Fitness aspects of transgenic Aedes fluviatilis mosquitoes expressing a Plasmodium-blocking molecule
Transgenic Research Associated with the International Society for Transgenic Technologies (ISTT) ISSN 0962-8819 Volume 19 Number 6 Transgenic Res (2010) 19:1129-1135 DOI 10.1007/ s11248-010-9375-8
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Author's personal copy Transgenic Res (2010) 19:1129–1135 DOI 10.1007/s11248-010-9375-8
BRIEF COMMUNICATION
Fitness aspects of transgenic Aedes fluviatilis mosquitoes expressing a Plasmodium-blocking molecule Maı´ra N. Santos • Paula M. Nogueira Fernando B. S. Dias • Denise Valle • Luciano A. Moreira
•
Received: 12 January 2010 / Accepted: 1 February 2010 / Published online: 13 February 2010 ! Springer Science+Business Media B.V. 2010
Abstract Vector-borne diseases cause millions of deaths every year globally. Alternatives for the control of diseases such as malaria and dengue fever are urgently needed and the use of transgenic mosquitoes that block parasite/virus is a sound strategy to be used within control programs. However, prior to use transgenic mosquitoes as control tools, it is important to study their fitness since different biological aspects might influence their ability to disseminate and compete with wild populations. We previously reported the construction of four transgenic Aedes fluviatilis mosquito lines expressing a Plasmodiumblocking molecule (mutated bee venom phospholipase A2–mPLA2). Presently we studied two aspects of their fitness: body size, that has been used as a fitnessrelated status, and the expression of major enzymes classes involved in the metabolism of xenobiotics,
including insecticides. Body size analysis (recorded by geometric wing morphometrics) indicated that both male and female mosquitoes were larger than the nontransgenic counterparts, suggesting that this characteristic might have an impact on their overall fitness. By contrast, no significant difference in the activity of enzymes related to metabolic insecticide resistance was detected in transgenic mosquitoes. The implication on fitness advantage of these features, towards the implementation of this strategy, is further discussed. Keywords Transgenic mosquito ! Fitness ! Insecticide ! Geometric morphometrics ! Phospholipase A2
Introduction M. N. Santos ! P. M. Nogueira ! L. A. Moreira (&) Malaria Laboratory, Centro de Pesquisas Rene´ Rachou (CPqRR/FIOCRUZ), Av. Augusto de Lima 1715, Barro Preto, Belo Horizonte, MG 30190-002, Brazil e-mail:
[email protected] F. B. S. Dias Laboratory of Triatomines and Epidemiology of Chagas Disease, CpqRR/FIOCRUZ, Av. Augusto de Lima, 1715, Barro Preto, Belo Horizonte, MG 30190-002, Brazil D. Valle Laboratory of Physiology and Control of Arthropod Vectors, Instituto Oswaldo Cruz (IOC/FIOCRUZ), Av. Brasil 4365, Manguinhos, Rio de Janeiro, RJ, Brazil
The impact of vector-borne diseases in the daily life of millions of people worldwide is disastrous. Malaria is a disease caused by the protozoan parasite Plasmodium spp., which is the foremost vector-borne disease worldwide, continues to worsen in many areas. There are now estimated 250 million cases of malaria worldwide and 3 billion people are at risk of infection, in 109 malarious countries (WHO 2008). The use of transgenic mosquitoes as an alternative control strategy has been attempted for at least 10 years and could be possibly used concomitantly with other already existent methods, either biological
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or even with insecticides. Important cornerstones have been met either as proof of principle for the transformation technique per se (Coates et al. 1999; Jasinskiene et al. 1998) or towards the practical application (Ito et al. 2002; Kokoza et al. 2000; Yoshida and Watanabe 2006). Fitness characteristics have been also studied for some of the successful transformation events, showing normally positive (Marrelli et al. 2007; Moreira et al. 2004) but sometimes negative (Catteruccia et al. 2003; Irvin et al. 2004) results. Fitness studies are a requisite to the release of any transgenic line in the field since the introduced strain should have at least equal chances to survive in comparison to the wild population (Marrelli et al. 2006). Normally, fitness cost occurs mostly in transgenic organisms carrying promoters that drive protein expression in all cells and less likely when these promoters are driving protein expression to specific tissues; but the cost can also occur due to an essential gene interruption (Lyman et al. 1996). Other important characteristics, such as insecticide resistance, are of utmost importance to be tested in transgenic lines before its actual use. Activation of detoxification enzymes is one of the main mechanisms of insecticide resistance. Known as metabolic resistance, it is based on the presence of higher levels or modified activity of three main enzyme classes, Esterases (EST), Multiple Function Oxidases (MFO) and glutathione S-Transferases (GST), total or partially hampering the insecticide to reach its target site in the central nervous system (Brogdon and McAllister 1998). MFO are potentially involved in the metabolism of all classes of insecticides, Esterases are frequently linked to the detoxification of carbamates, organophosphates and on a minor extension to pyrethroids, and GST have been associated with pyrethroids and organochlorines degradation (Brogdon and McAllister 1998; Hemingway and Ranson 2000; Montella et al. 2007). Here we explored some fitness-related characteristics of four transgenic lines of Ae. fluviatilis expressing the mutated phospholipase A2 enzyme (PLA2m) (Rodrigues et al. 2008) in relation to wing morphometrics, as a proxy for body size, as well as the resistance status to the organophosphate temephos and the activity of resistance related enzymes. Many studies, including this presented here, shall be of primordial matter before transgenic mosquitoes can
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be employed in the field as a disease or vector control strategy.
Materials, results and discussion Colonies of the non-transgenic Ae. fluviatilis and the four transgenic lines (4T, 8T, 9T and 10T) (Rodrigues et al. 2008) were reared under controlled temperature (27"C ± 1"C), humidity (70 ± 10%) and photoperiod 12:12 L/D. Adult mosquitoes were fed on 10% sucrose solution ad libitum and females fed on anesthetized mice for egg production. Larvae were fed with pellets of fish food (Goldfish Colour). Pupae were collected in small plastic cups and transferred into 2L cardboard cages. Transgenic mosquitoes, expressing the enhanced Green fluorescent protein (EGFP) were selected under an epi-fluorescence stereoscope. Heterozygote transgenic mosquitoes were used in all experiments by crossing, on every generation, transgenic males with virgin laboratory-colony females (Rodrigues et al. 2008). When the experiments described here were performed, transgenic colonies had been kept in the laboratory for more than 25 generations without any apparent sign of fitness loss, based on egg production and fertility. To study wing morphometry we used the right wings from both male and female mosquitoes (n = 30 of each sex and line). Mosquitoes were anesthetized on ice and had their wings removed with forceps under dissecting microscope. The wings were then fixed in white paper sheets with the aid of a transparent tape. Images were obtained with a digital camera (Nikon 4300) attached to a dissecting scope, at 409 magnification. Seven reference-points (landmarks) have been selected on wings (Fig. 1a) and were converted into coordinates with the aid of tpsDig version 2.1 software, (open Internet access) (Rohlf 1990). Firstly we analyzed the sexual dimorphism on size and wing conformation for both male and female matrixes and in all cases females were larger than males as previously reported (Jirakanjanakit et al. 2008) (data not shown). Further we evaluated the size variation and conformation of each sex, comparing the transgenic lines and the non- transgenic siblings. For the geometric morphometrics analysis we used the mathematical algorithm, Generalized Procrustes Analysis, which comprehends the optimum mathematical superposition of the
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Fig. 1 Morphometrics analysis of transgenic Aedes fluviatilis mosquitoes. a Selected landmarks on mosquito wings for morphometric analysis. b Centroid analysis for males of four transgenic lines (4T, 8T, 9T, 10T) and non-transgenic mosquitoes (NT). c same as above, females. NTs differed significantly from all transgenic lines for both males (P \ 0.05, Kruskal–Wallis) and females (P \ 0.0001, Kruskal–Wallis)
geometric coordinates. According to this process two variables were obtained: size, denominated Centroid Size, which estimates the isometric size and represents the central point of the polygon formed by the junction
of reference points, and the conformation variables (partial warps) (Rohlf 1993). Both size variables and conformation variables were obtained with the help of the software MOG, version 79. All softwares used in
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the morphometric analysis are freely available (http:// www.mpl.ird.fr/morphometrics). Size variation analysis was performed with the Kruskal–Wallis non-parametric test and size comparison among different transgenic lines was done with the Mann–Whitney U test. A multivariate regression was performed with the first conformation variable versus the size variable to check whether there was any allometric effect of size in the conformation parameter. Statistical analyses were performed through the softwares PAST, (http://life.bio.sunysb.edu/morph/) and Prism, version 3.0. Wing morphometrics has been reported as a more accurate method to detect body size in comparison to wing length because it takes into account the variation in wing size in all directions (Jirakanjanakit et al. 2007). It has been used in mosquitoes to detect effects of larval density (Jirakanjanakit et al. 2007), to study isofemale lines through generations (Jirakanjanakit et al. 2008) and to discriminate dengue vectors (Henry et al. 2009). Here we applied this technique for the first time in Ae. fluviatilis and also for transgenic mosquitoes as a proxy for body size. Analysis of wing size (centroid size, Fig. 1 b, c) indicated that both non-transgenic females (P \ 0.0001, Kruskal–Wallis) and males (P \ 0.05, Kruskal–Wallis) were significantly smaller in comparison to all transgenic lines. The linear regression multivariate analysis, applied to detect any relation between the wing conformation variation and size (allometry), showed that the variation of conformation is not influenced by wing size (females: F = 0.8653, r2 = 0.005932, P = 0.3538) (males: F = 0.007356, r2 = 0.00005073, P = 0.9318). Many examples of body size and higher fitness have been reported in mosquitoes in relation to malemating success (Ponlawat and Harrington 2009), survival (Maciel-De-Freitas et al. 2007) or even towards egg production and female feeding behavior (Takken et al. 1998; Voordouw and Koella 2007; Xue et al. 1995b). Hence, the larger body size of the four transgenic mosquito strains studied here could imply in a fitness advantage in the field, with an increased possibility of local dissemination among the local population. Bioassays with the organophosphate temephos, one main larvicide employed against mosquito vectors, were performed to verify if the presence of the transgenes could modify by any degree, the
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Transgenic Res (2010) 19:1129–1135 Table 1 Lethal concentrations (LC, in ng/100 ml) of transgenic Ae. fluviatilis mosquitoes to the organophosphate temephos and resistance ratios (RR) as compared to a nontransgenic strain, NT Strains
LC50
LC95
RR50
RR95
Slope
NT
0.0097
0.0269
1.0
1.0
3.7
4T
0.0131
0.0188
1.4
0.7
10.6
8T
0.0140
0.0185
1.4
0.7
13.7
9T
0.0131
0.0180
1.4
0.7
11.8
10T
0.0134
0.0241
1.4
0.9
6.4
resistance/susceptibility of transgenic mosquitoes to insecticides. Temephos dose–response assays were conducted essentially as described elsewhere (Braga et al. 2004), in accordance with the WHO protocol (WHO 1981). Resistance ratios (RR) were calculated by comparison with the lethal concentration obtained for non-transgenic Ae. fluviatilis strain (Table 1). At least four assays were run with each mosquito lineage and, in no case a statistically significant difference in lethal concentrations between the transgenic and non-transgenic lineages was detected, pointing to equivalency of fitness among all strains regarding insecticide resistance. This was corroborated by the RR values obtained, equivalent between the control and transgenic strains. The higher slope of transgenic strains is compatible with highly homogeneous samples, a result expected since each strain derived from a single transgenic mosquito. Biochemical assays were also conducted in order to evaluate the activity of enzymes potentially related to insecticide resistance. One-day old adult females, reared in the absence of any insecticide, were frozen at -70"C and further submitted to biochemical assays. The activities of Acetylcholinesterase, GSTs, ESTs, and MFOs were quantified according to the protocol previously described (Valle et al. 2006). EST activity was evaluated with three different substrates: a-naphthyl (referred to as a-EST), b-naphthyl (b-EST), and p–nitrophenyl (PNPA–EST) acetate. Results of enzymatic activities obtained from individual mosquitoes were corrected according to total protein. The percentage of individuals with enzymatic activity above the non-transgenic 99th percentile was calculated for each enzyme. According to this procedure, activities are classified as unaltered, if the rate is below 15%, incipiently altered, if value is between 15 and 50%, and altered when above 50% (Montella et al.
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Table 2 Activity of insecticide resistance-related enzymes in transgenic and non-transgenic Ae. fluviatilis mosquitoes ACE (%act)a
Mosquito
med
p99
n
med
p99
125 n
82.8 med
91.8 % [p99e
119 n
49.2 med
88 % [p99
n Non-transgenic Lines
4T
109
84.9
16
95
53.9
5
114
82.2
0
116
44
4
9T
151
84
0
89
45.8
3
10T
116
86
1
96
52.6
4
A-EST (nmol/mg ptn/min)
Non-transgenic
B-EST (nmol/mg ptn/min)
n
med
p99
n
med
p99
133 n
85.8 med
142.3 % [p99
131 n
222.08 med
388.3 % [p99
4T
118
80.2
0
117
222.3
2
8T
158
72.9
0
147
198
1
9T
155
79.9
0
158
198
0
10T
111
94
3
116
218.1
0
Mosquito
PNPA-EST (D abs/mg ptn/min)
GST (mmol/mg ptn/min)
n
med
p99
n
med
p99
125 n
4.9 med
8.8 % [p99
132 n
1.4 med
2.2 % [p99
4T
81
4.9
4
117
1.3
0
8T
120
4.7
4
79
1.6
1
9T
106
4.7
3
117
1.5
0
10T
96
5.4
6
118
1.5
1
Non-transgenic Lines
d
8T
Mosquito
Lines
c
MFO (nmoles cit/mg ptn)
b
Enzyme symbols are the same as in the text Enzyme activities are classified as unaltered (italics), incipiently altered (bold) or altered if the rate above non-transgenic 99th percentile is below 15%, between 15 and 50%, and above 50%, respectively a
Rate of activity in the presence of propoxur (% act) is shown
b
Number of specimens evaluated
c
Median of each enzymatic activity
d
Percentile 99 for non-transgenic line
e
Rate of the population with activity higher than the non-transgenic 99 percentile
2007; Valle et al. 2006). Except for the 4T line that exhibited an ‘‘incipiently altered’’ activity for Acetylcholinesterase (slightly above the threshold level), all the remaining enzyme activities for the four transgenic strains were classified as unaltered (Table 2). Since no significant differences relative to the non-transgenic line were noted in temephos susceptibility (Table 1),
and Acetylcholinesterase is involved in organophosphorus metabolism (Hemingway and Ranson 2000), we can speculate that this alteration has no biological relevance. Although no differences in insecticideresistance between transgenic and non-transgenic mosquitoes have been detected in the present study, large females have been previously associated to a
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lower DEET responsiveness as compared to smaller ones (Xue et al. 1995a). This indicates the importance of evaluating different aspects potentially associated with biological fitness and transmission of pathogens. The idea of including this type of study in fitness evaluation is pioneer, and is based on the existing possibility of the integration of transgene, for example, in a promoter region, enhancing detoxifying enzymes expression, or disrupting a gene linked to metabolism of insecticides. Because transposon can randomly integrate into the insect’s genome and due to position effects, other genes could have their expression differentially modified (Raffel and Muller 1940). One way to prevent this possible deleterious effect is to generate lines possessing site-specific insertions of the transgene by the use of phage integrase mechanism (Nimmo et al. 2006). Although we are aware that in natural populations the phenotypic behavior of transgenic lines could be completely different from laboratory conditions we think that studies to reduce any chances of unsuccessful implementation of this strategy in the field should definitely be extensively performed. Acknowledgments We would like to thank Walison E. de Jesus for technical support. L.A.M. and D.V. received research fellowships from CNPq. This work was partially funded by FAPEMIG.
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