Coetzee, Maureen

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A TAXONOMIC AND GENETICAL STUDY OF THE ANOPHELES COUSTAKI GROUP OF MOSQUITOES IN SOUTHERN AFRICAby Maureen CoetseeA Dissertation Submitted to the Faculty of Science, University of the Witwatersrand , Johannesburg, for the Degree of Master of ScienceJohannesburg, March 198 2ABSTRACTThe Anopheles coustani (Diptera:Culicidae) group of mosquitoes in southern Africa originally comprised a single species taxon A .coustani with tenebrosus and 2 lemannI as "varieties" thereof. Thirteen years ago (1968) the varieties were given full specific rank on morphological evidence. While the adults were easily distinguished, previous workers had not been able to find any significant differences betwaen the immature stages.In 1972 it was suggested, without evidence, that it was possible that the three forms are in fact morphs of one polymorphic population. Using both genetical and morphological techniques, the taxonomic and evolutionary status of these similar forms was reinvestigated in this present study.Examination of the polytene chromosomes of the fourth instar larvae provided evidence for a total of five species, and preliminary starch-gel electrophoresis studies confirmed these findings. The morphology of the adults and immatures was investigated and good discriminating characters were found in the pupae of all five species.These studies have shown that the hypothesis that a single polymorphic species is being dealt with is incorrect, and also revealed further sibling species in two of the taxa.As the members of the A.coustani group are highly anthropcphilic, these results are potentially pertjnant to studies of res. lal malaria transmission in Africa. They also demonstrate the utility of new approaches for the adequate study of a difficult complex of species in A nopheles.ivDECLARATION BY CANDIDATEI declare that this dissertation is my own work and that it has not been submitted before for any degree or examination in any other Universitv. Throuqhout this study I have been an employee of the Department of HeaThis work is dedicated to my husband Dolf who has been so patient and long-suffering./PREFACEThis study was stimulated by the need to study the morphology of the immature stages of the three species of the sub-genus Anopheles found in this country. It was only later that the genetics of the group was also considered. Current genetical studies of anophelines are proving to be almost as useful and enlightening in the study of evolutionary concepts as those carried out on memDers of tne genus Drosophila. This study contributes in a small way to our sum of knowledge of this important g e nus.I am deeply indebted to Dr. Botha de Meillon, who first awakened an interest in me for mosquitoes, and who has subsequently kept the flame alive, encouraging and supporting me whenever I looked like flagging. But for him, this dissertation would never have been writte n.I am most grateful to Prof. Hugh Paterson who has made all things possible. My tjme spent under his supervision was most imformative and very enjoyable. It was a privelege to come under his influence and so be exposed to all the newest ideas in this field of applied entomology.Many thanks are due to Mr. Chris Green and Mr. Rich Hunt of the Department of Medical Entomology, South A fricanviiInstitute for Medical Research, Johannesburg, who put up with me for one year and helped to further my education.I appreciate their generosity in che free use of thei ■ equipment, the material they supplied me with, and for discussions regarding the ideas and techniques employed in this study. Also from the above Institute, I thank Dr. J. Ledger, Head of the Entomology department, for his interest and for granting permission to use the facilities, Mrs. J. Segerman for her help with pinned specimens and Mr. G. Newlands, Ms. C. Martindale, Mr. I. Davidson and Mr. S. Henning for their interest in my work.For the collection of wild material I have been almost totally dependant on the co-operation of other people.In this respect I thank Dr. C. Hansford, Director of the National Institute for Tropical Diseases, Tzaneen, and the following members of his staff, m  alphabetical order:Mr. H. Cross, Mr. J. Cross, Ms. D. Durand, Mr. C. du Toit,Mr. D. Eckhardt, Mr. E. Jansen, Mr, K. Newbetry, Mr. A. Quan, Mr. W. Sekgobela, Mr J. Shipalane, Mr. D. Theron and Mr.G. van Eeden. Also Dr. P. Taylor of Blair Research Institute, Salisbury, Zimbabwe and Mr. J. le Grange of the Department of Health, Namibia.For helpful discussions I am indebted to Mr. C. Green,Mr. R. Hunt, Mr. K. Newberry and Ms. D. Durand. I thank Dr. D. Lambert for ideas on the presentation of this work.Last bu: not least, I thank my motner for all the support she has given me.TABLE OF CONTENTSTitle cageAbstract i iDeclaration by Candidate IVDedication vPreface VICHAPTER ONE GENERAL INTRODUCTION1.1 Identification....................................1.2 Sibling species..................................1 . 3 Objectives.........................................1.4 A brief history of the Anophe _s (Anopheles)coustani group of mosquitoes................CHAPTER TWOTHE CHROMOSOMAL ANALYSIS2.1 Introduction......................................2.2 Methods2.2.1 Mosquito collecting and rearingtechniques2.2.2 Salivary gland polytene chromosomestechnique....................................2.3 Cytological characteristics2.3.1 Chromosomal species.......................2.3.2 Floating inversion polymorphism-!.......2.4 Cross-mating experiments between the coustani sibling species....................................2.5 Discussion..........................................CHAPTER THREETHE ELECTROPHORETIC ANALYSISix3.1 Introduction........................................... ... 293.2 Methods................................................. ... 303.3 Results................................................. ... 313.4 Discussion................................................. 34CHAPTER FOURTHE MORPHOLOGICAL ANALYSIS4.1 Introduction........................................... ... 384.2 Adult identification4.2.1 Methods............................................. 394.2.2 Results............................................. 424.3 Pupal identification4.3.1 Methods............................................. 424.3.2 Results............................................. 43pgge 111522242.2.2 Salivary gland polytene chromosomes pagetechnique.......................................  1 12.3 Cytological characteristics2.3.1 Chromosomal species...........................  142.3.2 Floating inversion polymorphisms...........  152.4 Cross-mating experiments between the coustani sibling species.......... ...........................  222.5 Discussion.............................................. 24CHAPTER THREETHE ELECTROPHORETIC ANALYSIS3.1 Introduction...........................................  293.2 Methods.................................................  303.3 Results.................................................  313.4 Discussion.............................................. 34CHAPTER FOURTHE MORPHOLOGICAL ANALYSIS4.1 Introduction...........................................  384.2 Adult identification4.2.1 Methods..........................................  394.2.2 Results.......................................... 424.3 Pupal identification4.3.1 Methods...................................... . 424.3.2 Results.......................................... 43ix4.4.1 Methcds......................................4.4.2 Results..........................................•5.5 identif icaticn4.5.1 Introductior ....................................4.5.2 Scanning electron microscope technique....4.5.3 Results ...........................................4.6 Discussion..............................................CHAPTER FIVE CONCLUDING DISCUSSION5.1 Introduction...........................................3.2 A recent example of th I Ls ct pure morphological taxonomy...............................5.3 Species-specific characters.........................5.4 On identification.....................................APPENDIX ICOLLECTION RECORDS OF MATERIAL U S E D ....................APPENDIX IIDETAILED RECIPES FOR THE STAINS AND BUFFERS USED IN THE ELECTROPHORETIC ANALYSIS.........................APPENDIX IIIA SUMMARY OF THE STUDIES DONE ON THE IMMATURES OF A .COUSTAMI, A.ZIEHANNI AND A .TENEBROSUS, USING COMPUTER TECHNIQUES................................REFERENCESLIST OF FIGURES pageFig. 1 : The hind legs o£ the three speciesA .coustani , A .ziemanni and A .tenebrosus showing the differing amounts of pale areas..........................................Fi'. 2 : The chromosomal map of A.coustani Ashowing the fixed inversion differences with species B on the X arm and various floating inversions of both species on the autosomal arm;’.......................... 16'ig. 3 : The chi >somal map of A . 2 iemanni A shewing the inversion differences found in soecies B ..........................Pig. 4 : The :hr n ...nal map of A. ter.e::r ssus.......  18Fig. 5 : Fixed inversion differences on the Xchromosome of: four species belongingto the A.coustani g r o u p .................... 19Fig. 6 : The double heterozvgote loops on arm 3Rof A.coustani B .............................  23Fig. 7 Hybrid chromosomes of A.coustani B x A . ... 25'XI 1LIST OF FIGURES cageFig. 1 : The hind legs of the three speciesA .coustani, A . ziemanni and A .tenebrosus showing the differing amounts of pale areas.......................................... 11Fig. 2 : The chromosomal map of A.coustani Ashowing the fixed inversion differences with species B on the X arm and various floating inversions of both species on the autosomal arms.......................... 16Fig. 3 : The chromosomal map of A.ziemanni A showing the inversion differences found in species B ............. ............ 1"Fig* 4 : The chromosomal map of A .tenebrosus.......  18Fig. 5 : fixed inversion differences cn the Xchromosome of eour species belongingto the A .coustan.i g r o u p...................  19Fig. 6 : The double heterc<ygote loops on arm 3Rof A.coustani B .............................  23Fig. 7 : Hybrid chromosomes of A.coustani B x A.... 25IFig . 8X’ 1 1a) Spermatozoa emerging from thesquashed testes of a normal fertile A.coustani A male, b) The testes of aF 1 hybrid male A.coustani B x A with no evidence of spermatozoa being present, only early spermatids......................Fig. 9 : Esterase allozymes of the five speciesstudied.......................................Fig. 10 : Esterase allozymes of the two species of coustani with F1 hybrids in the middle,263233Fig. 11 : Examples of some ether enzyme systemstested......................................... 37Fig. 12 : The 15 spots measured on the wings, palps and legs of adults of A.coustani A and B ...............................................Fig. 13 : The wings of A .z iemanni A and B showing the difference in size of the apical pale spot................. ...................  41Fig. 14 : Pupal characters of the sibling species... 44Fig. 15 : The pupa of A . tenebrosus..................... 45xivPa2 eFig. 16 : The pupa of A . coustan i A ..................... ...46Fig. 17 : The pupa of A . 7. iemanni A ...... .............. ...47Fig. 18 : Larvai characters of the sibling species.. 50Fig. 19 : The larva of A .tenebrosus....................Fig. 20 : The larva of A .coustani A .......................52Fig. 21 : The larva of A . 2 iemanni A .......................53Fig. 22 : The egg of A .coustani A ...................... ...56Fig. 23 : The egg of A.coustani B ...................... ...56Fig. 24 : The egg of A . z iemanni A ...................... ...57Fig. 25 : The egg of A z iemanni B. This specimenhas collapsed slightly at the one end... 57Fig. 26 : The egg of A . tenebrosus...................... ...58Fig. 27 : a) The rosettes of A .tenebrosus. b) Therosettes of A.coustani B .................. ... 59Fig. 28 : Scatterplot of the computer analysis ofthe pupae........................................ 78XVFig. 29 : Scatterplot of the computer analysis of the larvae................................£a£e79XVFig. 29 : Scatterplot of the computer analysis of the larvae.................................joa^e79:XVILIST OF TABLESTable 1 : The numbers of 2La and 3Ra chromosome sequences found in the A .ziemanni B population.................................... 20Table 2 : The numbers of 2Ra and 3Rc chromosome sequences found in the A.coustani A populations..................................  22Table 3 : The number of specimens per species testedfor each enzyme sys e m .....................  31Table 4 : Frequency data for four enzyme systemsstudied.......................................  35Table 5 : Combined key for identification of families of the five species tenebrosus, coustani A coustani B, z iemanni A and ziemanni B... 61Table 6 : Keys to the larvae and pupae of Al'coustani A .tenebrosus and A . "ziemanni", whereby 91and 89% correct identification respectively was obtained.................................xv iLIST OF TABLES pageTable 1 : The numbers of 2La and 3Ra chromosome sequences found in the A .z iemannj B population.................................... 20Table 2 : The numbers of 2Ra and 3Rc chromosome sequences found in t!r e A.coustani A populations..................................  22Table 3 : The number of specimens per species testedfor each enzyme system..................... 31Table 4 : Frequency data for four enzyme systemsstudied.......................................  35Table 5 : Combined key for identification of families ot the five species tenebrosus, coustani A coustani 3, ziemanni A and ziemanni B ... 61Table 6 : Keys to the larvae and pupae of A'.'coustani",A .tenebrosus and A ."ziemanni", whereby 91and 39% correct identification respectivelywas obtained.................................CHAPTER ONEGENERAL INTRODUCTION 1 . 1 Identi f icationThe need for the accurate identification of mosquitoes was appreciated at an ear1;/ stage when observations showed that in any particular area malaria was transmitted by only one or two species of anophelines (Watson, 1921). This has led to a vast amount of literature being published on mosquito identification. In 193?, Edwards published a catalogue of mosquitoes of the world in which he recognised 140C species. The most recent catalogue published (Knight & Stone, 1977) recognises 2960 species, more than double that of Edwards'. While there were morphological differences, classical taxonomists could describe new species. Problems arose however, when taxonomic species were found to comprise of cryptic genetical species. These are commonly referred to as sibling species.Paterson (1975) states "... that medical entomological studies must be based on sound identification of genetical specie? *my emphasis) and gives an example of the resultant chaos if this is not adhered to, by quoting the1CHAPTER ONE GENERAL INTRODUCTIONI.1 Identi f icationThe need for the accurate identification of mosquitoes was appreciated at an early stage when observations showed that in any particular area malaria was transmitted by only one or two species of anophelines (Watson, 1921). This has led to a vast amount of literature being published on mosquito identification. In 1932, Ec'wards published a catalogue of mosquitoes of *-.he world in which he recogrised 1400 species. The most recent catalogue published (Knight & Stone, 1977) recognises 2960 species, more than double that of Edwards'. While there were morphological differences, classical taxc.iomists could describe new species. Problems arose however, when taxonomic species were found to comprise of cryptic genetical species. These are commonly referred to as sibling species.Paterson (1975) states "... that medical entomological studies must be based on sound identification of genetical species." (my emphasis) and gives an example of the resultant chaos if this is not adhered to, by quoting theAnopheles gambiae complex in relation to malaria control. Firstly, from the time of Ross' visit to West Africa until the discovery of the complex (Paterson, 1962, Davidson, 1962), malaria entomologists in Africa studied these important insects as if they were all members of a single species. As a consequence, it became necessary to collect once more all the biological data, but this time for each individual species. Secondly, the refusal by some authors to recognise the complex as late as 1974 served merely to confuse the picture. As the different members of the gambiae complex have different behavioural and physiological characters and consequently different vector capacities, it is obviously most important to identify correctly which member of the complex one is dealing with in malaria control programmes.Before starting on a study of species, it is necessary to state which concept of species one is adopting. In this case, becau e s-eciation is a genetical event, "species" is defined in genetical terms (as opposed to the taxonomic species concept which is concerned only with classification). According to the Recognition Concept (Paterson, 1978, 1980), species are populations of individual organisms which share a common specific- mate recognition system (SMRS) and habitat preference.The SMRS comprises a co-adapted signal-respcr.se reaction chain whose function is to ensure fertilization. The SMRS may take the form of visual, auditory, chemical orAuthor  Coetzee M  Name of thesis A Taxonomic and Genetical study of the Anopheles Coustani group of mosquitoes in Southern Africa  1982   PUBLISHER: University of the Witwatersrand, Johannesburg ©2013  LEGAL NOTICES:  Copyright Notice: All materials on the Un i ve r s i t y  o f  the  Wi twa te r s rand ,  Johannesbu rg  L ib ra ry  website are protected by South African copyright law and may not be distributed, transmitted, displayed, or otherwise published in any format, without the prior written permission of the copyright owner.  Disclaimer and Terms of Use: Provided that you maintain all copyright and other notices contained therein, you may download material (one machine readable copy and one print copy per page) for your personal and/or educational non-commercial use only.  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A taxonomic and genetical study of the Anopheles coustani group of mosquitoes in Southern Africa.

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