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Effects of antibiotics on fitness of the B biotype and a non-B biotype of the whitefly Bemisia tabaci Yong-Ming Ruan, Jing Xu & Shu-Sheng Liu*Institute of Insect Science, Zhejiang University, 268 Kaixuan Road, Hangzhou, 310029, China Key words: primary endosymbiont, secondary endosymbiont, tetracycline, ampicillin trihydrate, rifampicin, development time, survival, Homoptera, Aleyrodidae The whitefly, Bemisia tabaci Gennadius (Homoptera: Aleyrodidae), harbors primary and secondaryendosymbionts. Previous research showed that the invasive B biotype and an indigenous non-B biotype (named non-B ZHJ-1 population) of B. tabaci from Zhejiang, China, harbored differentendosymbionts. To investigate the function of these endosymbionts in the two biotypes of B. tabaci,we fed adult whiteflies with three antibiotics, tetracycline, ampicillin trihydrate, and rifampicin, andevaluated the fitness of their offspring on cotton plants. These three antibiotics did not remove theprimary endosymbiont Portiera aleyrodidarum but were capable of eliminating the secondary endo-symbionts. In the B biotype, treatments of adults with tetracycline or ampicillin trihydrate accelerateddevelopment and increased the survival of their offspring, while treatment of adults with rifampicinsignificantly retarded the development of their offspring but did not affect their survival. In the non-B ZHJ-1 population, treatments of adults with tetracycline or ampicillin trihydrate also acceleratedthe development of their offspring but did not significantly affect their survival, while treatment ofadults with rifampicin significantly retarded development and reduced the survival of their offspring.
These results suggest that removal of some secondary endosymbionts and/or reduction of the primaryendosymbiont from B. tabaci may produce both favorable and unfavorable effects on the fitness ofthe host insects.
2003). Endosymbionts of insects are usually categorized into two types, the primary (P-) endosymbionts, which are Bemisia tabaci (Gennadius) (Homoptera: Aleyrodidae) is harbored in specialized host cells called bacteriocytes and a worldwide pest of vegetable, ornamental, and field crops the secondary (S-) endosymbionts. S-endosymbionts are (Brown et al., 1995). Bemisia tabaci is a genetically diverse not restricted to bacteriocytes, but are present in almost all group, many populations of which have been named as biotypes (De Barro et al., 2000; Perring, 2001). Among Whiteflies contain orange-yellow, paired oval bac- the biotypes, the B biotype (also known as the silverleaf teriomes composed of bacteriocytes. These bacteriomes are whitefly, Bemisia argentifolii) has a broad host range, is visible at low magnification (Buchner, 1965). Polymerase highly fecund, and causes serious plant disorders not caused chain reaction (PCR) amplification and sequencing of by other B. tabaci biotypes (Barinaga, 1993).
the 16S ribosomal DNA (rDNA) genes showed that the Whiteflies and other homopterans have the unique P-endosymbiont is associated with all whitefly collections ability to feed exclusively on phloem sap, which is rich in examined and constitutes a distinct lineage within the carbohydrates and deficient in essential amino acids. This gamma subdivision of Proteobacteria (Clark et al., 1992; ability has been undoubtedly linked to the endosymbionts Thao & Baumann, 2004b). The P-endosymbiont of white- of these insects (Buchner, 1965; Douglas, 1989). Endosym- flies has been given the provisional designation ‘Candidatus bionts can supplement their host’s diet with essential amino Portiera aleyrodidarum’ (Thao & Baumann, 2004a). In acids and are essential for the development and reproduc- addition to Candidatus Portiera aleyrodidarum, B. tabaci tion of their insect hosts (Douglas, 1989; Moran et al., may also harbor several S-endosymbionts, such as ‘Candi-datus Hamiltonella defensa’ (Enterobacteriaceae), Wolbachia, *Correspondence: E-mail: shshliu@zju.edu.cn Arsenophonus, Cardinium (Bacteroidetes), and Fritschea 2006 The Authors Entomologia Experimentalis et Applicata 121: 159–166, 2006
Journal compilation 2006 The Netherlands Entomological Society bemisiae (Simkaniaceae) (Clark et al., 1992; Nirgianaki the growth, development, and survival of their offspring.
et al., 2003; Thao et al., 2003; Weeks et al., 2003; Thao & Our objective was to determine the effect of some of the Baumann, 2004a; Everett et al., 2005). Previous research endosymbionts on the fitness of the whitefly and to reveal indicated that different biotypes of B. tabaci may harbor any differential effect between biotypes of the whitefly.
different S-endosymbionts (Costa et al., 1995; Zchori-Fein& Brown, 2002; Nirgianaki et al., 2003).
Endosymbionts have a variety of effects on their hosts, such as resistance to parasitic wasps (Oliver et al., 2003, 2005; Ferrari et al., 2004), tolerance to heat stress (Montllor Two populations of B. tabaci were originally collected from et al., 2002), host plant specialization (Tsuchida et al., 2004), the suburbs of Hangzhou (30.2°N, 120.1°E), China, in and compensation for the loss of the P-endosymbiont 2003, and were identified as B biotype and non-B ZHJ-1 (Koga et al., 2003). Endosymbionts may play a direct population (Zang et al., 2005a). The B biotype population and/or indirect role in the evolution of their insect hosts.
was collected from common cabbage Brassica oleracea var.
Genetic changes in B. tabaci endosymbionts and/or capitata and the non-B ZHJ-1 population from cotton variations of the endosymbionts in different biotypes of G. hirsutum. Stock cultures of the two biotypes were reared B. tabaci may play a functional role in the evolution of on potted cotton (cultivar ‘Chuan-Mian no. 109’) in an biotypes of this whitefly species (Costa et al., 1995; Zchori- insectary at 25–30 °C with a photoperiod of L14:D10 and Fein & Brown, 2002; Nirgianaki et al., 2003).
60 – 80% r.h. Each culture was routinely sampled by PCR So far, only limited research has been conducted on the to monitor purity for the biotype every 2–3 generations.
relationship between B. tabaci and its endosymbionts(Costa et al., 1997, 1993a), partly due to the technical difficulty involved in culturing the majority of endo- For the extraction of whitefly DNA and biotype symbionts outside their hosts (Houk & Griffiths, 1980; identification by RAPD–PCR with H16 primer, we used Douglas, 1989; Wilkinson, 1998). Hence, the function of the methods of De Barro & Driver (1997). For diagnostic these endosymbionts has been examined in vivo using PCR detection of endosymbionts, we used the methods of antibiotics treatment, which can reduce the quantity of Zhou et al. (1998), Zchori-Fein & Brown (2002), and Thao endosymbionts or even eliminate them (Wilkinson, 1998).
& Baumann (2004a). Specific primers used are listed in In recent years, many populations of B. tabaci collected Table 1. All PCRs included a negative control (sterile water from various regions in China were identified as the instead of DNA) to spot any DNA contamination, and invasive B biotype (Luo et al., 2002). In 2003, we collected a positive control (DNA from previous sequencing) to a field sample of a non-B biotype of B. tabaci from cotton prevent false negatives. Because an antibiotic treatment Gossypium hirsutum L. (Malvaceae) in Zhejiang province could reduce endosymbionts to beyond PCR primer of China. This non-B biotype was identified by mito- limitations (Leonardo, 2004), PCRs for each primer set chondrial cytochrome oxidase-I (COI) gene sequencing, were performed using 1/100 standard amount of DNA to random amplification of polymorphic DNA (RAPD)– confirm sensitivity. Each primer set had scored positive for PCR, and the lack of squash silverleaf symptom, and has endosymbionts under standard PCR conditions.
been named as ‘non-B China-ZHJ-1 population’ (Zanget al., 2005a). A phylogenetic analysis using COI sequences of over 100 B. tabaci populations indicated that the non-B Antibiotic treatments were conducted using Parafilm ZHJ-1 population is indigenous to China (Zang et al., membrane sachets for direct feeding by adults (Costa et al., 1997). A glass tube (36 mm in diameter × 50 mm long, Using PCR survey to detect the presence of selected open at both ends) was covered at the top end by one layer endosymbionts in the B biotype and the ZHJ-1 population of Parafilm M membrane (Alcan Packaging, Chicago, IL, of B. tabaci from Zhejiang, we found that the two biotypes USA) stretched as thinly as possible. A drop of 0.2 ml diet harbored different endosymbionts (Ruan & Liu, 2005). The solution was placed on the outer surface of the stretched two biotypes harbored a common primary endosymbiont Parafilm and covered with another layer of stretched – P. aleyrodidarum, but their secondary endosymbionts Parafilm to enclose the solution between the Parafilm differed: H. defensa was detected in the B biotype but not layers without air bubbles (Mitsuhashi & Koyama, 1971; in the ZHJ-1 biotype, while Wolbachia and Arsenophonus Adams & van Emden, 1972). The tube was sealed with were detected in the ZHJ-1 biotype but not in B biotype.
Parafilm at the bottom and an 8 mm diameter hole was In this study, we used antibiotics to treat adult whiteflies made in the Parafilm for introducing adult whiteflies into of the B and non-B biotypes from Zhejiang, and examined the feeding chamber. The control diet solution was 0.005  Endosymbionts and fitness of Bemisia tabaci Table 1 PCR primer sets used in this study phosphate buffer (pH 7.0) with 25% sucrose (wt/vol), and significant at the 0.05 level, with ‘biotype’ and ‘antibiotic the antibiotics-treatment diet was the same basic solution treatment’ as the two categorical predictor variables, with the addition of 50 µg ml−1 tetracycline (Sigma, no.
followed by post hoc comparisons of means using Fisher T3258) (TEC), ampicillin trihydrate (Sigma, no. A6140) protected least significant difference (LSD) test. For each (AMP), or rifampicin (Sigma, no. R3501) (RF). Approxi- biotype, a 2 × 2 test of independence using a G-test was mately 50 newly emerged female or male adults were performed to compare the proportion of survival in each introduced into each feeding chamber, and the feeding antibiotic treatment with that in the control. All statistical chambers were placed in an environmental chamber (Sanyo calculations were carried out with STATISTICA 6 MLR-350HT, Gunma, Japan) at 28 °C with a photoperiod of L14:D10 and 60 – 80% r.h. The adult whiteflies werecollected after 48 h. Some of the adults collected were subjected to PCR detection for endosymbionts, and othersplaced on leaves of potted cotton plants using leaf-clip cages, with about 50 adults (approximately 1 : 1 sex ratio) The profiles of PCR amplification with primer H16 per cage. Adults were allowed to oviposit for 24 h and showed consistent and unique bands to distinguish the then removed from the cotton plants. The eggs laid on B biotype from the non-B ZHJ-1 individuals (Figure 1).
cotton leaves were counted under a dissecting microscope B biotype samples produced two distinct bands in the (Leica MZ6, Wetzlar, Germany) at 20 × magnification.
region between 300 bp and 500 bp. In contrast, ZHJ-1 Fifty to 60 eggs were left in each of the treatments for samples produced only one band in this region.
subsequent observations; extra eggs were removed usinga pair of microscopical forceps. The cotton plants wereplaced back in the environmental chamber for developmentof the offspring.
Body length of whiteflies was measured with an ocularscale under a stereo microscope (Olympus SZ-III, Tokyo,Japan) at 14 days after oviposition. Adult emergence waschecked daily until all adults had emerged. Some newlyemerged adults were subjected to PCR detection for endo-symbionts. The percentage survival from egg to adult wascalculated by dividing the number of empty pupal cases(from which adults had emerged) on the leaf with the initialnumber of eggs laid.
Figure 1 RAPD-PCR profiles using primer H16 to distinguish B biotype from the non-B ZHJ-1 Bemisia tabaci population. Lane 1: B biotype, Lane 2: ZHJ-1; Lane M contained 100 bp DNA ladder Body length and development time were first analyzed markers (1500, 1000, 900, 800, 700, 600, 500, 400, 300, and by GLM (general linear models) Factorial ANOVA, Figure 2 PCR screening for the presence of endosymbionts in the two biotypes of Bemisia tabaci. (A) 16S rDNA from Portiera aleyrodidarum; (B) 16S rDNA from Hamiltonnella defensa; (C) wsp gene from Wolbachia; (D) 16S-23S rDNA from Arsenophonus. Lane M contained DNA size markers (2000, 1000, 750, 500, 250, and 100 bp from top to bottom); Lane 1 is control treatment of B biotype adult, Lane 2 is AMP treatment of B biotype adult, Lane 3 is TEC treatment of B biotype adult, and Lane 4 is RF treatment of B biotype adult; Lane 5 is control treatment of ZHJ-1 adult, Lane 6 is AMP treatment of ZHJ-1 adult, Lane 7 is TEC treatment of ZHJ-1 adult, and Lane 8 is RF treatment of ZHJ-1 adult; Lane 9 is the negative control.
Diagnostic PCR screening for endosymbionts Fitness of Bemisia tabaci following antibiotic treatment Figure 2 shows the results of PCR screening for endo- Successful development from egg to adult was observed symbionts in the two biotypes of B. tabaci. Both biotypes in the offspring of the two biotypes of B. tabaci in all harbored P. aleyrodidarum. Hamiltonella defensa was antibiotic and control treatments. Results of two-way detected only in the B biotype, while Wolbachia and ANOVA on the mean body length indicated that this Arsenophonus were detected only in ZHJ-1. Following response variable was not significantly affected by biotype antibiotic treatment, P. aleyrodidarum was still detectable = 0.102, P = 0.75) or by the interaction between in the treated whiteflies and their offspring (Figure 2A).
However, H. defensa was undetectable in the treated B but was significantly affected by antibiotic treatment biotype adults (Figure 2B) and their offspring (data not = 53.23, P<0.001). In both biotypes, body length of shown), and Wolbachia and Arsenophonus were undetec- the offspring of adults treated with AMP was significantly table in the treated ZHJ-1 adults (Figure 2C,D) and their greater than that of the control, while body length in the offspring (data not shown). The results indicated that a treatment with RF was significantly shorter than that in the 48 h treatment with any of the three antibiotics at the control. Body length of offspring of the B biotype treated concentration of 50 µg ml−1 could eliminate H. defensa, with TEC was significantly greater than that of the control, Wolbachia, and Arsenophonus but not P. aleyrodidarum.
while body length of offspring of ZHJ-1 treated with TEC Endosymbionts and fitness of Bemisia tabaci Table 2 Effect of antibiotic treatment on fitness parameters of two biotypes of Bemisia tabaci 1Body length in mm of nymphs at 14 days after oviposition; the figures in parentheses indicate the number of survivors by that time. Values of mean ± SEM in the same column followed by the same letters do not differ significantly (P>0.05).
2Development time in days from egg to adult emergence; the figures in parentheses indicate the number of individuals that developed to adulthood. Values of mean ± SEM in the same column followed by the same letters do not differ significantly (P>0.05).
3Percentage survival from egg to adult emergence; differences between a given antibiotic treatment and control of the same biotype are indicated by ns (not significant), *P = 0.05, **P<0.01, or ***P<0.001.
did not differ significantly from that in the control treatments were relatively small and in some of the treatment levels of survival were low, no analysis of the As the data indicated that males and females differed in development time from egg to adult, data for the twosexes were analyzed separately. The development time of males was significantly affected by biotype (F 18.325, P<0.001) and antibiotic treatment (F Leonardo & Muiru (2003) and Tsuchida et al. (2004) P<0.001), as well as by the interaction between biotype showed that populations of the pea aphid, Acyrthosiphon and antibiotic treatment (F = 5.858, P = 0.001). Similarly, pisum, associated with different host plants or found in the development time of females was significantly affected different geographical locations may have different complements of symbionts. It is possible that different it was not significantly affected by biotype (F populations of the same biotype of B. tabaci may harbor P = 0.85) or the interaction between biotype and antibiotic different symbionts and may differ in their response to the antibiotic treatments used in this study. We tested only one treatments of adults with RF significantly prolonged population of each of the two biotypes of B. tabaci.
development time from egg to adult emergence of their off- Nevertheless, the comparison between the two popu- spring, while treatments of adults with AMP significantly lations has value, especially in view of the uniform identity reduced the development time of their offspring (Table 2).
of each of the two biotypes by molecular markers (COI and Treatment of ZHJ-1 adults with TEC significantly reduced ITS1) across its distribution range (De Barro et al., 2000; the development time of their offspring, but the same Luo et al., 2002; Wu et al., 2003; Zhang et al., 2005).
treatment of the B biotype did not have a significant effect(Table 2).
In the B biotype, treatments of adults with TEC or AMP All three antibiotics tested at a concentration of 50 µg ml−1 significantly increased the survival of their offspring, while were ineffective in removing P. aleyrodidarum from the the same treatment with RF did not have a significant effect whitefly adults and their offspring in both B and the non- (Table 2). In ZHJ-1, treatment of adults with TEC or AMP B biotypes of B. tabaci. Earlier work on aphids suggested did not have a significant effect on survival, while the same that penicillin, which acts on the cell wall, was ineffective treatment with RF significantly reduced survival of their in eliminating Buchnera (the P-endosymbionts of aphids) (Griffiths & Beck, 1974). The cell wall of Buchnera was The sex ratios varied widely between the treatments, reduced and the genome of Buchnera lacked some of the for both biotypes (Table 2). As the sample sizes for the genes for the biosynthetic pathway of the cell wall. Portiera aleyrodidarum from B. tabaci lacked a distinct cell wall insects. As the presence of the P-endosymbiont was not (Costa et al., 1993b). Therefore, ampicillin trihydrate that determined quantitatively, it was possible that a reduction inhibits bacterial cell-wall synthesis would selectively act of the P-endosymbiont after antibiotic treatment was on the S-endosymbionts without affecting P. aleyrodidarum.
also involved in the varying performance of the treated However, rifampicin inhibits DNA-dependent RNA poly- insects. This is the first report that antibiotic treatment merase in bacterial cells by binding its beta subunit, thus can increase fitness of the host insect for the target preventing transcription of messenger RNA (mRNA) and subsequent translation to proteins. Rifampicin diffuses Because the various cohorts of whiteflies in the various freely into tissues, living cells, and bacteria, making it treatments differed in development time, the measure- extremely effective against intracellular pathogens such ments of body length on the 14th day after oviposition as Mycobacterium tuberculosis (Shinnick, 1996; Campbell involved nymphs of varied ages among the treatments.
et al., 2001). Tetracycline affects endosymbionts by inhi- These data provided an additional measurement to show biting protein synthesis, and thus in theory these two the differences in development rate between treatments, antibiotics can completely remove P. aleyrodidarum. As but they did not reflect the actual effects of antibiotic treat- P-endosymbiont is essential for the survival of the whitefly ments on body size. Investigation of the effects on body and in our experiments survival of the offspring following size would require comparison of progeny at the same treatment of adults was observed in every treatment in developmental stage across the treatments.
both biotypes, our data indicate that TEC and RF Wilkinson (1998) reviewed studies on endosymbionts treatments did not eliminate P. aleyrodidarum in the test of aphids and made the suggestion that ‘treatment of insects. All three antibiotics were capable of removing the aphids with antibiotics to eliminate the symbiotic bacteria three S-endosymbionts considered in this study. Further has no direct deleterious effects on aphid biology’. In our evidence for these removals may be obtained through experiments, the performance of offspring produced observations by electron microscopy.
by adults treated with antibiotics was observed. If the So far, six different endosymbionts have been reported antibiotic treatments did not have direct effects on the from whiteflies, i.e., P. aleyrodidarum, H. defensa, Wolbachia, adult biology or even if they did have but the effects did Arsenophonus, Cardinium, and Fritschea bemisiae (Nir- not pass on to next generation, the retarded growth and gianaki et al., 2003; Thao et al., 2003; Weeks et al., 2003; development as well as reduced survival in the offspring of Thao & Baumann, 2004a; Everett et al., 2005). Recently RF-treated adults (Table 2) should be attributed to a direct a new S-endosymbiont, Rickettsia, was discovered from effect of the loss of the known S-endosymbionts and/or a B. tabaci (E Zchori-Fein, pers. comm.). In the present reduced quantity of P-endosymbionts. Costa et al. (1997) study on B. tabaci, we screened only the P-endosymbiont also reported adverse effect of RF treatment of B. tabaci and three S-endosymbionts. It is possible that more adults on the fitness of their offspring. The same specula- endosymbionts existed in the two B. tabaci populations tions may apply to the causes of the favorable effects from and that our antibiotic treatments did not remove all these After the adults of the B biotype and the non-B ZHJ-1 population received the antibiotic treatment, the offspring Effects of antibiotics on Bemisia tabaci they produced showed similar responses in growth and In this study with two biotypes of B. tabaci, treatments of development. However, the survival of their offspring adults with TEC or AMP accelerated the development rate differed: survival in the B biotype was significantly reduced of their offspring in both biotypes, as indicated by both the after treatment with AMP or TEC but was unaffected after body length at 14 days after oviposition and development treatment with RF, while survival in the ZHJ-1 was not time of the whole immature stage, and also increased significantly affected after treatment with AMP or TEC but the survival of the offspring in the B biotype (Table 2).
was significantly reduced after treatment with RF. These In contrast, treatment of adults with RF significantly two biotypes differ genetically and have no reproductive retarded the development of their offspring in both compatibility; they also differ in body size, competitive biotypes and reduced the survival of the offspring in the ability, diet breadth, and levels of resistance to some non-B biotype (Table 2). As profiles of diagnostic PCR insecticides (Zang et al., 2005a,b, 2006; also see the data of screening of the test insects showed that treatments the two biotypes in the control treatments in Table 2). It is with any of the three antibiotics removed the known likely that the varied responses to the antibiotic treatments S-endosymbionts, the results indicate that removal of in this study between the B and non-B biotypes may reflect these S-endosymbionts from B. tabaci may produce both intrinsic differences between them and thus warrant favorable and unfavorable effect on the fitness of the host Endosymbionts and fitness of Bemisia tabaci Douglas AE (1989) Mycetocyte symbiosis in insects. Biological Reviews of the Cambridge Philosophical Society 64: 409–434.
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