(Luận văn) wing morphometry of the females of culex pipiens and culex torrentium under different densiy conditions

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THANH TAN lu an n va Thesis title tn to WING MORPHOMETRY OF THE FEMALES OF CULEX p ie gh nl w PIPIENS AND CULEX TORRENTIUM UNDER DIFFERENT d oa BREEDING CONDITIONS an lu nf va BACHELOR THESIS z at nh oi lm ul Study Mode: Full- time Major: Bachelor in Environmental Science and Management z gm @ Faculty: International Training and Development Center Batch: K42- Advance Education Program m co l an Lu n va Thai Nguyen, January 21, 2015 ac th si DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Bachelor of Environmental Science and Management Program Student Nguyen Thanh Tan Student DTN1053110170 Thesis WING MORPHOMETRY OF THE FEMALES OF Name lu an n va ID gh tn to CULEX PIPIENS AND CULEX TORRENTIUM UNDER p ie Title oa nl w DIFFERENT DENSIY CONDITIONS Superviso Dr Rolf Niedringhaus (Oldenburg University, d lu Germany) nf va an rs z at nh oi Abstract: lm ul Associate Prof Dr Do Thi Lan Cx pipiens and Cx torrentium are two sibling mosquito species with a z @ close resemblance in morphology and ecology The morphological separation of gm co l these mosquitoes can only use for male, however, this technique is extremely difficult m and unsure Besides that, the discrimination of the female of the species was a hard an Lu burden Recently, Börstler et al., (2014) used a tool called wing morphometry n va ac th si which allowed to separate the two species However, it is expected that density/stresses on larval stage could modify the wing shape in a certain way Thus, having this project conducted This study aims to: 1) to test the reliability of the wing morphometry method for taxonomy of Cx pipiens and Cx torrentium under different density conditions; 2) bridge some gaps of the insufficient knowledge about the link of ecology resulted on the wing morphology of Cx pipiens and Cx torrentium To answer these questions, rearing mosquitoes, morphometric data collection and bivariate & multivariate lu an analysis were used The results of all methods are all in agreement and yielding n va the low classification success of mosquitoes under different density conditions tn to Concluding from this low classification success, the wing morphometry of ie gh p mosquitoes Cx pipiens and Cx torrentium were not stable or modified under oa nl w different density conditions The results implies a possibility to predict species occurrence in other parts of the breeding range, and to determine possible d lu Keyword nf va an breeding places in further surveys s Number z of page z at nh oi lm ul Geometric morphology, Culex Complexes, Cx pipiens, Cx torrentium, vein, landmarks, taxonomy, breeding conditions m co l Submission January 15, 2015 gm @ Date of an Lu n va ac th si ACKNOWLEDGEMENT First and foremost, I wish to express my sincere thanks to Prof Dr Ellen Kiel for given permission to accomplish my Bachelor thesis there, and also her constant motivating supervision during my studies in the research group of AG Gewässerökologie und Naturschutz, I would like to thank to my principal research advisor M.Sc Renke Lühken, who guided me wholeheartedly and uncomplicated support In particular, I place on record my gratitude to him for the multivariate analysis, and the identification of some mosquito species Without him, this work lu an cannot be done va n Furthermore, I want to deeply thank IAESTE organization, the administration to gh tn department of Oldenburg University, and the boards of Thai Nguyen University of p ie Agriculture and Forestry, for giving me this valuable and unforgettable chance studying and working in Germany, and more important providing me all necessary nl w d oa facilities, skills and knowledge to complete this bachelor thesis nf va an lu Especially thankful I am for the support of all staff and students in the research group of AG Gewässerökologie und Naturschutz, Oldenburg Fakultat V-Mathemetik lm ul und Naturwissenschaften Thanks a lot for your expert, valuable guidance and z at nh oi experiences during my working time there Finally yet importantly, I take this opportunity to record my sense of gratitude z m co l Thank you very much! gm @ to my families and friends who encourage and backing me unceasingly an Lu n va ac th si TABLE OF CONTENTS Page lu an n va p ie gh tn to d oa nl w nf va an lu z at nh oi lm ul z m co l gm @ an Lu n va ac th si LIST OF TABLES Page lu an n va p ie gh tn to d oa nl w nf va an lu z at nh oi lm ul z m co l gm @ an Lu n va ac th si LIST OF FIGURES Figure Distribution of the Cx pipiens complex and its sibling species (the map of Smith et al.,2004) 19 Figure Hypopygium of Cx torrentium(Becker et al., 2010) 20 Figure3 Aedeagus and paraproct of: (a) Cx p pipiens; (b) Cx torrentium;(Becker et al., 2010) .20 Figure Location of the study area for mosquito collections on the main campus –Haarentor of Oldenburg University, Germany (Google Map, 2014) 28 Figure A black bucket exposed near a water body containing dry hay infusion.………………………………………………………………………………17 Figure Two black buckets exposed near a pond containing dry hay lu infusion… ….17 an Figure All egg rafts were kept in rearing glass filled with the eutrophic hay infusion.30 va n Figure For each egg raft, 110 larvae were reared for 10 replicates of to tn experimental gh combinations…………… 18 were plugged tube p ie Figure All larval rearing racks are housed inside the rearing rooms, glass with cotton since the fifth 10 Emerged adults were pushed on a black off the wing d oa Figure nl w day……………………………………………….18 11 Using a tiny broom to scrape nf va an Figure lu net…………………… … …….19 scales……….…… ……… 19 lm ul Figure 12 Euparal 3C 239 (Waldeck GmbH & Co KG, Muenster) Figure 13.The positions of the 13 morphometric landmarks on the Culex complexes z at nh oi a) right wings b) left wings 33 Figure 14 The development stages of Cx pipiens and Cx torrentium a) Egg raft and z b) fourth instar larvae The photos were taken under the Leica MZ 7.5 gm @ Stereomicroscope and seen by Adobe Photoshop software version 7.0 .37 Figure 15 Principal component analysis of the shape variance of the mosquito female l left wings in a) low density (left) b) high density (right) Grey point indicates Cx co m pipiens and Black dots indicate Cx torrentium 44 an Lu Figure 16 Principal component analysis of the shape variance of the mosquito female right wings in a) low density b) high density Grey point indicates Cx pipiens and va Black dots indicate Cx torrentium 45 n ac th si Part INTRODUCTION 1.1 Research rationale The mosquitoes (Diptera: Culicidae) are one of the most life-threatening species on the planet because of their ability to transmit many deadliest diseases They are capable to bear the dangerous parasites or pathogens such as viruses, bacteria, protozoans, and nematodes from one vertebrate host and transmit them to another, lu which cause serious diseases such as malaria, yellow fever, dengue, as well as, an n va filariasis, Japanese encephalitis and a hundred others maladies by virtue of their blood- gh tn to sucking habits (Kettle 1995; Beaty and Marquardt 1996; Lehane 1991; Eldridge and Edman 2000, cited by Becker 2010) p ie According to the U.S Centers for Disease Control and Prevention, Ohio State nl w d oa University and Texas A&M University (March, 2007), mosquitoes and the diseases nf va an lu they spread have been killing more people than all the wars in the past Even nowadays, a great number of human beings in the world are still in danger of getting lm ul mosquito-borne diseases It was reported by the American Mosquito Control z at nh oi Association that every year, over one million people die from mosquito-borne diseases throughout the world Even though, humid tropics and subtropics are the favor habitats z gm @ for roughly three quarters of all mosquito species, mosquito-borne diseases occur not co l only in these regions (Becker, 2010) but in other region as well As a consequent of global trade and climate warming scenarios (IPPC), mosquito-borne diseases are m an Lu currently explosive worldwide, and have also influenced the sustainable development n va of the world, not only in socio-economy but also in politics The consequences of ac th si mosquitoes infection has been recorded in all continents, especially in South Africa and Asia In Viet Nam, only Dengue fever (a mosquito-borne disease) caused by Dengue virus, there were approximately 150,000 cases reported each year From those, it was about 50 to 100 people die, and most of them are children (The Eliminate Dengue Program) This trend is predicted to be increase dramatically in the far future, and become a horror for the country due to the fact that there are still no vaccine against dengue fever In order to effectively control the mosquitoes-borne diseases, we need to have a lu an good knowledge on mosquito’s systematics, morphology, and biology, which are still n va stayed limited in Viet Nam Some developed countries, in particular Germany, have to gh tn experienced the advancement in mosquito research, and control work Over 3500 p ie mosquito species have been reported, as well as a rich background and varied information exist which were well documented However, the ambition of having nl w d oa further comprehensive understanding of mosquitoes remain endless, particular in nf va an lu regard to biological and physiological distinctions, upon which the realities of subspecies depend on many variants causing remaining unsolved problems lm ul As not much threat as Anopheles and Aedes mosquitoes, the Culex (Cx.) z at nh oi mosquito is the third dangerous type of mosquito inhabiting our world Though it takes the blood meal from bird instead of human, it is believed to translate its assortment of z @ l gm diseases vector which is potentially fatal to mankind Floore & Tom (2002) describes Culex mosquitoes are painful and persistent biters, they prefer to attack wild birds in co m domestic and dark place, and rarely far away from human shelters, and living They an Lu use to be active only for a few weeks during the summer seasons when the females va n search for the warm surface water to lay their eggs Found in many temperate regions ac th si throughout urban and suburban area, Culex pipiens L is one of the most common and widespread mosquito species (Weitzel et al., 2009) From Raymond (1995) references, Cx pipiens and Cx torrentium are two sibling mosquito species with a close resemblance in morphology and ecology, which was first discovered in Britain in 1951 (Andersson & Jaenson 1987, Dahl 1988, Gillies & Gubbins 1982, Service 1968) The recent works (Hesson et al., 2013; Rudolf et al., 2013) argued that the two species has become widespread and common in central Europe and predominate in Germany Service (1968) indicated that the population size of Cx torrentium is about lu a third of that for Cx pipiens, from her specimens collection in some southern an n va localities in England Moreover, observations made by Service (1968), larvae of both gh tn to species usually see altogether in the same habitats, and no differences were recorded in the morphology of their immature stages Similarly, a comparative taxonomic study of p ie w Dahl C (1988) showed that neither the egg rafts nor larvae of both species can be oa nl distinguished It is, therefore, causing the confusion in many situations such as d distribution, vector status, and particular in diseases control program While Cx an lu nf va pipiens is the main vector, or carrier, of St Louis Encephalitis, West Nile Virus, lm ul Western Equine Encephalitis, Heartworm in dogs, and bird Malaria (Parreira et al., z at nh oi 2007; Papa et al., 2013; Radrova et al., 2013); Cx torrentium is a highly conveyor to Sindbis virus in Sweden, Norway, and Russia and turned out to be the main enzootic z vector for Sindbis in Sweden (Lundström 1994; Lundström et al., 1990, cited by gm @ Börstler et al., 2014) Although their main victim has been bird, they are considered to l an Lu and other mammals m co be a “bridge” vector as it was found to be able to transmit viruses from bird to humans n va ac th si and to 15 degree Celsius in the night time) As consequently, the survival rate decreased dramatically, especially in the last stage of pupation Moreover, feeding is also very important for the larval and pupal development, and it could be a reason for the mortality rate An abundance of literature mentioned about the food for larvae rearing mosquitoes Concluding from slower development of both species, eutrophic and food lacking consumption may be assumed (Table 2) As illustrated, the Cx pipiens required 16 to 19 days for larval development and to days were needed for the pupal development By the same longer taken, the Cx lu an torrentium required 10 to 18 days for larval completion, and maximum days for n va pupation It was longer because according to the manual for rearing mosquito of to gh tn Eugene et al., (1970), while he found that the Cx pipiens only need 8-13 days for p ie larval development and maximum days for pupation, and 5-10 days for larval development, and 2-3 days for pupal stages of Cx torrentium By the researcher’s nl w d oa observation, before and after fed them, there were a difference between the two nf va an lu groups, the groups of low density increased their mortality, whereas the high density increased their survival rate For the first group, the researcher think the larvae died is lm ul due to eutrophic (too much food) in water with the minimum of rearing individuals, z at nh oi and vice versa the mortality rate decreased in the high density group due to the food balance in water, because there were many larvae reared, the higher energy z gm @ consumption is expected This hypothesis is consistent for Ae Aegypti by co l Timmermann & Briegel (1998) as they tested the effect of starvation and feeding for m larvae With crowded conditions and minimal food supplies, most larvae were starved, an Lu even completely died Much less, the larvae grown in optimal conditions was also n va started to decrease the population after they fed Additionally, Reisen et al., (1984) ac th si found a delay in median ages at pupation dropped from 13 days to 17 days, and an incredible reduction from larval to adult survival which was original at 47% at larval survived to only 3% when he increased the inter-competition among larvae of Culex tarsalis Coquillett population These findings are consistent with the assumption and explanations mentioned above As a matter of fact, the effect of competition treatment was significant for both species The percentage of adults reared in the high density was only 9.77% survival rate, while it was 25.4 % for ones reared in low density This findings is logical with lu an the works of Jirakanjanakit et al., (2007), as he also found that a simple reduction in n va larval density couldconsiderably increase the rate of survivors, as well as the size of to gh tn individuals, turning them to better vectors The possibilitythat a mosquito, or any p ie vector competence can be influenced by its environment, consisting the breeding larval conditions were demonstrated in the works of Alto et al., (2005), or Bevins (2008) nl w d oa They altogether found that a significant higher of infection and dissemination with nf va an lu virus as result of higher density treatment Alto et al., (2005) did not deny that there were a strong effects of larval competition on vector competence for dengue virus in lm ul Ae Albopictus and Ae aegypti The researcher acknowledges that density-dependent z at nh oi competition among larvae is a key factor which is regulating the growth of mosquito populations z @ l gm From all the hypothesis and result above, it is concluded that the mortality rate as well as differences of mortality between groups were caused by the inter- and intra- co m environmental factors such as temperature, humidity, light intensity, feeding and an Lu competition in breeding conditions n va ac th si 5.1.2 Morphometric Differences between Treatment Groups The low classification success yielding from different breeding conditions by using multiple methods made the researcher believed that the environmental influences was modified both genetic and morphology of mosquitoes, especially the wing shape This hypothesis was not unfamiliar in some well-known publications, for example Debat et al., (2003), Hoffmann et al., (2005), Hoffmann et al., (2005), Aytekin et al., (2009) and Russell et al., (2011), when they confirmed that there were a significant change in the mosquito species, and more clearly yielding on the wing shape lu an This present study used both multivariate and bivariate wing morphometry n va analysis for the identification of the two sibling mosquitoes Cx pipiens and Cx to gh tn torrentium Contrasts were performed within each density treatment to determine p ie whether shape could be used to distinguish between high and low inter-competition oa nl w A bivariate morphometric analysis of mosquito species with variable as the d vein length r2/3 and r3 Both male and female, the right and left wings of the two lu nf va an species were used for analysis (Table 3) This present study found that the univariate wing morphometry analysis of index r2/3/r3 was 0.411 ± 0.088 (mean ± SD) in Cx lm ul torrentium, and was 0.294 ± 0.096 (mean ± SD) in Cx pipiens The average distance z at nh oi from the mean of the index among individuals were relative small, means that all specimens had high correlation to each other Though, these two siblings had a closer z @ gm index to each other, which may revealed a reliable character for their genus taxonomy, co l this result in the present study is consistent with other publications (Börstler et al., m 2014, Mohrig 1969) about the proportion of the index in both species in which Cx an Lu pipiens had higher value in the indices r2/3/r3 However, the findings did not fall closely va n in the range given by Börstler et al., (2014), as he found that the indices of wing ac th si morphometric was 0.185 in Cx pipiens, and 0.289 in Cx torrentium, or equally values were found by Mohrig (1969), in which Cx pipiens had the indices value of 0.167, and Cx torrentium was only 0.33 Still, the researcher think it can be a trustworthy index to discriminations of both species However, in accordance with the older literature such as Natvig (1948), Mohrig (1969) it cannot be reconstructed whether r2/3 was the only or the most variable vein, the researcher conducted a bivariate approach for taxonomic value of wing vein r 2/3 and r3 in Cx pipiensand Cx torrentium In fact, this characterseem to be neglected lu for both species more than 40 years (Börstler et al., 2014) This character has been an n va first defined by Natvig (1948) for Scandinavian populations of Culex pipiens and Cx gh tn to torrentium, and subsequently Mohrig (1969) for the populations in German Nevertheless, both of them used vein r2 as standard, the researcher used the p ie w differential vein r3 instead following Börstler et al., (2014), as the most up-to-date oa nl research on the discrimination of the two species But even so, the radial veins r and d r3 are almost equal length, therefore, the changes in both case is not significant The an lu nf va scatterplots of pairwise distribution of differential length of vein r3 vs r2/3 on the left lm ul and right wings of both species demonstrated the absent of any differences between z at nh oi female and male of both species in the vein r2/3 and r3 In both cases of male and female, the only difference between the groups was a slight shift on the vein length z r3 However, the shift on the wing morphology, which has already been discussed by gm @ Börstler et al., (2014), was the switch on the vein length r2/3 instead of r3 as this m co l present study found an Lu Because shape did not differ between the male and female, discriminant n va analysis of the two density treatments was used to test the reliability of the vein length ac th si r3 and r2 contrasts, or determine whether shape differed solely because of density According to Stephen & Juliano (2012), a higher successful classification were resulted as they categorized the specimens into the same breeding conditions, however, in my case study within both density, the two species were not better classified In fact, this result agreed with the findings of Service (1968) and Fedorova and Shaikevich (2007), when they also did not find a difference in the wing shape vein length of populations from Britain and Russia respectively However, the results from the study are inconsistent with other work on the effect of density rearing treatments lu on adult wing shape as Morales Vargas et al., (2010) found that altering larval an n va competition produced no significant changes in adult wing shape of Ae aegypti, in a gh tn to like manner Griffiths et al., (2005) reported that the wing shape in mosquitoes had been not affected by the latitude, or Stephen et al., (2012) found only change in semi- p ie w landmark position but not in overall shape, whereas the differences in my study was oa nl resulted from the altering of the reliable of the differential vein r2/3, and r3, as it used to d use for separate the two species nf va an lu The low classification success was not improved by the multivariate of lm ul landmarks positions using PCA, and female mosquitoes which was prefer to z at nh oi distinguish were the only use for this analysis The successful classification rate increased only slightly or even decreased compared with bivariate analysis z @ l gm In the same way, PCA analyses were in agreement with the bivariate analyses outcomes Both species spread on the same wide range of x and y-axis, in other words, co m both PC1 and PC2 did not capable to separate the two species well, even a stronger an Lu overlapping were seen in the expected groups of mosquito female right wings in low va n density or high density which were already sorted/separated analysis Success ac th si classification did not increase when the specimens were classified into different density treatments This visualizations of shape differences between the corresponding inter-competition groups in each species were not persistent with the findings from Börstler et al., (2014) They concedes the tendency of well classification for both Cx pipiens and Cx torrentium by using the evaluation of multi-, bi- and univariate wing morphometry In the univariate measure of only r2/3, they emphasizes that it can allow more than 90% of correct identification for mainly German specimens, and likewise in the bivariate analysis the correct classification can reach to more than 91% of all tested lu specimens Furthermore, PCA component of the right wing, as well as left wings an n va illustrated PC1 can be represented the part of shape variability that is most responsible gh tn to for species differences, and the classification accuracy of discriminant analysis exceeded 97%, whereas the researcher did not found these high rate of accurate p ie w classification of the two species in my experiment In case of Börstler et al., (2014), oa nl they conducted a varied and large population of Cx torrentium and Cx pipiens across d different Federal States of Germany, and also artificial breeding sites, but in the an lu nf va present study, artificial breeding site with a small area of Oldenburg university campus lm ul was the only conducted For that reason, it is claimed that in overall cases with varied z at nh oi specimens, the analysis of the vein length r2/3 and r3 still a reliable indicators for discriminating both species Alternatively, this experiment was only focused on a z small area with less specimens, therefore, the specimens can be more sensitive with co l gm @ the change of environmental factors m Although the findings were not correctly discriminating the species by using an Lu the vein length r2/3 and r3 as it used to be known in solving taxonomic problems, the n va author’s own view is the vein length r2/3 and r3 by themself could not be enough for ac th si discriminating the larval competition and density environment for Cx pipiens and Cx torrentium Lastly, it is concluded that the breeding condition on the larvae stages would influence the genetic or morphology of Cx pipiens and Cx torrentium, resulting the low successful classification of them by using only the vein length r2/3 and r3 5.2 Conclusion The results from the rearing larval mosquito experiment from the egg rafts of Cx pipiens and Cx torrentium was a complicated task Though there were enough lu an specimens for the study, the mortality rate of reared larvae was general relative high n va tn to (88.8%) due to the inter- and intra-environmental factors such as temperature, ie gh humidity, light intensity, feeding and competition in breeding conditions Because p some external factors were not controlled well in this study, the author suggested to oa nl w use Environmental chambers which can simulate a full range of environmental d conditions designed to test like temperatures, humidity, wind and light, etc From this lu nf va an study, it can be concluded these factors are very important for the development of mosquitoes, therefore, to better rear them in laboratory, these climatic conditions lm ul should be managed well Further, this study indicates that in today scenario of climate z at nh oi change, the climatic factors could affect to the mosquitoes population, and resulting problematic in taxonomic study of mosquitoes, as well as some possibly unexpected z l gm @ effects on the vector diseases control For this reason, the author argues that these artificial environmental co m characteristics in this study could make potential mosquito breeding places unsuitable an Lu resulting the change in both biology and morphology of the two species Cx pipiens va n and Cx torrentium In the course of this study, the discriminant analyses, the r2/3/r3 ac th si indices, the bivariate and even the multivariate analysis are all in agreement and yielding the low classification success of mosquitoes from the same or different breeding conditions Concluding from this low classification success, the wing morphometry of both male and females sibling mosquitoes Cx pipiens and Cx torrentium presented by the indices wing vein r2/3 and vein r2 were not stable when larval density change In broader words, this character modifiedunder different breeding conditions from the specimens of Oldenburg University’s campus, Germany This study bridges some gaps of the insufficient knowledge about the link of lu ecology resulted on the wing morphology of Cx pipiens and Cx torrentium The an n va results implies a possibility to predict species occurrence in other parts of the gh tn to breeding range, and to determine possible breeding places in further surveys Mosquitoes are commonly studied in the context of nuisance and the vector p ie w competence, but they are also an important part of the biological diversity The oa nl interactions between the mosquitoes and its breeding conditions with more d environmental factors, are not well studied For example, there are many more an lu nf va mosquito species across different genera that can act as vectors for other diseases lm ul (reviewed by Stephen & Juliano 2012), and a similar morphometric shape analysis on z at nh oi them might prove productive This research has to be done, before we should thought about mosquito-borne diseases interventions z Moreover, if larval rearing conditions of this species influence vector gm @ competence, then wing shape may be a useful indicator of vector competence in the l an Lu of a mosquito’s entire body, not just the wings m co superpictus Alternative approaches may involve morphometric analyses on the shape n va ac th si Reference 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NATO ASI Series G, Ecological Sciences No d Springer-Verlag, New York an lu nf va 29 Rudolf, M., C Czajka, J Börstler, C Melaun, H Jöst, H.v Thien, M Badusche, lm ul N Becker, J Schmidt-Chanasit, A Krüger, E Tannich, and S Becker 2013 First z at nh oi nationwide surveillance of Culex pipiens complex and Culex torrentium mosquitoes demonstrated the presence of Culex pipiens biotype pipiens/molestus z hybrids in Germany.PLoS ONE 8(9): e71832 gm @ 30 Russell, T L., Lwetoijera, D W., Knols, B G., Takken, W., Killeen, G F., & l m co Ferguson, H M (2011) Linking individual phenotype to density-dependent an Lu population growth: the influence of body size on the population dynamics of n va ac th si malaria vectors Proceedings of the Royal Society B: Biological Sciences, rspb20110153 31 Sanford MR, Demirci B, Marsden CD, Lee Y, Cornel AJ, et al., (2011) Morphological Differentiation May Mediate Mate-Choice between Incipient Species ofAnopheles gambiae s.s PLoS ONE 6(11): e27920 doi:10.1371/journal.pone.0027920 32 Savage, H M., &Kothera, L (2012) The Culex pipiens complex in the Mississippi River basin: identification, distribution, and bloodmeal hosts Journal of the lu American Mosquito Control Association, 28(4s), 93-99 an n va 33 Service MW, 1968 The taxonomy and biology of two sympatric sibling species of gh tn to Culex, C pipiens and C torrentium (Diptera, Culicidae) J ZoolSocLond 156: 313–323 p ie w 34 Shaikevich, E V (2007) PCR-RFLP of the COI gene reliably differentiates Cx f molestus and Cx torrentium of the Pipiens oa nl pipiens, Cx pipiens d Complex.EurMosq Bull, 23, 25-30 an lu nf va 35 Smith, J L., & Fonseca, D M (2004) Rapid assays for identification of members lm ul of the Culex (Culex) pipiens complex, their hybrids, and other sibling species 70(4), 339-345 z at nh oi (Diptera: Culicidae) The American journal of tropical medicine and hygiene, z 36 Stephens, C R.and Juliano, Steven A., (2012) Wing Shape as an Indicator of Larval gm @ Rearing Conditions for Aedesalbopictus and Aedesaegypti (Diptera: Culicidae) l an Lu illinoisstate.edu/fpbiosci/18 m co Faculty Publications – Biological Sciences Paper 18 Available at: http://ir.library n va ac th si 37 Stephens, C R., & Juliano, S A (2012) Wing Shape as an Indicator of Larval Rearing Conditions for Aedes albopictus and Ae aegypti (Diptera: Culicidae).Journal of medical entomology, 49(4), 927 38 The Eliminate Dengue Program Country project: Vietnam [Online].Available at: http://www.eliminatedengue.com/vietnam/faqs [Accessed: 06 September 2014] 39 Vinagradova EB, 2000 Culex pipiens pipiens Mosquitoes: Taxonomy, Distribution, Ecology, Physiology, Genetics, Applied Importance and Control Moscow, Russia: Pensoft lu 40 Vinogradova, E.B (1997) Mosquitoes of the Culex pipiens Species Complex in an n va Russia Proceedings of the Zoological Institute of the Russian Academy of gh tn to Sciences, 271, 307 pp 41 Weitzel, T., Collado, A., Jöst, A., Pietsch, K., Storch, V., & Becker, N (2009) p ie w Genetic differentiation of populations within the Culex pipiens complex and oa nl phylogeny of related species Journal of the American Mosquito Control d Association, 25(1), 6-17 an lu nf va 42 Werblow A, Klimpel S, Bolius S, Dorresteijn AWC, Sauer J, et al., (2014) lm ul Population Structure and Distribution Patterns of the Sibling Mosquito z at nh oi Species Culex pipiens and Culex torrentium (Diptera: Culicidae) Reveal Different Evolutionary Paths PLoS ONE 9(7): e102158.doi:10.1371/journal.pone.0102158 z m co l gm @ an Lu n va ac th si APPENDIX lu an n va p ie gh tn to d oa nl w nf va an lu z at nh oi lm ul z m co l gm @ an Lu n va ac th si