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The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci Complex in China
Journal of Integrative Agriculture
February 2012
2012, 11(2): 197-205
RESEARCH ARTICLE
The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci Complex in China LI Xiao-xi1, 2, LI Shao-jian1, XUE Xia1, Muhammad Z Ahmed1, 3, REN Shun-xiang1, Andrew G S Cuthbertson4 and QIU Bao-li1 Department of Entomology, South China Agricultural University, Guangzhou 510640, P.R.China Polytechnic College, Hebei University of Science and Technology, Shijiazhuang 050018, P.R.China 3 Department of Genetics, University of Pretoria, Pretoria 0002, South Africa 4 The Food and Environment Research Agency, Sand Hutton YO41 1LZ, United Kindom 1 2
Abstract The sweetpotato whitefly Bemisia tabaci (Homoptera: Aleyrodidae) is a destructive pest of agriculture and horticulture worldwide. Recent phylogenetic analysis using mitochondrial cytochrome oxidase 1 sequences indicates that this whitefly is a species complex including at least 24 morphologically indistinguishable but genetically distinct cryptic species. In this study, the inter-species crosses of Middle East-Asia Minor 1 (MEAM1), Mediterranean (MED) and Asia II 7 cryptic species, which were referred to as B, Q and Cv biotypes before, were conducted in two different devices, leaf cages (7 cm3) and cylinder cages (280 cm3), and at three temperatures of 22, 30 and 38°C. Results indicated that no female progeny were produced in the reciprocal cross between MEAM1×Asia II 7, between MED×Asia II 7 cryptic species neither in leaf cage nor in cylinder cages, while 0.81 and 1.37% of females in the offspring were recorded in the reciprocal cross between MEAM1×MED in leaf cage experiments. Approximately 0.95-0.98% female progeny were recorded in the reciprocal cross between MEAM1×MED at 30°C, 0.77% female progeny were recorded in the single cross direction between MEAM1 × MED at 22°C, and no female progeny were found in their reciprocal cross at 38°C in leaf cage. Our findings indicated that neither space dimension nor temperature have a significant effect on the hybridization of different B. tabaci cryptic species. Key words: Bemisia tabaci, cross mating, cryptic species, reproductive isolation, space dimension, temperature
INTRODUCTION The sweetpotato whitefly Bemisia tabaci (Homoptera: Aleyrodidae) is one of the most globally destructive pests of numerous agricultural crops and ornamental plants (Byrne and Bellows 1991; De Barro et al. 2005). The host plants of B. tabaci have been reported to be more than 600 species (Gelman et al. 2007), with billions of dollars damage caused yearly on crops due to its direct
feeding on the plant phloem sap, transmission of more than 110 plant pathogenic viruses, and excretion of honeydew that leads to stickiness and growth of sooty mold (Heinz 1996; Henneberry et al. 1997, 2000; Jones 2003; Inbar and Gerling 2008; Mahadav et al. 2008). The complex genetic diversity of B. tabaci has been well recognized since the late 1950’s (Bird 1957), and it was considered as a complex species including more than 24 biotypes identified by various techniques (Perring 2001). However, recent research based on
Received 15 February, 2011 Accepted 2 June, 2011 Correspondence QIU Bao-li, Tel: +86-20-85283717, Fax: +86-20-85280316, E-mail: [email protected]
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the comparison of mitochondrial COI (mtCOI) sequences and using Bayesian analysis indicated that B. tabaci is more likely to be a cryptic species complex rather than a complex species (Boykin et al. 2007; De Barro et al. 2011). Furthermore, Dinsdale et al. (2010) reported that there is a clear break in the frequency distributions of mtCOI sequence divergence at 3.5%, and such a break suggests a barrier to gene flow among different genetic groups of B. tabaci and is indicative of species level separation. On the other hand, although more and more phylogenetic evidence continues to be revealed that B. tabaci is a species complex, it is premature to draw this conclusion without supporting biological data. Thus, many investigations on the inter-specific cross mating have been carried out, in which most of them showed that no female progeny were produced in the inter-specific cross experiments (Perring et al. 1993; Bedford et al. 1994; Liu et al. 2007; Zang and Liu 2007; Luan et al. 2008; Xu et al. 2010; Wang et al. 2010; Sun et al. 2011; Wang et al. 2011; Liu et al. 2012), while in a few studies in which female progeny were recorded, either the progeny were found to be sterile or the biology of their survivorship and reproduction was not further studied (Ronda et al. 1999; De Barro and Hart 2000; Ma et al. 2004). In China, the first record of B. tabaci was in the 1940’s (Chou 1949), but it was not considered as a serious pest until outbreaks in the middle of the 1990’s due to the invasion of Middle East-Asia Minor 1 (MEAM1, previously referred to as biotype B) (Qiu et al. 2003b; Wu et al. 2003), and then the subsequent invasion of Mediterranean (MED, previously referred to as biotype Q) around 2003-2005 (Chu et al. 2006). According to the extensive field survey of Hu et al. (2011) and Guo et al. (2012), there are at least 13 indigenous cryptic species of B. tabaci in China, in addition to the alien MEAM1 and MED. Numerous crossing experiments have been conducted among these invasive and indigenous cryptic species in the past few years (Ruan et al. 2007; Zang and Liu 2007; Wang et al. 2010, 2011; Xu et al. 2010; Sun et al. 2011). All the data gained in the cross experiments indicated a pattern of reproductive isolation between different cryptic species of B. tabaci in China (Wang et al. 2011; Liu et al. 2012). Although numerous crossing studies have been conducted, suggesting that B. tabaci is a species complex with a certain number of cryptic species, most of
the experiments were done under fixed and optimal conditions including suitable temperature, photoperiod and humidity. It is noteworthy that previous studies have shown that the ecology and biology of B. tabaci can be affected by many biotic and abiotic factors. For example, temperature, humidity, host plant, natural enemies and other competitors in the same niche can have various effects on the development, survivorship and reproduction of B. tabaci (Berlinger et al. 1996; Tsai and Wang 1996; Wang and Tsai 1996; Qiu et al. 2005, 2007; Xu 2006; Iida et al. 2009). Thus, a question raised is, do these biotic and abiotic factors also have some positive or negative effects on the cross mating behavior of different B. tabaci cryptic species? This study undertakes the first validation of this question by setting up a series of experiments to investigate B. tabaci inter-specific cross mating. Firstly, we established the laboratory colonies of MEAM1, MED and Asia II 7 cryptic species collected in China. Their inter-specific cross matings were then investigated in two different test arenas and at three different temperatures. Our current work was not to repeat the previously reported investigations, but to evaluate the effect of space dimension and temperature on the crossing of different cryptic species of B. tabaci.
RESULTS Effects of space dimension on the cross mating of B. tabaci The results of inter-species cross among the three cryptic species of B. tabaci in leaf cages are shown in Table 1. Differences were found among the mean numbers of progeny of different cryptic species cross groups (F8, 141=2.08, P=0.0418) although most of the difference were not significant to each other. The sex ratio identification indicated that all the F1 progeny in the inter-specific crosses of MEAM1 ×Asia II 7 , Asia II 7 ×MEAM1 , MED ×Asia II 7 and Asia II 7 ×MED were males, whereas in the MEAM1 ×MED inter-species cross, two female progeny were recorded, and in the MED ×MEAM1 inter-specific cross, 5 female progeny were recorded in total. The female percentage in the offspring of MEAM1 ×MED and MED ×MEAM1 inter-species crosses were 0.81 and 1.37%, © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
respectively, while the female percentages among the three intra-specific crosses ranged from 54.2 to 65.6% and were not significantly different to each other. The few hybrid females produced from the MEAM1×MED cross were reared for 5 d to observe their reproduction. These hybrid females produced no F2 eggs, indicating that the rare F1 female adults were all sterile. The results of the inter-species crosses among the
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three cryptic species conducted using plastic cylinder cages are shown in Table 2. Similar to the leaf cage experiments, obvious differences were found among the mean numbers of progeny in the cross and control treatments (F8, 141=4.95, P<0.0001). However, in all the six inter-specific crosses, no female progeny was produced; in contrast 54-66% females were produced in the intra-specific control treatments.
Table 1 Progeny produced in the cross experiments among the B. tabaci MEAM1, MED, and Asia II 7 cryptic species undertaken in leaf cage experiments Cryptic species
n
Number of progeny per pair of adults (M±SE)1)
Asia II 7 ×MEAM1 Asia II 7 ×MED MEAM1 ×Asia II 7 MEAM1 ×MED MED ×MEAM1 MED ×Asia II 7 Asia II 7 ×Asia II 7 MEAM1 ×MEAM1 MED ×MED
20 20 20 20 20 20 10 10 10
14.0±4.2 ab 17.8±4.6 a 16.8±3.4 ab 11.9±1.5 b 14.6±2.0 ab 16.2±2.7 ab 16.1±2.9 ab 13.4±3.1 ab 15.2±3.5 ab
Female percentage in progeny per pair of adults (M±SE) 2) 0.0 0.0 0.0 0.813) 1.374) 0.0 54.2±4.1 a 65.6±2.5 a 59.7±4.8 a
1)
F 8, 141=2.08, P=0.0418. F2, 27=2.88, P=0.29. 3) In this inter-species cross, 247 F1 adults were produced in the 20 replicates, and only in 2 replicates one female progeny was found in each, the SE was not calculated due to the few positive data of female percentage recorded in the experiments. 4) For this inter-species cross, 292 F 1 adults in total were produced; in 4 of the 20 replicates 1, 1, 2 and 1 female progeny were produced, the remaining 16 replicates produced only males; the SE was not calculated. 2)
Table 2 Progeny produced in the crossing experiments among the B. tabaci MEAM1, MED, and Asia II 7 cryptic species conducted using plastic cylinder cages Cryptic species Asia II 7 ×MEAM1 Asia II 7 ×MED MEAM1 × Asia II 7 MEAM1 × MED MED ×Asia II 7 MED ×MEAM1 Asia II 7 ×Asia II 7 MEAM1 ×MEAM1 MED ×MED 1) 2)
n
Number of progeny per pair of adults (M±SE)1)
20 20 20 20 20 20 10 10 10
23.5±2.2 a 20.7±1.0 abc 19.6±1.7 abcd 15.8±1.2 d 16.3±1.5 d 22.6±1.1 ab 19.2±2.26 bcd 21.3±1.7 abc 18.0±1.3 cd
Female percentage in progeny per pair of adults (M±SE, %)2) 0.0 0.0 0.0 0.0 0.0 0.0 57.6±5.3 a 62.4±4.7 a 55.3±6.9 a
F8, 141=4.95, P<0.001. F2, 27=1.81, P=0.37.
Effects of temperature on the cross mating of B. tabaci cryptic species Temperature affected the numbers of progeny as well as sex ratios among the various crosses (Table 3). In general, the number of progeny was the highest at 30°C, followed by 22°C, with the lowest at 38°C, regardless of the type of inter- and intra-specific crosses. Significant differences were noted among the different crosses and intra-specific controls conducted at 22°C (F8, 141=31.64,
P<0.001) and at 38°C (F8, 141=13.46, P<0.001), but not among those at 30°C (F8, 141=1.71, P=0.27). As for the sex ratios of the progeny, female progeny were found in the crosses of MEAM1 ×MED with percentages of 0.77 and 0.98% at 22 and 30°C respectively, and female progeny were also found in the cross of MED ×MEAM1 with a percentage of 0.95% at 30°C. However, no females were recorded in the reciprocal crosses of MEAM1×Asia II 7 and MED×Asia II 7 at either 22, 30 or 38°C as well as in the cross of MEAM1×MED at 38°C .
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Table 3 Progeny produced in the cross experiments among the B. tabaci MEAM1, MED and Asia II 7 cryptic species conducted using leaf cages at different temperatures Cryptic species Asia II 7 ×MEAM1 Asia II 7 ×MED MEAM1 ×Asia II 7 MEAM1 ×MED MED ×Asia II 7 MED ×MEAM1 Asia II 7 ×Asia II 7 MEAM1 ×MEAM1 MED ×MED
n 20 20 20 20 20 20 10 10 10
Number of progeny per pair of adults under different temperatures (M±SE)1) 22°C 30°C 38°C 8.6±2.0 b 9.3±2.1 b 11.4±2.5 ab 13.1±1.4 a 14.9±1.7 a 10.2±1.2 ab 11.8±2.7 ab 13.7±2.5 a 14.3±1.6 a
21.3±3.8 a 23.8±5.0 a 24.7±4.6 a 25.6±2.8 a 23.9±3.2 a 26.4±4.1 a 25.6±2.6 a 27.6±4.4 a 29.5±3.2 a
8.1±2.3 b 8.2±2.6 b 9.4±2.5 ab 12.3±0.9 a 9.8±2.3 ab 8.2±1.3 b 12.6±1.1 a 11.1±2.3 ab 13.6±1.5 a
Female percentage in progeny per pair of adults under different temperatures (M±SE, %)2) 22°C 30°C 38°C 0.0 0.0 0.0 0.77 3) 0.0 0.0 56.7±2.9 a 65.4±3.3 a 60.2±5.2 a
0.0 0.0 0.0 0.98 4) 0.0 0.95 5) 56.2±2.3 a 62.8±3.7 a 55.6±4.9 a
0.0 0.0 0.0 0.0 0.0 0.0 52.4±3.8 a 55.1±4.6 a 57.2±2.1 a
F8, 141=31.64, P<0.001 for 20°C treatment. F8, 141=1.71, P=0.27 for 30°C treatment. F8, 141=13.46, P<0.001 for 38°C treatment. F2, 27=1.44, P=0.46 for 20°C treatment. F2, 27=0.986, P=0.82 for 30°C treatment. F2, 27=1.69, P=0.41 for 38°C treatment. 3) Two female progeny were found among the 261 adults in total. 4) Five female progeny were found among the 551 adults in total. 5) Five female progeny were found among the 528 adults in total. 1) 2)
Daily fecundity and progeny sex ratio of B. tabaci in the crossing experiments The average daily fecundity of MEAM1 and MED in their cross experiments increased continuously with time in the first 5 d after emergence (Fig. 1). In the interspecies cross of MEAM1 ×MED , 9 female progeny (2, 2 and 5 on day 2, 4 and 5, respectively) were produced in the 5-day investigation. In the inter-species cross of MED ×MEAM1 , 12 female progeny (3 and 9 on day 4 and 5, respectively) were recorded in 5 of 10 replicates in the 5-d investigation. In contrast, the numbers of female progeny produced per female in the intra-species treatments of MEAM1 and MED were substantially higher. In addition, female progeny were produced everyday in the intra-species treatments, while in the inter-species crosses female progeny were mostly produced in the last two days during the investigation.
DISCUSSION The destructive damage of B. tabaci on world agriculture and horticulture has been widely recognized. However, the enduring debate about the species status of B. tabaci has lasted for almost two decades. Crossing experiments are thought to be the most useful and necessary way to clarify the species status of B. tabaci. So far, convincing data has been obtained from sufficient cross experiments of different B. tabaci genetic groups to support the conclusion that B. tabaci is a
Fig. 1 The averaged number of female progeny produced per pair of adults daily in the cross of B. tabaci MEAM1 and MED cryptic species.
species complex containing a number of morphologically indistinguishable cryptic species (Costa et al. 1993; Perring et al. 1993; Bedford et al. 1994; Ronda et al. 1999; Luan et al. 2008; Crowder et al. 2010; Wang et al. 2010, 2011; Xu et al. 2010; Sun et al. 2011; Liu et al. 2012), a conclusion drawn from a phylogenetic view based on the mtCOI sequences of these B. tabaci cryptic species (Boykin et al. 2007; Dinsdale et al. 2010; De Barro et al. 2011). The behavior and efficiency of insect courtship and reproduction are quite complex, and our knowledge on the cross mating of different B. tabaci cryptic species is still limited. Most of the cross experiments previously reported were done in a fixed set of leaf cages,
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The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
no studies have been reported about the effects of the size of the test arena and temperature on the cross mating of B. tabaci, thus the results from the current study are expected to expand our knowledge in this area. In the present study, few female progeny were found in the cross of MEAM1 and MED when the hybridizations were conducted in leaf cages, while no female progeny were recorded in the crosses conducted using larger cylinder cages. The volumes of the cylinder cages used in our cross experiments are 40 times larger than that of a leaf cage, and we found that the heterogeneous individual adults of MEAM1 and MED B. tabaci in the cylinder cages were usually more further apart from each other, either on the same leaf or on different leaves during the 48 h observation, compared to their locations when enclosed in smaller leaf cages. This difference in terms of separation between individuals may have contributed to the different results obtained between trials using the two types of cages. Our findings were also consistent with the field surveys in Israel, in which B and Q populations were found to co-exist in the field in many locations, but no hybridization or geneflow occurred between the two (Horowitz et al. 2003; Khasdan et al. 2005; Elbaz et al. 2010). Meanwhile, the rare female offspring were mostly produced in the later period of observation (Fig. 1), which occurred possibly because heterogeneous copulations were only forced to happen later in the time interval, a speculation that seemed to be supported by the behavioral observation on these two species by Sun et al. (2011). Similar results have been reported between crosses of ZHJ1and ZHJ2 (Xu et al. 2010), between MEAM1 and MED (Sun et al. 2011), among MED, ZHJ1 and ZHJ2 (Wang et al. 2010), and among MEAM1, MED, Asian II 7, ZHJ1, ZHJ2, and ZHJ3 (Wang et al. 2011). In the studies above, all the data showed that more copulation events were recorded in group mating than in singlepair mating, indicating that the population density may affect the rate of copulation. In other words, the arena size may have some marginal effects on the mating activities of B. tabaci cryptic species. The rarer events of copulation in lower population densities may be due to the larger space for individual movements, although the effects of space or distance on the chemical attraction between whitefly heterosexual individuals has not been investigated to date.
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Temperature has been reported to have significant effects on the development, survivorship, and reproduction of B. tabaci, with 26-29°C being the optimum temperature (Wang and Tsai 1996; Qiu et al. 2003a). In our current study, the cross experiments of MEAM1 and MED were conducted at three temperatures, of which 30°C was deemed most suitable while 22 and 38°C were less suitable for the mating and reproduction of B. tabaci. It is assumed that adverse temperature may reduce the mating desire and behavior of different cryptic species. This inference would warrant further investigation through mating behavior observations of B. tabaci in the laboratory.
CONCLUSION To date, numerous studies have proved that B. tabaci is a species complex containing more than 24 morphologically indistinguishable cryptic species, and that all the cryptic species are completely reproductively isolated. Our results indicated that probability of copulation and hybridization in crossing experiments between cryptic species of B. tabaci may be marginally affected by the size of the test arena and temperature. Knowledge of such effects of experimental factors may help in understanding the variations obtained in this type of crossing studies.
MATERIALS AND METHODS Whitefly populations and host plants Adults of B. tabaci MEAM1 were collected in 2006 on eggplant Solanum melongena at the experimental farm of South China Agricultural University (SCAU) in Guangzhou, China. The Asia II 7 cryptic species was collected in 2005 from the ornamental plant laurel Codiaeum variegatum in Changban, Guangzhou, while the MED cryptic species was collected from pepper plant Capsicum frutescens from Nantong, Jiangsu, China, in 2007. All the three cryptic species were rared on their original host plants in separate cages at SCAU at ambient temperature, photoperiod and humidity. Their cryptic species were identified by mitochondrial CO1 sequencing according to Qiu et al. (2009). Cowpea (Vigna unguiculata cv. Baofeng) was used in the cross experiments. Cowpea is an important vegetable in China. Previous investigations in the laboratory and
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field showed that cowpea is a preferred host plant for B. tabaci MEAM1, MED and Asia II 7 cryptic species (Musa and Ren 2005; Xu 2006). Plants were first geminated in a plastic pot, and after 1 wk all the seedlings were transplanted into 1.3-L plastic pots containing sterilized soil-sand mixture in a whitefly-proof greenhouse. Plants were watered as required. Natural daylight was supplemented with sodium lamps to ensure a photoperiod of 16 L:8 D. Plants were used for experiments when they were two weeks old with 4-5 true leaves. Before use in the cross experiments, all the leaves from each plant were carefully examined under a 30× binocular stereoscope twice to ensure that only insectfree plants were used.
Collection of the newly emerged virgin adults of B. tabaci To ensure that the B. tabaci adults were virgin for the cross experiments, in the evening prior to the day of initiating cross experiments, plant leaves with whitefly pupae were cut by scissors and whitefly pupae were put into glass tubes (5 cm length×0.5 cm diameter) individually. The sexes of newly emerged virgin adults were identified according to the description of Gill et al. (1990) the following morning.
Effects of size of test arena on the cross mating of B. tabaci Two sets of experiments were used for the whitefly crosses in the current study. In the first set, a leaf cage was used. The dimension of the leaf cage was 2 cm in height×3 cm in diameter (Fig. 2-A). The leaf cages were fixed to the under surface of leaves with clips. The internal volume of a leaf cage was approximately 7 cm3. In the second set, a plastic column (10 cm in height×6 cm in diameter) was used to make a cylinder cage for the cross experiments (Fig. 2-B). These plastic cylinders were covered with nylon gauze on the top, bottom and side, with a T shape opening in the nylon gauze at the bottom to allow placement of the test plant. Each cylinder cage enclosed a whole plant of cowpea planted in a pot. The internal volume of the cylinder cage was approximately 280 cm3. Single pair cross matings were conducted in all the experiments. In each replicate using either a leaf cage or cylinder cage, one pair of newly emerged virgin female and male were introduced. The adults were collected after 48 h of mating and reproduction and stored in 95% ethanol at -20ºC for later species identification if they produced female offspring. The F 1 eggs were counted and allowed to develop through to adults on the leaves. Then the individuals were sexed and also counted for the calculation of F1 survivorships if they emerged as adults. In each direction of one cross combination, i.e., MEAM1 ×MED or MED ×
Fig. 2 Sketch diagrams of the leaf cage and the plastic cylinder used in the current study. A, leaf cage. 1, leaf petiole; 2, leaf undersurface; 3, leaf cage; 4, nylon gauze for ventilation; 5, hole for introducing whiteflies. B, plastic cylinder. 1, whole plastic cylinder; 2 and 3, nylon gauze for ventilation; 4, hole for introducing whiteflies and the cover; 5, cowpea plant.
MEAM1 , 20 replicates of inter-species crosses were conducted, and 10 replicates of intra-specific matings were also conducted as control. When there were female F 1 adults recorded, each of them was introduced into a leaf cage on a new cowpea plant with a male, 5 d later the leaves were checked under a binocular stereoscope to see if there were F2 eggs. All the experiments except the molecular identification were conducted at (26±1)ºC, with photoperiod of 16 L:8 D and (75±10)% RH.
Effects of temperature on the cross mating of B. tabaci Crossing experiments were conducted at three temperatures of 22, 30 and 38ºC, respectively, to examine whether
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The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
inter-specific crossings would be affected by temperature. The newly emerged females and males were collected as described above. Leaf cages were used for the experiments. The protocols for conducting inter-specific crosses (20 replicates in each cross) and intra-species controls (10 replicates in each control treatment) were the same as above. All the experiments in this section were done with a photoperiod of 16 L:8 D and (75±10)% RH.
Daily fecundity and progeny sex ratio of B. tabaci in the cross experiments Ten pairs of newly emerged females and males from different cryptic species were introduced into 10 leaf cages, one pair per cage, and reared for 5 d (the first 5 d after emergence) to check the daily fecundity and the sex ratio of the F 1 progeny. The pair of adults in each replicate were transferred to a new leaf cage and the number of eggs deposited in each replicate was recorded every 24 h. All the eggs were allowed to develop to adults on leaves for sex ratio calculation. Six pairs of intra-specific mating of MEAM1 and MED were used as controls.
Data analysis For the cross experiments, the fecundity of the F 0 generation, the sex ratios of the F1 generation in the intraand inter-species cross experiments were analyzed using a one-way ANOVA, and the means were separated using the least significant difference test (LSD) at P=0.05. All the proportion data were transformed by square root to meet the requirements of normality and homogeneity of variances before analysis. All data were analyzed using the SAS Systems Release 8.01 for Windows (SAS Institute, 2001).
Acknowledgements This research was funded by the National Basic Research Program of China (2009CB119203), the National Natural Science Foundation of China (31071732) and the Foundation of Lingnan Fruit Innovation Team of Guangdong Province, China (2009-14).
References De Barro P J, Hart P J. 2000. Mating interactions between two biotypes of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) in Australia. Bulletin of Entomological Research, 90, 103-112. De Barro P J, Trueman J W H, Frohlich D R. 2005. Bemisia argentifolii is a race of B-tabaci (Hemiptera: Aleyrodidae) the molecular genetic differentiation of B-tabaci populations around the world. Bulletin of
203
Entomological Research, 95, 193-203. De Barro P J, Liu S S, Boykin L M, Dinsdale A B. 2011. Bemisia tabaci: A statement of species status. Annual Review of Entomology, 56, 1-19. Bedford I D, Briddon R W, Brown J K, Rosell R C, Markham P G. 1994. Geminivirus transmission and biological characterization of Bemisia tabaci (Gennadius) biotypes from different geographic regions. Annals of Applied Biology, 125, 311-325. Berlinger M J, Lehmann-Sigura N, Taylor R A J. 1996. Survival of Bemisia tabaci adults under different climatic conditions. Entomologia Experimentalis et Applicata, 80, 511-519. Bird J. 1957. A Whitefly Transmitted Mosaic of Jatropha G o s s y p i f o l i a ( Te c h n i c a l p a p e r ) . A g r i c u l t u r a l Experimental Station, University of Puerto Rico. Mayagüez, Puerto Rico. 22, 1-35. Boykin L M, Shatters Jr R G , Rosell R C, McKenzie C L, Bagnall R A, McKenzie C L, De Barro P J, Frohlich D R. 2007. Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of mitochondrial COI DNA sequences. Molecular Phylogenetics and Evolution, 44, 1306-1319. Byrne D N, Bellows Jr T S. 1991. Whitefly biology. Annual Review of Entomology, 36, 431-457. Chou I. 1949. List of whitefly species in China. Entomologia Sinica, 3, 1-18. (in Chinese) Chu D, Zhang Y J, Brown J K, Cong B, Xu B Y, Wu Q J, Zhu G R. 2006. The introduction of the exotic Q biotype of Bemisia tabaci from the Mediterranean region into China on ornamental crops. Florida Entomologist, 89, 168-174. Costa H S, Brown J K, Sivasupramaniam S, Bird J. 1993. Regional distribution, insecticide resistance and reciprocal crosses between the ‘A’ and ‘B’ biotypes of Bemisia tabaci. Insect Science and Its Application, 14, 255-266. Crowder D W, Sitvarin M I, Carriere Y. 2010. Mate discrimination in invasive whitefly species. Journal of Insect Behavior, 23, 364-380. Dinsdale A, Cook L, Riginos C, Buckley Y M, De Barro P. 2010. Refined global analysis of Bemisia tabaci (Hemiptera: Ternorrhyncha: Aleyrodoidea: Aleyrodidae) mitochondrial cytochrome oxidase 1 to identify species level genetic boundaries. Annals of the Entomological Society of America, 103, 196-208. Elbaz M, Lahav N, Morin S. 2010. Evidence for pre-zygotic reproductive barrier between the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae). Bulletin of Entomological Research, 100, 581-590. Gelman D B, Pszczolkowski M A, Blackburn M B, Ramaswamy S B. 2007. Ecdysteroids and juvenile hormones of whiteflies, important insect vectors for plant viruses. Journal of Insect Physiology, 53, 274284. Gill R J. 1990. The morphology of whiteflies. In: Gerling D,
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The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
ed., Whiteflies: Their Bionomics, Pest Status and Management. Intercept Ltd., Andover. pp. 13-46. Guo X J, Rao Q, ZHANG F, Luo C, Zhang H Y, Gao X W. 2012. Diversity and genetic differentiation of the whitefly Bemisia tabaci species complex in China based on mtDNA CO1 and cDNA-AFLP analysis. Journal of Integrative Agriculture, 11, 206-214. Heinz K. 1996. Predators and parasitoids as biological control agents of Bemisia in greenhouses. In: Gerling D, Mayer R T, eds., Bemisia, 1995: Taxonomy, Biology, Damage Control and Management. Intercept Ltd., Andover, Hants, UK. pp. 435-449. Henneberry T J, Toscano N C, Perring T M, Faust R M. 1997. Preface. In: Henneberry T J, Toscano N C, Perring T M, Faust R M, eds., Silverleaf Whitefly, Supplement to the Five-Year National Research and Action Plan: Progress, Review, Technology Transfer, and New Research and Action Plan (1997-2001). USDA, Washington D.C. p. 2. Henneberry T J, Jech L F, Hendrix D L. 2000. Bemisia argentifolii (Homoptera: Aleyrodidae) honeydew and honeydew sugar relationships to sticky cotton. Southwestern Entomologist, 25, 1-14. Horowitz A R, Denholm I, Gorman K, Cenis J L, Kontsedalov S, Ishaaya I. 2003. Biotype Q of Bemisia tabaci identified in Israel. Phytoparasitica, 31, 94-98. Hu J, De Barro P, Zhao H, Wang J, Nardi F, Liu S S. 2011. An Extensive field survey combined with a phylogenetic analysis reveals rapid and widespread invasion of two alien whiteflies in China. PLoS ONE, 6, e16061. Iida H, Kitamura T, Honda K. 2009. Comparison of egghatching rate, survival rate and development time of the immature stage between B and Q biotypes of Bemisia tabaci (Gennadius) (Homoptera:Aleyrodidae) on various agricultural crops. Applied Entomology and Zoology, 44, 267-273. Inbar M, Gerling D. 2008. Plant-mediated interactions between whiteflies, herbivores, and natural enemies. Annual Review of Entomology, 53, 431-448. Jones D R. 2003. Plant viruses transmitted by whitefly. European Journal of Plant Pathology, 109, 195-219. Khasdan V, Levin I, Rosner A, Morin S, Kontsedalov S, Maslenin L, Horowitz A R. 2005. DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics. Bulletin of Entomological Research, 95, 605-613. Liu S S, De Barro P J, Xu J, Luan J B, Zang L S, Ruan Y M, Wan F H, 2007. Asymmetric mating interactions drive widespread invasion and displacement in a whitefly. Science, 318, 1769-1772. Liu S S, Colvin J, De Barro P J. 2012. Species concepts as applied to the whitefly Bemisia tabaci systematics: how many species are there? Journal of Integrative Agriculture, 11, 176-186. Luan J B, Ruan Y M, Zang L, Liu S S. 2008. Precopulation
204
intervals, copulation frequencies, and initial progeny sex ratios in two biotypes of whitefly, Bemisia tabaci. Entomologia Experimentalis et Applicata, 129, 316324. Ma D, Hadjistylli M, Gorman K, Denholm I, Devine G. 2004. Pre- and post-zygotic breeding incompatibilities between B and Q biotypes of Bemisia tabaci. In: Proceeding of the 2nd European Whitefly Symposium. 5-9 October, 2004, Cavtat, Croatia. pp. 13-14. Mahadav A, Gerling D, Gottlieb Y, Czosnek H, Ghanim M. 2008. Parasitization by the wasp Eretmocerus mundus induces transcription of genes related to immune response and symbiotic bacteria proliferation in the whitefly Bemisia tabaci. BMC Genomics, 9, 342. Musa P D, Ren S X. 2005. Development and reproduction of Bemisia tabaci (Homoptera: Aleyrodidae) on three bean species. Insect Science, 12, 25-30. Perring T M. 2001. The Bemisia tabaci species complex. Crops Protection, 20, 725-737. Perring T M, Copper A D, Rodriguez R J, Farrar C A, Bellows T S. 1993. Identification of a whitefly species by genomic and behavioral studies. Science, 259, 74-77. Qiu B L, De Barro P J, Ren S X. 2005. Development, survivorship and reproduction of Eretmocerus sp. nr. furuhashii (Hymenoptera: Aphelinidae) parasitizing Bemisia tabaci (Hemiptera: Aleyrodidae) on glabrous and non-glabrous host plants. Bulletin of Entomological Research, 95, 313-319. Qiu B L, De Barro P J, Ren S X, Xu C X. 2007. Effect of temperature on the life history of Eretmocerus sp. nr. furuhashii, a parasitoid of Bemisia tabaci. BioControl, 52, 733-746. Qiu B L, Chen Y P, Liu L, Peng W L, Li X X, Ahmed M Z, Mathur V, Du Y Z, Ren S X. 2009. Identification of three major Bemisia tabaci biotypes in China based on morphological and DNA polymorphisms. Progress in Natural Science, 19, 713-718. Qiu B L, Ren S X, Mandour N S, Lin L. 2003a. Effect of temperature on the development and reproduction of Bemisia tabaci B biotype (Homoptera: Aleyrodidae). Entomologia Sinica, 10, 43-49. Qiu B L, Ren S X, Wen S Y, Mandour N S. 2003b. Biotype identification of the populations of Bemisia tabaci (Homoptera: Aleyrodidae) in China using RAPD-PCR. Acta Entomologica Sinica, 46, 605-608. Ronda M A, Adan A, Cifuentes D, Cenis J L, Beitia F. 1999. Laboratory evidence of interbreeding between biotypes of Bemisia tabaci (Homoptera: Aleyrodidae) present in Spain. In: V11th International Plant Vir us Epidemiology Symposium-Plant Virus Epidemiology: Current status and Future Prospects. Aguadulce, 1116 April, 1999, Spain. pp. 83-84. Ruan Y M, Luan J B, Zang L S, Liu S S. 2007. Observing and recording copulation events of whiteflies on plants using a video camera. Entomologia Experimentalis et Applicata, 124, 229-233.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
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SAS Institute. 2001. SAS/STAT User’s Guide. SAS Institute, Cary, NC. Sun D B, Xu J, Luan J B, Liu S S. 2011. Reproductive incompatibility between the B and Q biotypes of the whitefly Bemisia tabaci in China: genetic and behavioral evidence. Bulletin of Entomological Research, 101, 211-220. Tasi J H, Wang K. 1996. Development and reproduction of Bemisia argentifolii (Homoptera: Aleyrodidae) on five host plants. Environmental Entomology, 25, 810-816. Wang K H, Tsai J H. 1996. Temperature effect on development and reproduction of silverleaf whitefly (Homoptera: Aleyrodidae). Annals of the Entomological Society of America, 89, 375-384. Wang P, Ruan Y M, Liu S S. 2010. Crossing experiments and behavioral observations reveal reproductive incompatibility among three putative species of the whitefly Bemisia tabaci. Insect Science, 17, 508-516. Wang P, Sun D B, Qiu B L, Liu S S. 2011. The presence of six cryptic species of the whitefly Bemisia tabaci complex in China as revealed by crossing experiments.
LI Xiao-xi et al.
Insect Science, 18, 67-77. Wu X X, Li Z X, Hu D X, Shen Z R. 2003. Identification of Chinese populations of Bemisia tabaci (Gennadius) by analyzing the ribosomal ITS1 sequence. Progress in Natural Science, 13, 276-281. Xu C X. 2006. The intra- and inter-species competition of whitefly Bemisia tabaci and two-spotted mite Tetranychus urticae. Ph D thesis, South China Agriculutural University, Guangzhou. pp. 5-48. (in Chinese) Xu J, De Barro P J, Liu S S. 2010. Reproductive incompatibility among genetic groups of Bemisia tabaci supports the proposition that the whitefly is a cryptic species complex. Bulletin of Entomological Research, 100, 359-366. Zang L S, Liu S S. 2007. A comparative study on mating behavior between the B biotype and a non-B populations of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) from Zhejiang, China. Journal of Insect Behavior, 20, 157-171. (Managing editor SUN Lu-juan)
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2012, 11(2): 197-205
RESEARCH ARTICLE
The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci Complex in China LI Xiao-xi1, 2, LI Shao-jian1, XUE Xia1, Muhammad Z Ahmed1, 3, REN Shun-xiang1, Andrew G S Cuthbertson4 and QIU Bao-li1 Department of Entomology, South China Agricultural University, Guangzhou 510640, P.R.China Polytechnic College, Hebei University of Science and Technology, Shijiazhuang 050018, P.R.China 3 Department of Genetics, University of Pretoria, Pretoria 0002, South Africa 4 The Food and Environment Research Agency, Sand Hutton YO41 1LZ, United Kindom 1 2
Abstract The sweetpotato whitefly Bemisia tabaci (Homoptera: Aleyrodidae) is a destructive pest of agriculture and horticulture worldwide. Recent phylogenetic analysis using mitochondrial cytochrome oxidase 1 sequences indicates that this whitefly is a species complex including at least 24 morphologically indistinguishable but genetically distinct cryptic species. In this study, the inter-species crosses of Middle East-Asia Minor 1 (MEAM1), Mediterranean (MED) and Asia II 7 cryptic species, which were referred to as B, Q and Cv biotypes before, were conducted in two different devices, leaf cages (7 cm3) and cylinder cages (280 cm3), and at three temperatures of 22, 30 and 38°C. Results indicated that no female progeny were produced in the reciprocal cross between MEAM1×Asia II 7, between MED×Asia II 7 cryptic species neither in leaf cage nor in cylinder cages, while 0.81 and 1.37% of females in the offspring were recorded in the reciprocal cross between MEAM1×MED in leaf cage experiments. Approximately 0.95-0.98% female progeny were recorded in the reciprocal cross between MEAM1×MED at 30°C, 0.77% female progeny were recorded in the single cross direction between MEAM1 × MED at 22°C, and no female progeny were found in their reciprocal cross at 38°C in leaf cage. Our findings indicated that neither space dimension nor temperature have a significant effect on the hybridization of different B. tabaci cryptic species. Key words: Bemisia tabaci, cross mating, cryptic species, reproductive isolation, space dimension, temperature
INTRODUCTION The sweetpotato whitefly Bemisia tabaci (Homoptera: Aleyrodidae) is one of the most globally destructive pests of numerous agricultural crops and ornamental plants (Byrne and Bellows 1991; De Barro et al. 2005). The host plants of B. tabaci have been reported to be more than 600 species (Gelman et al. 2007), with billions of dollars damage caused yearly on crops due to its direct
feeding on the plant phloem sap, transmission of more than 110 plant pathogenic viruses, and excretion of honeydew that leads to stickiness and growth of sooty mold (Heinz 1996; Henneberry et al. 1997, 2000; Jones 2003; Inbar and Gerling 2008; Mahadav et al. 2008). The complex genetic diversity of B. tabaci has been well recognized since the late 1950’s (Bird 1957), and it was considered as a complex species including more than 24 biotypes identified by various techniques (Perring 2001). However, recent research based on
Received 15 February, 2011 Accepted 2 June, 2011 Correspondence QIU Bao-li, Tel: +86-20-85283717, Fax: +86-20-85280316, E-mail: [email protected]
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the comparison of mitochondrial COI (mtCOI) sequences and using Bayesian analysis indicated that B. tabaci is more likely to be a cryptic species complex rather than a complex species (Boykin et al. 2007; De Barro et al. 2011). Furthermore, Dinsdale et al. (2010) reported that there is a clear break in the frequency distributions of mtCOI sequence divergence at 3.5%, and such a break suggests a barrier to gene flow among different genetic groups of B. tabaci and is indicative of species level separation. On the other hand, although more and more phylogenetic evidence continues to be revealed that B. tabaci is a species complex, it is premature to draw this conclusion without supporting biological data. Thus, many investigations on the inter-specific cross mating have been carried out, in which most of them showed that no female progeny were produced in the inter-specific cross experiments (Perring et al. 1993; Bedford et al. 1994; Liu et al. 2007; Zang and Liu 2007; Luan et al. 2008; Xu et al. 2010; Wang et al. 2010; Sun et al. 2011; Wang et al. 2011; Liu et al. 2012), while in a few studies in which female progeny were recorded, either the progeny were found to be sterile or the biology of their survivorship and reproduction was not further studied (Ronda et al. 1999; De Barro and Hart 2000; Ma et al. 2004). In China, the first record of B. tabaci was in the 1940’s (Chou 1949), but it was not considered as a serious pest until outbreaks in the middle of the 1990’s due to the invasion of Middle East-Asia Minor 1 (MEAM1, previously referred to as biotype B) (Qiu et al. 2003b; Wu et al. 2003), and then the subsequent invasion of Mediterranean (MED, previously referred to as biotype Q) around 2003-2005 (Chu et al. 2006). According to the extensive field survey of Hu et al. (2011) and Guo et al. (2012), there are at least 13 indigenous cryptic species of B. tabaci in China, in addition to the alien MEAM1 and MED. Numerous crossing experiments have been conducted among these invasive and indigenous cryptic species in the past few years (Ruan et al. 2007; Zang and Liu 2007; Wang et al. 2010, 2011; Xu et al. 2010; Sun et al. 2011). All the data gained in the cross experiments indicated a pattern of reproductive isolation between different cryptic species of B. tabaci in China (Wang et al. 2011; Liu et al. 2012). Although numerous crossing studies have been conducted, suggesting that B. tabaci is a species complex with a certain number of cryptic species, most of
the experiments were done under fixed and optimal conditions including suitable temperature, photoperiod and humidity. It is noteworthy that previous studies have shown that the ecology and biology of B. tabaci can be affected by many biotic and abiotic factors. For example, temperature, humidity, host plant, natural enemies and other competitors in the same niche can have various effects on the development, survivorship and reproduction of B. tabaci (Berlinger et al. 1996; Tsai and Wang 1996; Wang and Tsai 1996; Qiu et al. 2005, 2007; Xu 2006; Iida et al. 2009). Thus, a question raised is, do these biotic and abiotic factors also have some positive or negative effects on the cross mating behavior of different B. tabaci cryptic species? This study undertakes the first validation of this question by setting up a series of experiments to investigate B. tabaci inter-specific cross mating. Firstly, we established the laboratory colonies of MEAM1, MED and Asia II 7 cryptic species collected in China. Their inter-specific cross matings were then investigated in two different test arenas and at three different temperatures. Our current work was not to repeat the previously reported investigations, but to evaluate the effect of space dimension and temperature on the crossing of different cryptic species of B. tabaci.
RESULTS Effects of space dimension on the cross mating of B. tabaci The results of inter-species cross among the three cryptic species of B. tabaci in leaf cages are shown in Table 1. Differences were found among the mean numbers of progeny of different cryptic species cross groups (F8, 141=2.08, P=0.0418) although most of the difference were not significant to each other. The sex ratio identification indicated that all the F1 progeny in the inter-specific crosses of MEAM1 ×Asia II 7 , Asia II 7 ×MEAM1 , MED ×Asia II 7 and Asia II 7 ×MED were males, whereas in the MEAM1 ×MED inter-species cross, two female progeny were recorded, and in the MED ×MEAM1 inter-specific cross, 5 female progeny were recorded in total. The female percentage in the offspring of MEAM1 ×MED and MED ×MEAM1 inter-species crosses were 0.81 and 1.37%, © 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
respectively, while the female percentages among the three intra-specific crosses ranged from 54.2 to 65.6% and were not significantly different to each other. The few hybrid females produced from the MEAM1×MED cross were reared for 5 d to observe their reproduction. These hybrid females produced no F2 eggs, indicating that the rare F1 female adults were all sterile. The results of the inter-species crosses among the
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three cryptic species conducted using plastic cylinder cages are shown in Table 2. Similar to the leaf cage experiments, obvious differences were found among the mean numbers of progeny in the cross and control treatments (F8, 141=4.95, P<0.0001). However, in all the six inter-specific crosses, no female progeny was produced; in contrast 54-66% females were produced in the intra-specific control treatments.
Table 1 Progeny produced in the cross experiments among the B. tabaci MEAM1, MED, and Asia II 7 cryptic species undertaken in leaf cage experiments Cryptic species
n
Number of progeny per pair of adults (M±SE)1)
Asia II 7 ×MEAM1 Asia II 7 ×MED MEAM1 ×Asia II 7 MEAM1 ×MED MED ×MEAM1 MED ×Asia II 7 Asia II 7 ×Asia II 7 MEAM1 ×MEAM1 MED ×MED
20 20 20 20 20 20 10 10 10
14.0±4.2 ab 17.8±4.6 a 16.8±3.4 ab 11.9±1.5 b 14.6±2.0 ab 16.2±2.7 ab 16.1±2.9 ab 13.4±3.1 ab 15.2±3.5 ab
Female percentage in progeny per pair of adults (M±SE) 2) 0.0 0.0 0.0 0.813) 1.374) 0.0 54.2±4.1 a 65.6±2.5 a 59.7±4.8 a
1)
F 8, 141=2.08, P=0.0418. F2, 27=2.88, P=0.29. 3) In this inter-species cross, 247 F1 adults were produced in the 20 replicates, and only in 2 replicates one female progeny was found in each, the SE was not calculated due to the few positive data of female percentage recorded in the experiments. 4) For this inter-species cross, 292 F 1 adults in total were produced; in 4 of the 20 replicates 1, 1, 2 and 1 female progeny were produced, the remaining 16 replicates produced only males; the SE was not calculated. 2)
Table 2 Progeny produced in the crossing experiments among the B. tabaci MEAM1, MED, and Asia II 7 cryptic species conducted using plastic cylinder cages Cryptic species Asia II 7 ×MEAM1 Asia II 7 ×MED MEAM1 × Asia II 7 MEAM1 × MED MED ×Asia II 7 MED ×MEAM1 Asia II 7 ×Asia II 7 MEAM1 ×MEAM1 MED ×MED 1) 2)
n
Number of progeny per pair of adults (M±SE)1)
20 20 20 20 20 20 10 10 10
23.5±2.2 a 20.7±1.0 abc 19.6±1.7 abcd 15.8±1.2 d 16.3±1.5 d 22.6±1.1 ab 19.2±2.26 bcd 21.3±1.7 abc 18.0±1.3 cd
Female percentage in progeny per pair of adults (M±SE, %)2) 0.0 0.0 0.0 0.0 0.0 0.0 57.6±5.3 a 62.4±4.7 a 55.3±6.9 a
F8, 141=4.95, P<0.001. F2, 27=1.81, P=0.37.
Effects of temperature on the cross mating of B. tabaci cryptic species Temperature affected the numbers of progeny as well as sex ratios among the various crosses (Table 3). In general, the number of progeny was the highest at 30°C, followed by 22°C, with the lowest at 38°C, regardless of the type of inter- and intra-specific crosses. Significant differences were noted among the different crosses and intra-specific controls conducted at 22°C (F8, 141=31.64,
P<0.001) and at 38°C (F8, 141=13.46, P<0.001), but not among those at 30°C (F8, 141=1.71, P=0.27). As for the sex ratios of the progeny, female progeny were found in the crosses of MEAM1 ×MED with percentages of 0.77 and 0.98% at 22 and 30°C respectively, and female progeny were also found in the cross of MED ×MEAM1 with a percentage of 0.95% at 30°C. However, no females were recorded in the reciprocal crosses of MEAM1×Asia II 7 and MED×Asia II 7 at either 22, 30 or 38°C as well as in the cross of MEAM1×MED at 38°C .
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Table 3 Progeny produced in the cross experiments among the B. tabaci MEAM1, MED and Asia II 7 cryptic species conducted using leaf cages at different temperatures Cryptic species Asia II 7 ×MEAM1 Asia II 7 ×MED MEAM1 ×Asia II 7 MEAM1 ×MED MED ×Asia II 7 MED ×MEAM1 Asia II 7 ×Asia II 7 MEAM1 ×MEAM1 MED ×MED
n 20 20 20 20 20 20 10 10 10
Number of progeny per pair of adults under different temperatures (M±SE)1) 22°C 30°C 38°C 8.6±2.0 b 9.3±2.1 b 11.4±2.5 ab 13.1±1.4 a 14.9±1.7 a 10.2±1.2 ab 11.8±2.7 ab 13.7±2.5 a 14.3±1.6 a
21.3±3.8 a 23.8±5.0 a 24.7±4.6 a 25.6±2.8 a 23.9±3.2 a 26.4±4.1 a 25.6±2.6 a 27.6±4.4 a 29.5±3.2 a
8.1±2.3 b 8.2±2.6 b 9.4±2.5 ab 12.3±0.9 a 9.8±2.3 ab 8.2±1.3 b 12.6±1.1 a 11.1±2.3 ab 13.6±1.5 a
Female percentage in progeny per pair of adults under different temperatures (M±SE, %)2) 22°C 30°C 38°C 0.0 0.0 0.0 0.77 3) 0.0 0.0 56.7±2.9 a 65.4±3.3 a 60.2±5.2 a
0.0 0.0 0.0 0.98 4) 0.0 0.95 5) 56.2±2.3 a 62.8±3.7 a 55.6±4.9 a
0.0 0.0 0.0 0.0 0.0 0.0 52.4±3.8 a 55.1±4.6 a 57.2±2.1 a
F8, 141=31.64, P<0.001 for 20°C treatment. F8, 141=1.71, P=0.27 for 30°C treatment. F8, 141=13.46, P<0.001 for 38°C treatment. F2, 27=1.44, P=0.46 for 20°C treatment. F2, 27=0.986, P=0.82 for 30°C treatment. F2, 27=1.69, P=0.41 for 38°C treatment. 3) Two female progeny were found among the 261 adults in total. 4) Five female progeny were found among the 551 adults in total. 5) Five female progeny were found among the 528 adults in total. 1) 2)
Daily fecundity and progeny sex ratio of B. tabaci in the crossing experiments The average daily fecundity of MEAM1 and MED in their cross experiments increased continuously with time in the first 5 d after emergence (Fig. 1). In the interspecies cross of MEAM1 ×MED , 9 female progeny (2, 2 and 5 on day 2, 4 and 5, respectively) were produced in the 5-day investigation. In the inter-species cross of MED ×MEAM1 , 12 female progeny (3 and 9 on day 4 and 5, respectively) were recorded in 5 of 10 replicates in the 5-d investigation. In contrast, the numbers of female progeny produced per female in the intra-species treatments of MEAM1 and MED were substantially higher. In addition, female progeny were produced everyday in the intra-species treatments, while in the inter-species crosses female progeny were mostly produced in the last two days during the investigation.
DISCUSSION The destructive damage of B. tabaci on world agriculture and horticulture has been widely recognized. However, the enduring debate about the species status of B. tabaci has lasted for almost two decades. Crossing experiments are thought to be the most useful and necessary way to clarify the species status of B. tabaci. So far, convincing data has been obtained from sufficient cross experiments of different B. tabaci genetic groups to support the conclusion that B. tabaci is a
Fig. 1 The averaged number of female progeny produced per pair of adults daily in the cross of B. tabaci MEAM1 and MED cryptic species.
species complex containing a number of morphologically indistinguishable cryptic species (Costa et al. 1993; Perring et al. 1993; Bedford et al. 1994; Ronda et al. 1999; Luan et al. 2008; Crowder et al. 2010; Wang et al. 2010, 2011; Xu et al. 2010; Sun et al. 2011; Liu et al. 2012), a conclusion drawn from a phylogenetic view based on the mtCOI sequences of these B. tabaci cryptic species (Boykin et al. 2007; Dinsdale et al. 2010; De Barro et al. 2011). The behavior and efficiency of insect courtship and reproduction are quite complex, and our knowledge on the cross mating of different B. tabaci cryptic species is still limited. Most of the cross experiments previously reported were done in a fixed set of leaf cages,
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The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
no studies have been reported about the effects of the size of the test arena and temperature on the cross mating of B. tabaci, thus the results from the current study are expected to expand our knowledge in this area. In the present study, few female progeny were found in the cross of MEAM1 and MED when the hybridizations were conducted in leaf cages, while no female progeny were recorded in the crosses conducted using larger cylinder cages. The volumes of the cylinder cages used in our cross experiments are 40 times larger than that of a leaf cage, and we found that the heterogeneous individual adults of MEAM1 and MED B. tabaci in the cylinder cages were usually more further apart from each other, either on the same leaf or on different leaves during the 48 h observation, compared to their locations when enclosed in smaller leaf cages. This difference in terms of separation between individuals may have contributed to the different results obtained between trials using the two types of cages. Our findings were also consistent with the field surveys in Israel, in which B and Q populations were found to co-exist in the field in many locations, but no hybridization or geneflow occurred between the two (Horowitz et al. 2003; Khasdan et al. 2005; Elbaz et al. 2010). Meanwhile, the rare female offspring were mostly produced in the later period of observation (Fig. 1), which occurred possibly because heterogeneous copulations were only forced to happen later in the time interval, a speculation that seemed to be supported by the behavioral observation on these two species by Sun et al. (2011). Similar results have been reported between crosses of ZHJ1and ZHJ2 (Xu et al. 2010), between MEAM1 and MED (Sun et al. 2011), among MED, ZHJ1 and ZHJ2 (Wang et al. 2010), and among MEAM1, MED, Asian II 7, ZHJ1, ZHJ2, and ZHJ3 (Wang et al. 2011). In the studies above, all the data showed that more copulation events were recorded in group mating than in singlepair mating, indicating that the population density may affect the rate of copulation. In other words, the arena size may have some marginal effects on the mating activities of B. tabaci cryptic species. The rarer events of copulation in lower population densities may be due to the larger space for individual movements, although the effects of space or distance on the chemical attraction between whitefly heterosexual individuals has not been investigated to date.
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Temperature has been reported to have significant effects on the development, survivorship, and reproduction of B. tabaci, with 26-29°C being the optimum temperature (Wang and Tsai 1996; Qiu et al. 2003a). In our current study, the cross experiments of MEAM1 and MED were conducted at three temperatures, of which 30°C was deemed most suitable while 22 and 38°C were less suitable for the mating and reproduction of B. tabaci. It is assumed that adverse temperature may reduce the mating desire and behavior of different cryptic species. This inference would warrant further investigation through mating behavior observations of B. tabaci in the laboratory.
CONCLUSION To date, numerous studies have proved that B. tabaci is a species complex containing more than 24 morphologically indistinguishable cryptic species, and that all the cryptic species are completely reproductively isolated. Our results indicated that probability of copulation and hybridization in crossing experiments between cryptic species of B. tabaci may be marginally affected by the size of the test arena and temperature. Knowledge of such effects of experimental factors may help in understanding the variations obtained in this type of crossing studies.
MATERIALS AND METHODS Whitefly populations and host plants Adults of B. tabaci MEAM1 were collected in 2006 on eggplant Solanum melongena at the experimental farm of South China Agricultural University (SCAU) in Guangzhou, China. The Asia II 7 cryptic species was collected in 2005 from the ornamental plant laurel Codiaeum variegatum in Changban, Guangzhou, while the MED cryptic species was collected from pepper plant Capsicum frutescens from Nantong, Jiangsu, China, in 2007. All the three cryptic species were rared on their original host plants in separate cages at SCAU at ambient temperature, photoperiod and humidity. Their cryptic species were identified by mitochondrial CO1 sequencing according to Qiu et al. (2009). Cowpea (Vigna unguiculata cv. Baofeng) was used in the cross experiments. Cowpea is an important vegetable in China. Previous investigations in the laboratory and
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field showed that cowpea is a preferred host plant for B. tabaci MEAM1, MED and Asia II 7 cryptic species (Musa and Ren 2005; Xu 2006). Plants were first geminated in a plastic pot, and after 1 wk all the seedlings were transplanted into 1.3-L plastic pots containing sterilized soil-sand mixture in a whitefly-proof greenhouse. Plants were watered as required. Natural daylight was supplemented with sodium lamps to ensure a photoperiod of 16 L:8 D. Plants were used for experiments when they were two weeks old with 4-5 true leaves. Before use in the cross experiments, all the leaves from each plant were carefully examined under a 30× binocular stereoscope twice to ensure that only insectfree plants were used.
Collection of the newly emerged virgin adults of B. tabaci To ensure that the B. tabaci adults were virgin for the cross experiments, in the evening prior to the day of initiating cross experiments, plant leaves with whitefly pupae were cut by scissors and whitefly pupae were put into glass tubes (5 cm length×0.5 cm diameter) individually. The sexes of newly emerged virgin adults were identified according to the description of Gill et al. (1990) the following morning.
Effects of size of test arena on the cross mating of B. tabaci Two sets of experiments were used for the whitefly crosses in the current study. In the first set, a leaf cage was used. The dimension of the leaf cage was 2 cm in height×3 cm in diameter (Fig. 2-A). The leaf cages were fixed to the under surface of leaves with clips. The internal volume of a leaf cage was approximately 7 cm3. In the second set, a plastic column (10 cm in height×6 cm in diameter) was used to make a cylinder cage for the cross experiments (Fig. 2-B). These plastic cylinders were covered with nylon gauze on the top, bottom and side, with a T shape opening in the nylon gauze at the bottom to allow placement of the test plant. Each cylinder cage enclosed a whole plant of cowpea planted in a pot. The internal volume of the cylinder cage was approximately 280 cm3. Single pair cross matings were conducted in all the experiments. In each replicate using either a leaf cage or cylinder cage, one pair of newly emerged virgin female and male were introduced. The adults were collected after 48 h of mating and reproduction and stored in 95% ethanol at -20ºC for later species identification if they produced female offspring. The F 1 eggs were counted and allowed to develop through to adults on the leaves. Then the individuals were sexed and also counted for the calculation of F1 survivorships if they emerged as adults. In each direction of one cross combination, i.e., MEAM1 ×MED or MED ×
Fig. 2 Sketch diagrams of the leaf cage and the plastic cylinder used in the current study. A, leaf cage. 1, leaf petiole; 2, leaf undersurface; 3, leaf cage; 4, nylon gauze for ventilation; 5, hole for introducing whiteflies. B, plastic cylinder. 1, whole plastic cylinder; 2 and 3, nylon gauze for ventilation; 4, hole for introducing whiteflies and the cover; 5, cowpea plant.
MEAM1 , 20 replicates of inter-species crosses were conducted, and 10 replicates of intra-specific matings were also conducted as control. When there were female F 1 adults recorded, each of them was introduced into a leaf cage on a new cowpea plant with a male, 5 d later the leaves were checked under a binocular stereoscope to see if there were F2 eggs. All the experiments except the molecular identification were conducted at (26±1)ºC, with photoperiod of 16 L:8 D and (75±10)% RH.
Effects of temperature on the cross mating of B. tabaci Crossing experiments were conducted at three temperatures of 22, 30 and 38ºC, respectively, to examine whether
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The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
inter-specific crossings would be affected by temperature. The newly emerged females and males were collected as described above. Leaf cages were used for the experiments. The protocols for conducting inter-specific crosses (20 replicates in each cross) and intra-species controls (10 replicates in each control treatment) were the same as above. All the experiments in this section were done with a photoperiod of 16 L:8 D and (75±10)% RH.
Daily fecundity and progeny sex ratio of B. tabaci in the cross experiments Ten pairs of newly emerged females and males from different cryptic species were introduced into 10 leaf cages, one pair per cage, and reared for 5 d (the first 5 d after emergence) to check the daily fecundity and the sex ratio of the F 1 progeny. The pair of adults in each replicate were transferred to a new leaf cage and the number of eggs deposited in each replicate was recorded every 24 h. All the eggs were allowed to develop to adults on leaves for sex ratio calculation. Six pairs of intra-specific mating of MEAM1 and MED were used as controls.
Data analysis For the cross experiments, the fecundity of the F 0 generation, the sex ratios of the F1 generation in the intraand inter-species cross experiments were analyzed using a one-way ANOVA, and the means were separated using the least significant difference test (LSD) at P=0.05. All the proportion data were transformed by square root to meet the requirements of normality and homogeneity of variances before analysis. All data were analyzed using the SAS Systems Release 8.01 for Windows (SAS Institute, 2001).
Acknowledgements This research was funded by the National Basic Research Program of China (2009CB119203), the National Natural Science Foundation of China (31071732) and the Foundation of Lingnan Fruit Innovation Team of Guangdong Province, China (2009-14).
References De Barro P J, Hart P J. 2000. Mating interactions between two biotypes of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) in Australia. Bulletin of Entomological Research, 90, 103-112. De Barro P J, Trueman J W H, Frohlich D R. 2005. Bemisia argentifolii is a race of B-tabaci (Hemiptera: Aleyrodidae) the molecular genetic differentiation of B-tabaci populations around the world. Bulletin of
203
Entomological Research, 95, 193-203. De Barro P J, Liu S S, Boykin L M, Dinsdale A B. 2011. Bemisia tabaci: A statement of species status. Annual Review of Entomology, 56, 1-19. Bedford I D, Briddon R W, Brown J K, Rosell R C, Markham P G. 1994. Geminivirus transmission and biological characterization of Bemisia tabaci (Gennadius) biotypes from different geographic regions. Annals of Applied Biology, 125, 311-325. Berlinger M J, Lehmann-Sigura N, Taylor R A J. 1996. Survival of Bemisia tabaci adults under different climatic conditions. Entomologia Experimentalis et Applicata, 80, 511-519. Bird J. 1957. A Whitefly Transmitted Mosaic of Jatropha G o s s y p i f o l i a ( Te c h n i c a l p a p e r ) . A g r i c u l t u r a l Experimental Station, University of Puerto Rico. Mayagüez, Puerto Rico. 22, 1-35. Boykin L M, Shatters Jr R G , Rosell R C, McKenzie C L, Bagnall R A, McKenzie C L, De Barro P J, Frohlich D R. 2007. Global relationships of Bemisia tabaci (Hemiptera: Aleyrodidae) revealed using Bayesian analysis of mitochondrial COI DNA sequences. Molecular Phylogenetics and Evolution, 44, 1306-1319. Byrne D N, Bellows Jr T S. 1991. Whitefly biology. Annual Review of Entomology, 36, 431-457. Chou I. 1949. List of whitefly species in China. Entomologia Sinica, 3, 1-18. (in Chinese) Chu D, Zhang Y J, Brown J K, Cong B, Xu B Y, Wu Q J, Zhu G R. 2006. The introduction of the exotic Q biotype of Bemisia tabaci from the Mediterranean region into China on ornamental crops. Florida Entomologist, 89, 168-174. Costa H S, Brown J K, Sivasupramaniam S, Bird J. 1993. Regional distribution, insecticide resistance and reciprocal crosses between the ‘A’ and ‘B’ biotypes of Bemisia tabaci. Insect Science and Its Application, 14, 255-266. Crowder D W, Sitvarin M I, Carriere Y. 2010. Mate discrimination in invasive whitefly species. Journal of Insect Behavior, 23, 364-380. Dinsdale A, Cook L, Riginos C, Buckley Y M, De Barro P. 2010. Refined global analysis of Bemisia tabaci (Hemiptera: Ternorrhyncha: Aleyrodoidea: Aleyrodidae) mitochondrial cytochrome oxidase 1 to identify species level genetic boundaries. Annals of the Entomological Society of America, 103, 196-208. Elbaz M, Lahav N, Morin S. 2010. Evidence for pre-zygotic reproductive barrier between the B and Q biotypes of Bemisia tabaci (Hemiptera: Aleyrodidae). Bulletin of Entomological Research, 100, 581-590. Gelman D B, Pszczolkowski M A, Blackburn M B, Ramaswamy S B. 2007. Ecdysteroids and juvenile hormones of whiteflies, important insect vectors for plant viruses. Journal of Insect Physiology, 53, 274284. Gill R J. 1990. The morphology of whiteflies. In: Gerling D,
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
The Effects of Space Dimension and Temperature on the Cross Mating of Three Cryptic Species of the Bemisia tabaci
ed., Whiteflies: Their Bionomics, Pest Status and Management. Intercept Ltd., Andover. pp. 13-46. Guo X J, Rao Q, ZHANG F, Luo C, Zhang H Y, Gao X W. 2012. Diversity and genetic differentiation of the whitefly Bemisia tabaci species complex in China based on mtDNA CO1 and cDNA-AFLP analysis. Journal of Integrative Agriculture, 11, 206-214. Heinz K. 1996. Predators and parasitoids as biological control agents of Bemisia in greenhouses. In: Gerling D, Mayer R T, eds., Bemisia, 1995: Taxonomy, Biology, Damage Control and Management. Intercept Ltd., Andover, Hants, UK. pp. 435-449. Henneberry T J, Toscano N C, Perring T M, Faust R M. 1997. Preface. In: Henneberry T J, Toscano N C, Perring T M, Faust R M, eds., Silverleaf Whitefly, Supplement to the Five-Year National Research and Action Plan: Progress, Review, Technology Transfer, and New Research and Action Plan (1997-2001). USDA, Washington D.C. p. 2. Henneberry T J, Jech L F, Hendrix D L. 2000. Bemisia argentifolii (Homoptera: Aleyrodidae) honeydew and honeydew sugar relationships to sticky cotton. Southwestern Entomologist, 25, 1-14. Horowitz A R, Denholm I, Gorman K, Cenis J L, Kontsedalov S, Ishaaya I. 2003. Biotype Q of Bemisia tabaci identified in Israel. Phytoparasitica, 31, 94-98. Hu J, De Barro P, Zhao H, Wang J, Nardi F, Liu S S. 2011. An Extensive field survey combined with a phylogenetic analysis reveals rapid and widespread invasion of two alien whiteflies in China. PLoS ONE, 6, e16061. Iida H, Kitamura T, Honda K. 2009. Comparison of egghatching rate, survival rate and development time of the immature stage between B and Q biotypes of Bemisia tabaci (Gennadius) (Homoptera:Aleyrodidae) on various agricultural crops. Applied Entomology and Zoology, 44, 267-273. Inbar M, Gerling D. 2008. Plant-mediated interactions between whiteflies, herbivores, and natural enemies. Annual Review of Entomology, 53, 431-448. Jones D R. 2003. Plant viruses transmitted by whitefly. European Journal of Plant Pathology, 109, 195-219. Khasdan V, Levin I, Rosner A, Morin S, Kontsedalov S, Maslenin L, Horowitz A R. 2005. DNA markers for identifying biotypes B and Q of Bemisia tabaci (Hemiptera: Aleyrodidae) and studying population dynamics. Bulletin of Entomological Research, 95, 605-613. Liu S S, De Barro P J, Xu J, Luan J B, Zang L S, Ruan Y M, Wan F H, 2007. Asymmetric mating interactions drive widespread invasion and displacement in a whitefly. Science, 318, 1769-1772. Liu S S, Colvin J, De Barro P J. 2012. Species concepts as applied to the whitefly Bemisia tabaci systematics: how many species are there? Journal of Integrative Agriculture, 11, 176-186. Luan J B, Ruan Y M, Zang L, Liu S S. 2008. Precopulation
204
intervals, copulation frequencies, and initial progeny sex ratios in two biotypes of whitefly, Bemisia tabaci. Entomologia Experimentalis et Applicata, 129, 316324. Ma D, Hadjistylli M, Gorman K, Denholm I, Devine G. 2004. Pre- and post-zygotic breeding incompatibilities between B and Q biotypes of Bemisia tabaci. In: Proceeding of the 2nd European Whitefly Symposium. 5-9 October, 2004, Cavtat, Croatia. pp. 13-14. Mahadav A, Gerling D, Gottlieb Y, Czosnek H, Ghanim M. 2008. Parasitization by the wasp Eretmocerus mundus induces transcription of genes related to immune response and symbiotic bacteria proliferation in the whitefly Bemisia tabaci. BMC Genomics, 9, 342. Musa P D, Ren S X. 2005. Development and reproduction of Bemisia tabaci (Homoptera: Aleyrodidae) on three bean species. Insect Science, 12, 25-30. Perring T M. 2001. The Bemisia tabaci species complex. Crops Protection, 20, 725-737. Perring T M, Copper A D, Rodriguez R J, Farrar C A, Bellows T S. 1993. Identification of a whitefly species by genomic and behavioral studies. Science, 259, 74-77. Qiu B L, De Barro P J, Ren S X. 2005. Development, survivorship and reproduction of Eretmocerus sp. nr. furuhashii (Hymenoptera: Aphelinidae) parasitizing Bemisia tabaci (Hemiptera: Aleyrodidae) on glabrous and non-glabrous host plants. Bulletin of Entomological Research, 95, 313-319. Qiu B L, De Barro P J, Ren S X, Xu C X. 2007. Effect of temperature on the life history of Eretmocerus sp. nr. furuhashii, a parasitoid of Bemisia tabaci. BioControl, 52, 733-746. Qiu B L, Chen Y P, Liu L, Peng W L, Li X X, Ahmed M Z, Mathur V, Du Y Z, Ren S X. 2009. Identification of three major Bemisia tabaci biotypes in China based on morphological and DNA polymorphisms. Progress in Natural Science, 19, 713-718. Qiu B L, Ren S X, Mandour N S, Lin L. 2003a. Effect of temperature on the development and reproduction of Bemisia tabaci B biotype (Homoptera: Aleyrodidae). Entomologia Sinica, 10, 43-49. Qiu B L, Ren S X, Wen S Y, Mandour N S. 2003b. Biotype identification of the populations of Bemisia tabaci (Homoptera: Aleyrodidae) in China using RAPD-PCR. Acta Entomologica Sinica, 46, 605-608. Ronda M A, Adan A, Cifuentes D, Cenis J L, Beitia F. 1999. Laboratory evidence of interbreeding between biotypes of Bemisia tabaci (Homoptera: Aleyrodidae) present in Spain. In: V11th International Plant Vir us Epidemiology Symposium-Plant Virus Epidemiology: Current status and Future Prospects. Aguadulce, 1116 April, 1999, Spain. pp. 83-84. Ruan Y M, Luan J B, Zang L S, Liu S S. 2007. Observing and recording copulation events of whiteflies on plants using a video camera. Entomologia Experimentalis et Applicata, 124, 229-233.
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.
205
SAS Institute. 2001. SAS/STAT User’s Guide. SAS Institute, Cary, NC. Sun D B, Xu J, Luan J B, Liu S S. 2011. Reproductive incompatibility between the B and Q biotypes of the whitefly Bemisia tabaci in China: genetic and behavioral evidence. Bulletin of Entomological Research, 101, 211-220. Tasi J H, Wang K. 1996. Development and reproduction of Bemisia argentifolii (Homoptera: Aleyrodidae) on five host plants. Environmental Entomology, 25, 810-816. Wang K H, Tsai J H. 1996. Temperature effect on development and reproduction of silverleaf whitefly (Homoptera: Aleyrodidae). Annals of the Entomological Society of America, 89, 375-384. Wang P, Ruan Y M, Liu S S. 2010. Crossing experiments and behavioral observations reveal reproductive incompatibility among three putative species of the whitefly Bemisia tabaci. Insect Science, 17, 508-516. Wang P, Sun D B, Qiu B L, Liu S S. 2011. The presence of six cryptic species of the whitefly Bemisia tabaci complex in China as revealed by crossing experiments.
LI Xiao-xi et al.
Insect Science, 18, 67-77. Wu X X, Li Z X, Hu D X, Shen Z R. 2003. Identification of Chinese populations of Bemisia tabaci (Gennadius) by analyzing the ribosomal ITS1 sequence. Progress in Natural Science, 13, 276-281. Xu C X. 2006. The intra- and inter-species competition of whitefly Bemisia tabaci and two-spotted mite Tetranychus urticae. Ph D thesis, South China Agriculutural University, Guangzhou. pp. 5-48. (in Chinese) Xu J, De Barro P J, Liu S S. 2010. Reproductive incompatibility among genetic groups of Bemisia tabaci supports the proposition that the whitefly is a cryptic species complex. Bulletin of Entomological Research, 100, 359-366. Zang L S, Liu S S. 2007. A comparative study on mating behavior between the B biotype and a non-B populations of the whitefly, Bemisia tabaci (Hemiptera: Aleyrodidae) from Zhejiang, China. Journal of Insect Behavior, 20, 157-171. (Managing editor SUN Lu-juan)
© 2012, CAAS. All rights reserved. Published by Elsevier Ltd.