THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY NGUYEN THANH TAN Thesis title WING MORPHOMETRY OF THE FEMALES OF CULEX PIPIENS AND CULEX TORRENTIUM UNDER DIFFERENT BREEDING CONDITIONS BACHELOR THESIS Study Mode: Full- time Major: Bachelor in Environmental Science and Management Faculty: International Training and Development Center Batch: K42- Advance Education Program Thai Nguyen, January 21, 2015 n DOCUMENTATION PAGE WITH ABSTRACT Thai Nguyen University of Agriculture and Forestry Degree Bachelor of Environmental Science and Management Program Student Nguyen Thanh Tan Name Student DTN1053110170 ID Thesis WING MORPHOMETRY OF THE FEMALES OF Title CULEX PIPIENS AND CULEX TORRENTIUM UNDER DIFFERENT DENSIY CONDITIONS Superviso Dr. Rolf Niedringhaus (Oldenburg University, rs Germany) Associate. Do Thi Lan Abstract: Cx. pipiens and Cx.
torrentium are two sibling mosquito species with a close resemblance in morphology and ecology. The morphological separation of these mosquitoes can only use for male, however, this technique is extremely difficult and unsure. Besides that, the discrimination of the female of the 2 species was a hard burden. Recently, Börstler et al., (2014) used a tool called wing morphometry n 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. To answer these questions, rearing mosquitoes, morphometric data collection and bivariate & multivariate analysis were used. The results of all methods are all in agreement and yielding the low classification success of mosquitoes under different density conditions.
Concluding from this low classification success, the wing morphometry of mosquitoes Cx. pipiens and Cx. torrentium were not stable or modified under different density conditions. The results implies a possibility to predict species occurrence in other parts of the breeding range, and to determine possible breeding places in further surveys.
Keyword Geometric morphology, Culex Complexes, Cx. torrentium, vein, landmarks, taxonomy, s breeding conditions Number of page Date of January 15, 2015 Submission n ACKNOWLEDGEMENT First and foremost, I wish to express my sincere thanks to Prof. 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. 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 cannot be done. Furthermore, I want to deeply thank IAESTE organization, the administration department of Oldenburg University, and the boards of Thai Nguyen University of Agriculture and Forestry, for giving me this valuable and unforgettable chance studying and working in Germany, and more important providing me all necessary facilities, skills and knowledge to complete this bachelor thesis. 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 und Naturwissenschaften.
Thanks a lot for your expert, valuable guidance and experiences during my working time there. Finally yet importantly, I take this opportunity to record my sense of gratitude to my families and friends who encourage and backing me unceasingly. Thank you very much! n TABLE OF CONTENTS Page n LIST OF TABLES Page n LIST OF FIGURES Figure 1. Distribution of the Cx.
pipiens complex and its sibling species (the map of Smith et al. Hypopygium of Cx. torrentium(Becker et al. Aedeagus and paraproct of: (a) Cx.
torrentium;(Becker et al. Location of the study area for mosquito collections on the main campus –Haarentor of Oldenburg University, Germany (Google Map, 2014). A black bucket exposed near a water body containing dry hay infusion. Two black buckets exposed near a pond containing dry hay infusion….
All egg rafts were kept in rearing glass filled with the eutrophic hay infusion. For each egg raft, 110 larvae were reared for 10 replicates of 2 experimental combinations……………. All larval rearing racks are housed inside the rearing rooms, glass tube were plugged with cotton since the fifth day………………………………………………. Emerged adults were pushed on a black net…………………….
Using a tiny broom to scrape off the wing scales………. Euparal 3C 239 (Waldeck GmbH & Co. KG, Muenster) Figure 13.The positions of the 13 morphometric landmarks on the Culex complexes a) right wings b) left wings. The development stages of Cx.
pipiens and Cx. torrentium a) Egg raft and b) fourth instar larvae. The photos were taken under the Leica MZ 7.5 Stereomicroscope and seen by Adobe Photoshop software version 7. Principal component analysis of the shape variance of the mosquito female left wings in a) low density (left) b) high density (right).
Grey point indicates Cx. pipiens and Black dots indicate Cx. 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 Black dots indicate Cx. 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, which cause serious diseases such as malaria, yellow fever, dengue, as well as, filariasis, Japanese encephalitis and a hundred others maladies by virtue of their blood- sucking habits (Kettle 1995; Beaty and Marquardt 1996; Lehane 1991; Eldridge and Edman 2000, cited by Becker 2010). According to the U.
Centers for Disease Control and Prevention, Ohio State University and Texas A&M University (March, 2007), mosquitoes and the diseases 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 mosquito-borne diseases. It was reported by the American Mosquito Control 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 for roughly three quarters of all mosquito species, mosquito-borne diseases occur not 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 currently explosive worldwide, and have also influenced the sustainable development of the world, not only in socio-economy but also in politics. The consequences of n 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 good knowledge on mosquito’s systematics, morphology, and biology, which are still stayed limited in Viet Nam. Some developed countries, in particular Germany, have experienced the advancement in mosquito research, and control work. Over 3500 mosquito species have been reported, as well as a rich background and varied information exist which were well documented.
However, the ambition of having further comprehensive understanding of mosquitoes remain endless, particular in regard to biological and physiological distinctions, upon which the realities of subspecies depend on many variants causing remaining unsolved problems. As not much threat as Anopheles and Aedes mosquitoes, the Culex (Cx.) 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 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 domestic and dark place, and rarely far away from human shelters, and living.
They use to be active only for a few weeks during the summer seasons when the females search for the warm surface water to lay their eggs. Found in many temperate regions n throughout urban and suburban area, Culex pipiens L. is one of the most common and widespread mosquito species (Weitzel et al. 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. 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 a third of that for Cx. pipiens, from her specimens collection in some southern localities in England. Moreover, observations made by Service (1968), larvae of both 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 Dahl C.
(1988) showed that neither the egg rafts nor larvae of both species can be distinguished. It is, therefore, causing the confusion in many situations such as distribution, vector status, and particular in diseases control program. pipiens is the main vector, or carrier, of St. Louis Encephalitis, West Nile Virus, Western Equine Encephalitis, Heartworm in dogs, and bird Malaria (Parreira et al., 2007; Papa et al., 2013; Radrova et al.
torrentium is a highly conveyor to Sindbis virus in Sweden, Norway, and Russia and turned out to be the main enzootic vector for Sindbis in Sweden (Lundström 1994; Lundström et al., 1990, cited by Börstler et al. Although their main victim has been bird, they are considered to be a “bridge” vector as it was found to be able to transmit viruses from bird to humans and other mammals. n Taxonomic studies on these two species has been developed by morphologic methods since 1900s, however, it is often evaluated to be difficult, time-consuming, and often limited with certainty by characters of the male genitalia. In 1988, Dahl et al., argued that almost all morphological characters of males are variable and overlapping.
This, together with the fact that in epidemiological studies, understanding the discrimination of adult females is very important as it is always known to be human blood- sucking and transmitted sickness & diseases. The gaps in our knowledge about the quantitative, spatial and temporal distribution of both species stay remained, and have been confused in many former studies, and not separated in many publications, for example, Schäfer et al. pipiens/torrentium, even neglected the existence of Cx. torrentium in some other publications (e.
Rydzdanicz & Lonc, 2003 cited by Börstler et al. According to literature made by Börstler et al., 2014, wing morphology was first mentioned in Mahrig (1969) publication based on the wing vein r2/3 and vein r2, of Cx. pipiens females, but no longer be rejected the reliability of this character by Service (1968) for specimens from Great Britain, as did Fedorova and Shaikevich (2007) for Muscovite specimens. Only recently, an advanced method was developed, as known as a multiplex real-time PCR protocol to distinguish various species and biotypes of Culex from both single and pooled specimens from mainly Germany.
Börstler et al., (2014) recognized this method on his discrimination of Cx. pipiens and Cx. torentium, is fast clarify, cheaper, and more convenient without using a molecular laboratory, or genetic test. From his findings, he pointed that the length of wing radial vein r2/3 as a morphometric n character of mosquito is reliable for the wing differences between the two sibling species of Culex.