (2) maximum width of body (BW), (3) length of antenna (ANT), (4) length of antennal segment III (ANT III), (5) length of antennal segment VI (ANT VI), (6) length of proboscis (PROB), (7) length of ultimate rostral segments (URS), (8) length of fore femur (FEM), (9) length of siphunculus (SIPH), and (10) length of cauda (CAU). Host transfer experiments Nymphs from all three clones were subjected to reciprocal host transfer to record the effects of induction of new host plant on the performance of aphids in a new food environment. Individual nymphs, 0–12 h old, were allowed to settle on apical leaves of potted plants of field hosts and laboratory hosts. These aphids were used to produce experimental nymphs for the second generation. If successful, a third generation was produced. The following three experiments were set up using parental clones of L. pseudobrassicae from the three host plant species: (1) ‘Expt. I’ for L. pseudobrassicae aphids from B. campestris as field host, (2) ‘Expt. II’ for L. pseudobrassicae aphids from B. juncea as field host, and (3) ‘Expt. III’ for L. pseudobrassicae aphids from R. indica as field host. Simultaneous transfers were made for the aphids of L. pseudobrassicae clones from the respective field host plant to other host plants to be tested for colonization success. In each case of host transfer, twenty observations were made on colonization success (CS), the survival and reproduction by viviparous aphids on a host plant leading to the establishment of a colony, was recorded. Aphids that either failed to develop to the adult stage in the first generation
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sensitive to 2 μg. Each of the morphological, ecological and biological characters that were measured from the wingless aphids from different L. pseudobrassicae clones were compared using Tukey's multiple range test. Comparison of frequency of success and failure in colonization by L. pseudobrassicae aphids on different host plant species in the host transfer experiments was tested by chi-squared test. Statistical programme ‘Origin 7’ was used for the analysis of data. Results Ecological variations The L. pseudobrassicae clone reared on B. juncea showed a mean growth rate of 60.95 aphids/day, which is higher by approximately 37% and 95% compared to the clones reared on B. campestris and R. indica, respectively (Fig. 1a). A similar trend in performance with respect to carrying capacity (K) by different clones of L. pseudobrassicae was recorded on their respective host plants (Fig. 1b). Carrying capacity of R. indica host plant for L pseudobrassicae aphids was recorded to be the significantly lower (73 aphids) than that of B. campestris (973 aphids) and B. juncea (1350 aphids). Despite the low carrying capacity of R. indica for L. pseudobrassicae aphids, time taken to achieve this value (Tk) on this host plant species was not significantly different from that of B. juncea, which showed a higher mean carrying capacity (Fig. 1c). Biological variations Results presented in Table 1 suggest that the wingless aphids of L. pseudobrassicae clones from B. campestris and B. juncea were twice as large or larger in size by fresh weight than those reared on R. indica. This was true for aphids at birth and at the final molt. Larger aphids had a longer development time, exhibited longer generation time and reproduced for longer time than the smaller aphids reared on R. indica. Aphids from B. campestris clones produced more offspring (mean ± sem = 44.50 ± 7.47) than those reared on B. juncea (mean ± sem = 29.40 ± 2.31), and these produced significantly more offspring compared to adult females reared on R. indica (mean ± sem = 19.60 ± 3.37). The intrinsic rate of increase (Rmax) of L. pseudobrassicae aphids on the three plant species was nearly the same. However, the net reproductive rate (Ro) was highest in the clone reared on B. juncea and the lowest in the clone from R. indica. Ro in the L. pseudobrassicae clone from B. juncea was higher (mean ± sem = 59.87 ± 6.04) than the R. indica (mean ± sem = 24.46 ± 3.76) and B. campestris clones (mean ± sem = 45.20 ± 5.42) (Table 1).
Fig. 1. Mean values of growth rate (GR) (a), carrying capacity (K) (b) and time to attain carrying capacity (Tk) (c) in apterous L. pseudobrassicae populations from three host plant species, B. campestris (Bc), B. juncea (Bj) and R. indica (Ri). Bars indicate standard error. Different letters accompanying bars denote significant differences between mean values by Tukey's multiple range test at P b 0.05.
or failed to reach the third generation were considered to be ‘failure’ in colonization. Data analysis Data of the third generation aphids, wherever available, were used to compare results. This was done to allow time for acclimatization to the laboratory rearing conditions. All microscopic measurements were converted to mm using a stage micrometer. All weights in this study were taken in a Mettler microbalance
Table 1 Mean values of biological attributes recorded in apterous aphids of L. pseudobrassicae clones from three different host plant species. Biological attributes BW (mg) AW (mg) DT (days) GT (days) RT (days) F (no.) Rmax R0
Mean ± SEM⁎ of measurements from three host plant species (n = 20) B. campestris
B. juncea
R. indica
0.037 ± 0.000 a 0.49 ± 0.004 a 7.0 ± 0.211 a 7.95 ± 0.189 a 9.60 ± 1.392 a 44.50 ± 7.47 a 0.28 ± 0.002 a 45.20 ± 5.42 a
0.04 ± 0.000 a 0.53 ± 0.031 a 7.98 ± 0.171 b 8.60 ± 0.163 b 13.80 ± 1.289 b 29.40 ± 2.31 b 0.28 ± 0.007 a 59.87 ± 6.04 b
0.02 ± 0.001 b 0.25 ± 0.007 b 5.90 ± 0.100 c 6.10 ± 0.233 c 8.40 ± 1.447 a 19.60 ± 3.374 c 0.26 ± 0.001 b 24.46 ± 3.76 c
⁎ Different letters with mean values in a row indicate significant differences between the treatments by Tukey's multiple range test at P b 0.05. BW = birth weight, AW = weight at the final molt, DT = developmental time, GT = generation time, RT = reproductive duration, F = fecundity, Rmax = intrinsic rate of increase, R0 = net reproductive rate.
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Morphological variations Results presented in Table 2 show that the wingless aphids of L. pseudobrassicae clones from B. campestris and B. juncea were bigger and their antennae, ultimate rostral segments, siphunculi and fore femora were significantly longer than the aphids from R. indica clone. Aphid clones from B. campestris and B. juncea, however, did not show distinguishable differences in morphometry. Host transfer experiments Results presented in Table 3 show the success rates of colonization of L. pseudobrassicae clones when transferred from one host plant species to another host plant species of same genus or different genera of Cruciferae. When transferred to B. juncea and R. indica plants, L. pseudobrassicae clone aphids from B. campestris did not survive or perished in the third generation after initial success in the first generation and partial success in the second generation (Expt. I). When L. pseudobrassicae clones from B. juncea were transferred to B. campestris, the colonization success rate of aphids on the transferred hosts declined from 100% to 40% in the third generation and aphids did not survive the third generation on R. indica plants (Expt. II). When transferred to B. campestris and B. juncea plants, L. pseudobrassicae clones from the weed host, R. indica, failed to colonize and perished (Expt. III). Discussion This study has shown that L. pseudobrassicae populations have developed distinguishable ecological and biological variations in relation to different species of host plants. Reciprocal transfer of L. pseudobrassicae populations between host plant species was nearly impossible. Morphological variation in L. pseudobrassicae clone aphids between the two crop species of Brassica was not evident but the R. indica clone aphids showed significant differences in several characters in comparison to conspecifics from Brassica host plants. As a consequence, L. pseudobrassicae may be regarded as a genetically Table 2 Mean (mm) values of morphological characters in apterous aphids of L. pseudobrassicae clones from three host plant species. Morphological Characters BL BW ANT ANT III ANT VI PROB URS CAU SIPH FEM
Mean⁎ (range: minimum and maximum) of measurements from different host plants (n = 20) B. campestris
B. juncea
R. indica
1.87 a (1.55–2.37) 1.08 a (0.87–1.35) 1.39 a (1.10–1.79) 0.35 a (0.30–0.41) 0.43 a (0.47–0.65) 0.38 a (0.36–0.42) 0.10 a (0.09–11) 0.19 a (0.15–0.26) 0.24 a (0.20–0.26) 0.41 a (0.44–0.56)
1.99 a (1.81–2.03) 1.30 b (1.22–1.44) 1.25 a (1.11–1.37) 0.34 a (0.26–0.39) 0.39 a (0.33–0.52) 0.41 a (0.37–0.48) 0.10 a (0.09–0.11) 0.16 b (0.11–0.19) 0.22 a (0.20–0.24) 0.39 a (0.35–0.41)
1.38 b (1.26–1.59) 0.79 c (0.74–1.00) 0.77 b (0.70–1.18) 0.19 b (0.15–0.23) 0.28 b (0.24–0.37) 0.32 b (0.30–0.41) 0.08 b (0.07–0.09) 0.15 b (0.15–0.19) 0.18 b (0.15–0.20) 0.23 b (0.19–0.35)
⁎ Different letters with mean values in a row indicate significant differences between the treatments by Tukey's multiple range test at P b 0.05. BL = Body length, BW = maximum width of body, ANT = length of antenna, ANT III = antennal segment III, ANT VI = antennal segment VI, PROB = length of proboscis, URS = length of ultimate rostral segment, SIPH = length of siphunculus, CAU = length of cauda, FEM = length of fore femur.
Table 3 Success rate of colonization by apterous aphids of L. pseudobrassicae clones as a result of host transfer (host plants are denoted as: B. campestris = Bc, B. juncea = Bj, R. indica = Ri). Expt.
Field host
Laboratory host
I.
Bc
II.
Bj
III.
Ri
Bc Bj Ri Bj Bc Ri Ri Bc Bj
% success⁎ (n: based on colonization success in the respective generation) 1st generation
2nd generation
3rd generation
100 (20) 100 (20) 80 (16) 100 (20) 90 (18) 80 (16) 100 (20) 0 (0) 0 (0)
100 (20) 30 (6) 20 (4) 100 (20) 70 (14) 30 (6) 100 (20) – –
100 0 0 100 40 0 90 – –
(20) (0) (0) (20) (14) (0) (18)
χ2
NS ⁎⁎ ⁎⁎ NS ⁎⁎ ⁎⁎ NS – –
⁎ 0 = initially survived but failed to complete development or reproduce in that generation; – no aphid survived; χ2 Chi-square significance values for surviving aphids over the three generations: NS = P N 0.05. ⁎⁎ P b 0.01.
heterogeneous species infesting various host plants at different rates (Jaenike, 1981; Diehl and Bush, 1984).This implies that L. pseudobrassicae populations from B. campestris will not infect B. juncea or R. indica plants, and vice versa. This result agrees with earlier findings that described host plant-based variations in L. pseudobrassicae and several other aphid species from different parts of the world. According to Agarwala and Das (1998), L. pseudobrassicae aphids from radish hosts had shorter siphunculi, longer developmental time and lower intrinsic rate of increase compared to conspecifics from the Indian mustard host, although no host transfer results were provided. The present study has extended the knowledge of intra-specific variation in L. pseudobrassicae in relation to host plants. Ruiz-Montoya et al. (2005) described the morphological variation of populations of Brevicoryne brassicae associated with two host species, Brassica oleracea L. and B. campestris, occurring in the same habitat. Peppe and Lomonaco (2003) described host race formation in Myzus persicae on two Cruciferae host species, B. olearacea and Raphanus sativus L. Guldemond et al. (1994) and Agarwala and Das (2007) described the host races of Aphis gossypii aphids on different host plant species from Europe and Asia. Similarly, biotypes and sibling species were distinguished in Cryptomyzus galeopsidis (Guldemond, 1990) and Schizaphis graminum (Wilhoit and Mittler, 1991). Gorur (2005) and Gorur et al. (2005) showed that prevalent genotypic variations and phenotypic plasticity in A. fabae aphids and other herbivorous insects favor the selection of new host plants. These examples indicate that host plant divergence in insect populations favors the formation of host plant-specific races. Aphids and other phytophagous insects are known by intraspecific phenotypic variations in response to temperature, seasonal variation, natural enemies and host quality (Dixon et al., 1982; Agarwala, 2007). It is widely accepted that the fitness of an aphid clone is determined by plant nutritional quality and aphid nutritional biology (reviewed in Powell et al., 2006). Several studies indicate that reproductive decisions in aphids are made on the basis of plant cues, and that cue-based control of aphid reproduction is one of the determinants of fitness (Via, 1991; Caillaud and Via, 2000; Gabrys and Tjallingii, 2002). It is, therefore, argued that host plant preference and performance in aphids are correlated and that they directly influence the process of host plant selection in sympatric condition (Via and Hawthorne, 2002). Acknowledgments Authors are thankful to the Indian Council of Agricultural Research, New Delhi for financial support. Thanks are also extended to Prithwi Jyoti Bhowmik of the authors' laboratories for helps in the preparation of the manuscript.
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Journal of Asia-Pacific Entomology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / j a p e
Morphological, ecological and biological variations in the mustard aphid, Lipaphis pseudobrassicae (Kaltenbach) (Hemiptera: Aphididae) from different host plants B.K. Agarwala ⁎, Kalpana Das, Parichita Raychoudhury Ecology and Biosystematics Laboratories, Department of Zoology, Tripura University, Suryamaninagar 799 130, Tripura, India
a r t i c l e
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Article history: Received 9 January 2009 Revised 17 March 2009 Accepted 20 March 2009 Keywords: Aphids Lipaphis pseudobrassicae Host plant specialization
a b s t r a c t Genetic and morphological differentiation of insect populations in relation to the use of different host plants is an important phenomenon that leads to ecological specialization. In this study, we describe variations in morphology, and in ecological and biological parameters of Lipaphis pseudobrassicae (Kaltenbach) clones associated with three host species of Cruciferae, Brassica juncea (L.) var. rai sarson Czern and Cross (brown mustard), Brassica campestris L. var. sarson Prain (yellow mustard), and Rorippa indica (L.) Hiern (wild herb). This study was aimed at obtaining evidence regarding phenotypic differentiation induced by, or associated with, the use of distinct host species. Ten morphological characters, 4 growth parameters and 8 biological functions were investigated in wingless aphids collected from plants of the three host species. Aphids from B. campestris and B. juncea clones were bigger in size, heavier in weight and showed higher growth rates and fecundity than the clones from R. indica. Between the two crop plants, clones from B. juncea showed significantly higher growth rates than the clones from B. campestris. Transfer of L. pseudobrassicae populations from B. campestris to B. juncea and R. indica and vice versa resulted in poor performance. Results indicate that the average phenotype of L. pseudobrassicae individuals inhabiting different host plant species differs as a consequence of the contrasting feeding environments the host species provide. © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2009 Published by Elsevier B.V. All rights reserved.
Introduction Recent morphological, biochemical and host plant preference studies have shown that several polyphagous and oligophagous aphid species consist of genetically different forms, i.e. host races, or even appear to represent complexes of several separate species. In Schizaphis graminum (Rondani), several biotypes were distinguished on the basis of their performance on different cereals, a difference that is supported by studies of DNA (Beregovoy et al., 1988; Powers et al., 1989; Wilhoit and Mittler, 1991). Similarly, host races and sibling species were distinguished in Cryptomyzus galeopsidis (Kaltenbach) and Aphis gossypii Glover (Guldemond, 1990, Guldemond et al., 1994). More recently, host plant-based clonal diversity was recorded in the polyphagous species, Myzus persicae (Sulzer) (Peppe and Lomonaco, 2003) and Aphis gossypii (Agarwala and Das, 2007), and in the oligophagous species, Brevicoryne brassicae (RuizMontoya et al., 2005). Gorur et al. (2005, 2007) reported high levels of phenotypic plasticity in Aphis fabae Scopoli when reared on broad bean and nasturtium host plants. They suggested that the presence of genotypic and phenotypic variations in natural populations
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facilitate host plant specialization, host race formation and sympatric speciation in herbivore insects (Gorur, 2005). These results clearly indicate that obligate species of herbivores like aphids have the potential to adapt to new host plants and to develop phenotypic and genotypic changes in new food environments. Often the genetic and morphological differences and variations in fitness characters, such as intrinsic rate of natural increase, relative growth rate, fecundity and development time of insect populations, in relation to the use of different host plants cause ecological specialization. Host plant specialized populations of a species can lead to speciation in sympatric conditions (RuizMontoya et al. 2005; Gorur et al. 2005, 2007). Aphid – host plant relationship was examined in Lipaphis pseudobrassicae (Kaltenbach), an oligophagous species of cruciferous plants, in Brassica campestris L. var. sarson Prain (yellow mustard), B. juncea (L.) var. rai sarson Czern and Coss (brown mustard) and a wild herb, Rorippa indica (L.) Hiern. Host transfer experiments, involving reciprocal transfer of aphid clones from field host to laboratory hosts, were conducted to test the host specialization hypothesis. Earlier Agarwala and Das (1998) showed that L. pseudobrassicae aphids [=L. erysimi (Kaltenbach)] collected from radish and aphids collected from Indian mustard differed in length of siphunculi, developmental time and intrinsic rate of increase. These results were not supported by host transfer experiments.
1226-8615/$ – see front matter © Korean Society of Applied Entomology, Taiwan Entomological Society and Malaysian Plant Protection Society, 2009 Published by Elsevier B.V. All rights reserved. doi:10.1016/j.aspen.2009.03.002
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Materials and methods Insect materials Wingless aphids of L. pseudobrassicae were collected in the winter of 2006–2007 from three plant species, Brassica campestris var. sarson, B. juncea var. rai sarson and Rorippa indica. Aphids were collected from the university farm and refuse area at Suryamaninagar, West Tripura, north-east India (23.50°N, 91.25°E). Fieldcollected wingless (apterae) parthenogenetic viviparous aphid females representing three clones, one from each plant species, were used as the source of stock culture in the greenhouse conditions (20° ± 2 °C temperature and b16 h photoperiod) on their respective host plants species. Each culture was comprised of a sufficient number of potted plants of a species infested with aphids. All pots were held in water trays on benches illuminated with photo-simulator lamps. Aphids from the stock culture were used to raise the host plantspecific clones in a rearing cabinet (20° ± 2 °C temperature, 65–70% relative humidity and 16:8 h photoperiod). For cloning, fourth stadium nymphs from different mothers reared on different plants of the same species were used to obtain several sister clones. An individual nymph was placed on the apical part of the pot-grown 3–4 leaf stage plants of similar age. Each potted plant was covered with nylon gauze cage to prevent contamination. Aphids were allowed to settle on each potted plant and then to increase in number. Observations were made at frequent intervals till the clonal population peaked and then started to decline. This practice was repeated at least 20 times for all host plant species. Clones were monitored individually on a daily basis and any winged aphid produced was removed to avoid contamination of clones. Fresh plants were periodically substituted for any that had deteriorated. This practice allowed an uninterrupted supply of aphids from the three host plant species during this study. Ecological variations Variations in population growth rate and carrying capacity were determined for L. pseudobrassicae clones from the three plant species. Twenty replicates were used in each study. Population growth rate (GR), the change in the number of individuals of a population per unit time, was recorded in the rising phase and was calculated using the equation (Odum, 1971): GR = (Nt − N0) / Δt, where N0 is the number of aphids initially released on a potted plant, Nt is the number of aphids recorded at the maximum count of the plant, and Δt is the difference of time between N0 and Nt. Carrying capacity (K), the upper limit of population size of an organism that is acceptable to a given environmental condition, was determined using the equation: K = Σ (Nmax − Nmin) / Nmax, where K is the carrying capacity of the individual host plant, and Nmax and Nmin are the maximum and minimum number of aphids, respectively, present in the population at the beginning and at the peak of growth (Stiling, 2006). Time taken to reach the carrying capacity (Tk) was calculated by the equation: Tk = Σ no. of days to K / n, where n is the number of observations (Agarwala and Das, 2007). Biological variations The following biological attributes of L. pseudobrassicae from the three host plant species were determined: For determining live weight, developmental time, generation time, reproductive time and fecundity, new born aphids were individually weighed and then placed on detached leaves in leaf cages (Blackman, 1987). One nymph was placed per leaf per cage, in a low temperature incubator at 22° ± 1 °C and 16:8 h photo-
period. All nymphs were allowed to become adults and reproduce. Nymphs at final molt (b12 h old) were individually weighed and observed for the duration of pre-reproduction, reproduction and post-reproduction periods. The number of nymphs born to individual adult apterous aphids were counted, and all but one were removed. The remaining aphid was allowed to develop into the next generation. Leaves were changed every 24 h to maintain the vigor of experimental culture. Each aphid was observed for three succeeding generations. As a result of this procedure, birth weight (BW), adult weight at the final molt (AW), developmental time from the birth of a nymph to its final molt (DT), generation time from the birth of a nymph to the time of onset of reproduction by this nymph (GT), reproductive duration from the birth of first nymph to the last nymph by an apterous female (RD), and fecundity (F) were recorded. Twenty replicates were used from each host plant species. The intrinsic rate of increase (Rmax), a measure of rate of increase of a population under controlled conditions, was calculated using the method of Krebs (1985): Rmax = logе(R0) / G, where, R0 is the net reproductive rate and G is the mean length of a generation. G is determined by the equation: G = Σlx bx / R0, where lx is the proportion of females surviving, bx is the number of female offspring born per female during its reproductive time and x is the age of the female adult. The net reproductive rate (R0), the multiplication rate of an organism per generation in terms of number of female offspring produced by a cohort of females, i.e. adult wingless aphids of a colony, was calculated using the following equation (Krebs, 1985): R0 = Σlx bx. Morphological variations Twenty adults of similar age were individually collected from the clones of three host plant species. These were prepared as whole mounted specimens for microscopic examination following the method of Eastop and van Emden (1972). Ten different characters of taxonomic importance were measured with an eyepiece micrometer at 400× magnification using a light microscope: (1) length of body (BL), (2) maximum width of body (BW), (3) length of antenna (ANT), (4) length of antennal segment III (ANT III), (5) length of antennal segment VI (ANT VI), (6) length of proboscis (PROB), (7) length of ultimate rostral segments (URS), (8) length of fore femur (FEM), (9) length of siphunculus (SIPH), and (10) length of cauda (CAU). Host transfer experiments Nymphs from all three clones were subjected to reciprocal host transfer to record the effects of induction of new host plant on the performance of aphids in a new food environment. Individual nymphs, 0–12 h old, were allowed to settle on apical leaves of potted plants of field hosts and laboratory hosts. These aphids were used to produce experimental nymphs for the second generation. If successful, a third generation was produced. The following three experiments were set up using parental clones of L. pseudobrassicae from the three host plant species: (1) ‘Expt. I’ for L. pseudobrassicae aphids from B. campestris as field host, (2) ‘Expt. II’ for L. pseudobrassicae aphids from B. juncea as field host, and (3) ‘Expt. III’ for L. pseudobrassicae aphids from R. indica as field host. Simultaneous transfers were made for the aphids of L. pseudobrassicae clones from the respective field host plant to other host plants to be tested for colonization success. In each case of host transfer, twenty observations were made on colonization success (CS), the survival and reproduction by viviparous aphids on a host plant leading to the establishment of a colony, was recorded. Aphids that either failed to develop to the adult stage in the first generation
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sensitive to 2 μg. Each of the morphological, ecological and biological characters that were measured from the wingless aphids from different L. pseudobrassicae clones were compared using Tukey's multiple range test. Comparison of frequency of success and failure in colonization by L. pseudobrassicae aphids on different host plant species in the host transfer experiments was tested by chi-squared test. Statistical programme ‘Origin 7’ was used for the analysis of data. Results Ecological variations The L. pseudobrassicae clone reared on B. juncea showed a mean growth rate of 60.95 aphids/day, which is higher by approximately 37% and 95% compared to the clones reared on B. campestris and R. indica, respectively (Fig. 1a). A similar trend in performance with respect to carrying capacity (K) by different clones of L. pseudobrassicae was recorded on their respective host plants (Fig. 1b). Carrying capacity of R. indica host plant for L pseudobrassicae aphids was recorded to be the significantly lower (73 aphids) than that of B. campestris (973 aphids) and B. juncea (1350 aphids). Despite the low carrying capacity of R. indica for L. pseudobrassicae aphids, time taken to achieve this value (Tk) on this host plant species was not significantly different from that of B. juncea, which showed a higher mean carrying capacity (Fig. 1c). Biological variations Results presented in Table 1 suggest that the wingless aphids of L. pseudobrassicae clones from B. campestris and B. juncea were twice as large or larger in size by fresh weight than those reared on R. indica. This was true for aphids at birth and at the final molt. Larger aphids had a longer development time, exhibited longer generation time and reproduced for longer time than the smaller aphids reared on R. indica. Aphids from B. campestris clones produced more offspring (mean ± sem = 44.50 ± 7.47) than those reared on B. juncea (mean ± sem = 29.40 ± 2.31), and these produced significantly more offspring compared to adult females reared on R. indica (mean ± sem = 19.60 ± 3.37). The intrinsic rate of increase (Rmax) of L. pseudobrassicae aphids on the three plant species was nearly the same. However, the net reproductive rate (Ro) was highest in the clone reared on B. juncea and the lowest in the clone from R. indica. Ro in the L. pseudobrassicae clone from B. juncea was higher (mean ± sem = 59.87 ± 6.04) than the R. indica (mean ± sem = 24.46 ± 3.76) and B. campestris clones (mean ± sem = 45.20 ± 5.42) (Table 1).
Fig. 1. Mean values of growth rate (GR) (a), carrying capacity (K) (b) and time to attain carrying capacity (Tk) (c) in apterous L. pseudobrassicae populations from three host plant species, B. campestris (Bc), B. juncea (Bj) and R. indica (Ri). Bars indicate standard error. Different letters accompanying bars denote significant differences between mean values by Tukey's multiple range test at P b 0.05.
or failed to reach the third generation were considered to be ‘failure’ in colonization. Data analysis Data of the third generation aphids, wherever available, were used to compare results. This was done to allow time for acclimatization to the laboratory rearing conditions. All microscopic measurements were converted to mm using a stage micrometer. All weights in this study were taken in a Mettler microbalance
Table 1 Mean values of biological attributes recorded in apterous aphids of L. pseudobrassicae clones from three different host plant species. Biological attributes BW (mg) AW (mg) DT (days) GT (days) RT (days) F (no.) Rmax R0
Mean ± SEM⁎ of measurements from three host plant species (n = 20) B. campestris
B. juncea
R. indica
0.037 ± 0.000 a 0.49 ± 0.004 a 7.0 ± 0.211 a 7.95 ± 0.189 a 9.60 ± 1.392 a 44.50 ± 7.47 a 0.28 ± 0.002 a 45.20 ± 5.42 a
0.04 ± 0.000 a 0.53 ± 0.031 a 7.98 ± 0.171 b 8.60 ± 0.163 b 13.80 ± 1.289 b 29.40 ± 2.31 b 0.28 ± 0.007 a 59.87 ± 6.04 b
0.02 ± 0.001 b 0.25 ± 0.007 b 5.90 ± 0.100 c 6.10 ± 0.233 c 8.40 ± 1.447 a 19.60 ± 3.374 c 0.26 ± 0.001 b 24.46 ± 3.76 c
⁎ Different letters with mean values in a row indicate significant differences between the treatments by Tukey's multiple range test at P b 0.05. BW = birth weight, AW = weight at the final molt, DT = developmental time, GT = generation time, RT = reproductive duration, F = fecundity, Rmax = intrinsic rate of increase, R0 = net reproductive rate.
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Morphological variations Results presented in Table 2 show that the wingless aphids of L. pseudobrassicae clones from B. campestris and B. juncea were bigger and their antennae, ultimate rostral segments, siphunculi and fore femora were significantly longer than the aphids from R. indica clone. Aphid clones from B. campestris and B. juncea, however, did not show distinguishable differences in morphometry. Host transfer experiments Results presented in Table 3 show the success rates of colonization of L. pseudobrassicae clones when transferred from one host plant species to another host plant species of same genus or different genera of Cruciferae. When transferred to B. juncea and R. indica plants, L. pseudobrassicae clone aphids from B. campestris did not survive or perished in the third generation after initial success in the first generation and partial success in the second generation (Expt. I). When L. pseudobrassicae clones from B. juncea were transferred to B. campestris, the colonization success rate of aphids on the transferred hosts declined from 100% to 40% in the third generation and aphids did not survive the third generation on R. indica plants (Expt. II). When transferred to B. campestris and B. juncea plants, L. pseudobrassicae clones from the weed host, R. indica, failed to colonize and perished (Expt. III). Discussion This study has shown that L. pseudobrassicae populations have developed distinguishable ecological and biological variations in relation to different species of host plants. Reciprocal transfer of L. pseudobrassicae populations between host plant species was nearly impossible. Morphological variation in L. pseudobrassicae clone aphids between the two crop species of Brassica was not evident but the R. indica clone aphids showed significant differences in several characters in comparison to conspecifics from Brassica host plants. As a consequence, L. pseudobrassicae may be regarded as a genetically Table 2 Mean (mm) values of morphological characters in apterous aphids of L. pseudobrassicae clones from three host plant species. Morphological Characters BL BW ANT ANT III ANT VI PROB URS CAU SIPH FEM
Mean⁎ (range: minimum and maximum) of measurements from different host plants (n = 20) B. campestris
B. juncea
R. indica
1.87 a (1.55–2.37) 1.08 a (0.87–1.35) 1.39 a (1.10–1.79) 0.35 a (0.30–0.41) 0.43 a (0.47–0.65) 0.38 a (0.36–0.42) 0.10 a (0.09–11) 0.19 a (0.15–0.26) 0.24 a (0.20–0.26) 0.41 a (0.44–0.56)
1.99 a (1.81–2.03) 1.30 b (1.22–1.44) 1.25 a (1.11–1.37) 0.34 a (0.26–0.39) 0.39 a (0.33–0.52) 0.41 a (0.37–0.48) 0.10 a (0.09–0.11) 0.16 b (0.11–0.19) 0.22 a (0.20–0.24) 0.39 a (0.35–0.41)
1.38 b (1.26–1.59) 0.79 c (0.74–1.00) 0.77 b (0.70–1.18) 0.19 b (0.15–0.23) 0.28 b (0.24–0.37) 0.32 b (0.30–0.41) 0.08 b (0.07–0.09) 0.15 b (0.15–0.19) 0.18 b (0.15–0.20) 0.23 b (0.19–0.35)
⁎ Different letters with mean values in a row indicate significant differences between the treatments by Tukey's multiple range test at P b 0.05. BL = Body length, BW = maximum width of body, ANT = length of antenna, ANT III = antennal segment III, ANT VI = antennal segment VI, PROB = length of proboscis, URS = length of ultimate rostral segment, SIPH = length of siphunculus, CAU = length of cauda, FEM = length of fore femur.
Table 3 Success rate of colonization by apterous aphids of L. pseudobrassicae clones as a result of host transfer (host plants are denoted as: B. campestris = Bc, B. juncea = Bj, R. indica = Ri). Expt.
Field host
Laboratory host
I.
Bc
II.
Bj
III.
Ri
Bc Bj Ri Bj Bc Ri Ri Bc Bj
% success⁎ (n: based on colonization success in the respective generation) 1st generation
2nd generation
3rd generation
100 (20) 100 (20) 80 (16) 100 (20) 90 (18) 80 (16) 100 (20) 0 (0) 0 (0)
100 (20) 30 (6) 20 (4) 100 (20) 70 (14) 30 (6) 100 (20) – –
100 0 0 100 40 0 90 – –
(20) (0) (0) (20) (14) (0) (18)
χ2
NS ⁎⁎ ⁎⁎ NS ⁎⁎ ⁎⁎ NS – –
⁎ 0 = initially survived but failed to complete development or reproduce in that generation; – no aphid survived; χ2 Chi-square significance values for surviving aphids over the three generations: NS = P N 0.05. ⁎⁎ P b 0.01.
heterogeneous species infesting various host plants at different rates (Jaenike, 1981; Diehl and Bush, 1984).This implies that L. pseudobrassicae populations from B. campestris will not infect B. juncea or R. indica plants, and vice versa. This result agrees with earlier findings that described host plant-based variations in L. pseudobrassicae and several other aphid species from different parts of the world. According to Agarwala and Das (1998), L. pseudobrassicae aphids from radish hosts had shorter siphunculi, longer developmental time and lower intrinsic rate of increase compared to conspecifics from the Indian mustard host, although no host transfer results were provided. The present study has extended the knowledge of intra-specific variation in L. pseudobrassicae in relation to host plants. Ruiz-Montoya et al. (2005) described the morphological variation of populations of Brevicoryne brassicae associated with two host species, Brassica oleracea L. and B. campestris, occurring in the same habitat. Peppe and Lomonaco (2003) described host race formation in Myzus persicae on two Cruciferae host species, B. olearacea and Raphanus sativus L. Guldemond et al. (1994) and Agarwala and Das (2007) described the host races of Aphis gossypii aphids on different host plant species from Europe and Asia. Similarly, biotypes and sibling species were distinguished in Cryptomyzus galeopsidis (Guldemond, 1990) and Schizaphis graminum (Wilhoit and Mittler, 1991). Gorur (2005) and Gorur et al. (2005) showed that prevalent genotypic variations and phenotypic plasticity in A. fabae aphids and other herbivorous insects favor the selection of new host plants. These examples indicate that host plant divergence in insect populations favors the formation of host plant-specific races. Aphids and other phytophagous insects are known by intraspecific phenotypic variations in response to temperature, seasonal variation, natural enemies and host quality (Dixon et al., 1982; Agarwala, 2007). It is widely accepted that the fitness of an aphid clone is determined by plant nutritional quality and aphid nutritional biology (reviewed in Powell et al., 2006). Several studies indicate that reproductive decisions in aphids are made on the basis of plant cues, and that cue-based control of aphid reproduction is one of the determinants of fitness (Via, 1991; Caillaud and Via, 2000; Gabrys and Tjallingii, 2002). It is, therefore, argued that host plant preference and performance in aphids are correlated and that they directly influence the process of host plant selection in sympatric condition (Via and Hawthorne, 2002). Acknowledgments Authors are thankful to the Indian Council of Agricultural Research, New Delhi for financial support. Thanks are also extended to Prithwi Jyoti Bhowmik of the authors' laboratories for helps in the preparation of the manuscript.
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