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Filmed as University Microfilms International 300 North Zeeb Road Ann Arbor. Michigan 48106 USA St John's Road. Tyler's Green High Wycombe. Bucks. England HP10 8HR I \ \ 77-25,256 LEWANDOWSKI, Henry Bernard, Jr., 1948DETERMINATION OF THE IMPORTANT NATURAL POTENTIAL VECTORS OF DOG HEARTWORM IN MICHIGAN. Michigan State University, Ph.D., 1977 Entomology Xerox University M icrofilm s r Ann Arbor, M ichigan 48106 DETERMINATION OF THE IMPORTANT NATURAL POTENTIAL VECTORS OF DOG HEARTWORM IN MICHIGAN by Henry B. Lewandowskl, Jr. A DISSERTATION Submitted to Michigan State University In partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Entomology 1977 ABSTRACT DETERMINATION OF THE IMPORTANT NATURAL POTENTIAL VECTORS OF DOG HEARTWORM IN MICHIGAN by Henry B. Lewandowskl, Jr. In Michigan, the dog heartworm, Dirofilaria immitls (Leidy), Is now recognized as a serious pest. with increased frequency. Cases of this disease are being reported Because of a lack of field studies, little was known about the natural maintenance of this parasite in Michigan. The objectives of this study were to: 1) determine which species of mosquitoes in Michigan are attracted to dogs and are present in sufficient numbers to make them suspect as potential vectors of t). immitis; 2) determine which species may carry the parasite under natural conditions by examining field-captured mosquitoes for the presence of infective larvae; 3) determine if I), immitis develops to the infective stage in species of mosquitoes found to be the best potential natural vectors; and 4) transmit I), immitis from dog to dog to prove that Michigan strains of suspect mosquito species are capable of transmitting dog heartworm. In 1974 and 1975 mosquitoes were collected in dog-baited and CDC miniature light traps. They were identified to species and over 43,000 were crushed in groups of 25 so that infective I), immitis could be isolated from field-captured specimens. Infective larvae of D . immitis and D . tenuis were stained for acid phosphatase activity to determine if this histochemical stain could be used to identify larvae obtained from field captured mosquitoes. Aedes stlmulans, A. vexans, Anopheles Henry B. Lewandowski, Jr. quadrimaculatus. Mansonla perturbans, and Culex piplena were selected to study the development of _D. Immitis larvae In Michigan strains of these mosquito species. Transmission of dog heartworm to non-infected dogs was attempted with Aedes stimulans, A. vexans and Anopheles quadrimaculatus. Field studies showed that Aedes cinereus. A. fitchli, A. stimulans A . trlseriatus, A. trivittatus, Anopheles quadrimaculatus. A. walker1, Culex pipiens and Mansonla perturbans were attracted to dogs and collected most frequently. These species appeared to be the best potential vectors of dog heartworm in Michigan. Laboratory studies showed that Anopheles quadrimaculatua to be a very efficient host of D. immitis larvae. A. vexana is also a suitable host while larvae complete development in Culex pipiens but this species is a very poor host. Aedes stimulans and Mansonla perturbans are unacceptable hosts of •D. immitis larvae. The hlstochemical stain proved to have no value for the purpose of identifying infective larvae Isolated from field-captured mosquitoes. Results of this study indicate that Anopheles quadrimaculatus, A. walkeri and Aedes vexans are likely to be the most Important mosquitoes Involved in the natural maintenance of I), immitis in Michigan To Connie 11 ACKNOWLEDGEMENTS I am deeply Indebted to my advisor. Dr. Gary R. Hooper for his advice and support throughout this research. His kindness and sense of humor Is something for which he will always have my respect. always available to offer his help. He was Dr. H. D. Newson deserves special commendation for allowing me to utilize the facilities In his laboratory. He was always available for advice and treated me as his own student. A sincere thinks Is also extended to the other members of my Graduate Committee: Drs. George W. Bird, James E. Bath and Jeffrey F. Williams. Each of these committee members have made important contributions to this work and their enthusiasm for the project, willingness to help and friendship made my graduate studies at Michigan State University a most enjoyable experience. Many other members of the university staff sacrificed much of their time to help me: Drs. Robert Meyers and Susan Stein, Clinical Veterinarians for the Laboratory Animal Care Service aided In the care and handling of the animals used In these experiments; Ms. Jean Beemsterboer, Laboratory Research Aid, drew many blood samples from Scotch and other dogs who refused to donate their blood willingly; Mary Eddy, Laboratory Animal Care Service, turned my docile experimental dogs Into mlschevlous pets; and Mr. Fred Howe, Assistant Director, Laboratory Animal Care Service, provided much help with regards to obtaining and maintaining the animals used In this study. Ill iv A special thanks goes to Messrs. Jose B. Velarde who helped obtain materials and construct some of the equipment used in this project; George Dennis who compiled the Information on the distribution of Michigan mosquitoes; Patrick H. Hafner, David F. Laroux and James W. Crossman who conducted dog heartworm surveys; and Bradley Risk and David Harris who helped in the handling of mosquitoes brought to the laboratory. I am grateful to Dr. Wayne Crans, Rutgers University* for his advice given at the beginning of this project and Dr. Ming M. Wong, California Primate Center, for providing specimens of Dirofilarls tenuis. My friends Mori Zalm, Heidi Kaska and Dr. Dennis McGroarty contributed to hours of interesting scientific discussion. I also want to thank my parents, Mr. and Mrs. Henry Lewandowski for their constant encouragement. Most of all, my wife Connie deserves a tremendous amount of credit for making many sacrifices which helped me obtain my Ph. D. TABLE OF CONTENTS P*ge LIST OF T A B L E S.................................................. vii LIST OF F I G U R E S ............................................... vlii LIST OF APPENDICES............................................ x INTRODUCTION .................................................. 1 OBJECTIVES .................................................... 3 LITERATURE REVIEW Classification and Evolution . . . . . . . ............... Vector Determination .................................... The Dog as a H o s t ........................................ Development In the Mosquito............................... Fate of the Infective L a r v a e ............................. Development In the D o g .................................. Dally and Seasonal Periodicity........................... Alternate Vertebrate Hoots ............................... Geographic Distribution .................................. Importance in M i c h i g a n .................................. 4 5 5 7 8 9 10 11 12 15 MATERIALS AND METHODS Site S e l e c t i o n .......................................... Description of the S i t e s ................................ Collection Methods ...................................... Identification and Examination of Mosquitoes for the Presence of Infective L a r v a e ......... Problems In Mosquito Identification....................... Differentiation of D. imdtls l a r v a e ..................... Selection of Mosquitoes for Lsboratory Studies . . . . . . . Developmental Trials .................................... Obtaining the Infective Blood Meal ....................... Determination of Microfilaremia or Dilution of Infected B l o o d .............................................. Transmission Trials ...................................... 30 30 RESULTS AND DISCUSSION Field Collections........................................ Unlvoltlne m o s q u i t o e s ............................... 34 36 v 17 20 21 24 28 28 29 29 29 TABLE OF CONTENTS, CONT’D Page Multivoltine mosquitoes breeding in temporary or fluctuating water situations ................... Multivoltine mosquitoes breeding In permanent water s i t u a t i on s .......................... Examination of Mosquitoes for the Presence of Infective Larvae ..................................... Differentiation of D . Immitis Larvae ..................... Developmental Trials .................................... Transmission Trials ...................................... 40 60 68 73 74 81 GENERAL DISCUSSION A e d e s .............................................. 84 A n o p h e l e s .......................................... 95 C u l e x .............................................. 98 Culiseta.............................................. 102 Mansonla . ......... 103 Psorophora......... 103 SUMMARY AND CONCLU S I O N S ......................................... 104 APPENDICES...................................................... 107 LIST OF REFERENCES...............................................146 vi LIST OF TABLES Tables Pages 1. 1974 and 1975 dog-baited collection totals.................. 35 2. 1975 CDC miniature light trap collection t o t a l s ............ 37 3. Ilosquitoes pooled in 1974 from CDC miniature light trap and dog-baited trap collections ........................... 69 4. Mosquitoes pooled in 1975 frosi CDC miniature light trap and dog-baited trap collections ........................... 70 5. Nematodes, possibly Dirofliaris immitis, extracted from mosquitoes................................................ 71 6. Summary of Developmental T r i a l s ........................... 75 7. Importance of various mosquitoes as vectors of dog heartworm In the Lansing, Michigan a r e a ..................... 106 vii LIST OF FIGURES Figures 1. Pages Distribution of dog heartworm in the continental United States.................................................... 14 Seven sites In the Lansing, Michigan area where adult and larval mosquitoes were collected In 1974, 1975, ar1 1976 . . . 19 3. CDC miniature light t r a p .................................. 24 4. Dog-baited t r a p .......................................... 26 5. Apparatus used to restrain dogs during mosquito feedings . . . 32 6. Seasonal Incidence of Aedes fitchii-stimulans at several locations In the Lansing, Michigan area during June through early October, 1975 38 Important mosquito species collected in a dog-belted trap located in an open situation at Site 1 during June through August, 1974 .............................................. 41 Important mosquito species collected In a dog-baited trap located In a woodland situation at Site 1 during June through August,. 1974 ...................... 43 Important mosquito species collected In a dog-baited trap at Site 5 during June through early October, 1975 45 Seasonal incidence of Mansonla perturbans In the Lansing, Michigan area during June through early October, 1975 . . . . 47 Seasonal Incidence of Aedes cinereus and some members of the Aedes communis complex (See Barr, 1 9 5 8 ) ............... 50 Seasonal Incidence of Aedes stictlcus at Site 5 In the Lansing, Michigan area during June through early October, 1975 ........... ...................... 52 Seasonal Incidence of Aedes trlserlatus at Site 4 In the Lansing, Michigan area during June through early October, 1975 ............................................ 54 2. 7. 8. 9. 10. 11. 12. 13. viii LIST OF FIGURES, CONT’D. Figures 34. 15. 16. 17. 18. Pages Seasonal incidence of Aedes trivittatus at 2 locations in the Lansing, Michigan area during June through early October, 1975 ............................................ 56 Seasonal incidence of Aedes vexans in the Lansing, Michigan area during June through early October, 1975 . . . . 58 Seasonal incidence of Anopheles quadrlnaculatus in the Lansing, Michigan area during June through early October, 1975 . . . . . ................................... 61 Seasonal incidence of Anopheles walkeri in the Lansing, Michigan area during June through early October, 1975 . . . . 63 Seasonal incidence of Culex pipiens in the Lansing, Michigan area during June through early October, 1975 . . . . 65 19-24. Distribution of Aedes atropalpus. A. canadensis, A. cinereus, A. fitchli. A. excrucians. and A. punctor. respectively, in Michigan........................... 86 25-30. Distribution of Aedes solllcitans. A. sticticus, A. stinulans. A. trlseriatus. A. trivittatus. and A. vexans, respectively, in M i c h i g a n ................ 91 31-36. Distribution of Anopheles earlei. A. punctipennis A. quadriaacule tus, A. walker1 . Culex pipiens. and C. restuans. respectively, in M i c h i g a n .............. 96 37-42. Distribution of Culex sallnar lus. C. tarsal is. £. territans. Culiseta inornate. Mansonla perturbans, and Psorophora ferox, respectively, in Michigan ........... ix 100 LIST OF APPENDICES Appendices Pages A. Mosquitoes collected In a dog-baited trap at Site 1 in a woodland area, 1 9 7 4 ................................... 107 B. Mosquitoes collected in an open area, 1974 in a dog-baited trap at Site 1 108 C. CDC trap collectionsat Site 2, 1975 ...................... 109 D. CDC trap collections at Site 3-A, 1975 .............. 110 E. CDC trap collections at Site 3-B, 1975 ................ Ill F. Mosquitoes collected in a dog-baited trapatSite4,1975 . G. CDC trap collectionsat Site 4, 1975 ...................... 113 H. Mosquitoes collected 114 I. CDC trap collectionsat Site 5, 1975 ...................... J-0. P. in a dog-baited trapatSite5,1975 Representative studyarea weather data - Capital City A i r p o r t ................... Developmental trial: Aedes stimulans Trial 1 112 115 116-121 ........... 122 Q. Bevel opmantel trial: Mansonla perturbans Trial1 ........... 123 R. Developmental trial: Mansonla perturbans Trial2 ........... 124 S. Developmental trial: Mansonla perturbans Trial3 ........... 125 T. Developmental trial: Mansonla perturbans Trial 4 U. Developamntal trial: Aedes vexans Trial1 .................. 127 V. Developmental trial: Aedes vexans Trial2 .................. 128 W. Developmental trial: Aedes vexans Trial3 .................. 129 X. Developmental trial: Aedes vexans Trial4 ............... Y. Developmental trial: Anopheles quadrimaculatus Trial 1 x 126 130 . . 131 LIST OF APPENDICES, CONT'D Appendices Pages Z. Developmental trial: Anopheles quadrimaculatus Trial 2 . . 132 AA. Developmental trial: Anopheles quadrimaculatus Trial 3 . . 133 BB. Developmental trial: Anopheles quadrimaculatua Trial 4 . . 134 CC. Developmental trial: Anopheles quadrimaculatus Trial 5 . . 135 DD. Developmental trial: Cule» pipiens Trial 1 136 EE. Developmental trial: Culex pipiens Trial 2 137 FF. Developmental trial: Culex pipiens Trial 3 138 GG. Concentration of Microfilariae at the time of the infective blood m e a l ...................................... 139 HH. Transmission trial: Aedes stimulans Trial 1 .............. II. Transmission trial: Aedes vexans Trials 1 - 4 ................ 144 JJ. Transmission trial: Anopheles quadrimaculatus Trials 1 and 2 .................................................... 145 xl 143 INTRODUCTION One of the main causative agents of canine filariaais ia Dirofilaria lnealtla (Leidy)* commonly referred to as the dog heartworm. This parasite was described by Leidy in 1850 (Leidy, 1850) and is placed in the phylum Nematoda, superfamily Filarioidea. Adult males and females live in the heart and pulmonary artery of canine hosts. Females produce active embryos called microfilariae which are found in a dog's circulatory system. Microfilariae, or first stage largae, are Ingested by mosquitoes taking a blood meal from an Infected dog. From the mosquito mldgut, microfilariae migrate to the Malpighian tubule where they inhabit the distal cells of these excretory organs for approximately 6 or 7 days. These larvae break out of the cells to complete development in the lumen of the Malpighian tubules where the first and second molts occur. The third larval stage is infective to dogs and development to this stage requires about 12-14 days In suitable hosts. Infective larvae escape from the labium of the mosquito host while the infected Insect takes a blood meal and enter the dog through the wound created by the mosquito proboscis. Apparently they are unable to penetrate the vertebrate host unless the skin is broken. Kume and Itagakl (1955) were the first to trace the development of I), immitis larvae in subcutaneous tissues of the dog. Two additional molts occur in the dog about 10 and 65 days after inoculation (Orlhel, 1961). 1 Soon 2 after the final molt young adults travel, via the circulatory system, to the heart and pulmonary artery of the definitive host. Besides the usual occurence In dogs, various authors have found D. immitis in red foxes, beavers, coyotes, wolves, dingoes, gibbons, cats, seals, tigers, jaguars, sea lions and man. No human deaths have been reported due to D. immitis infections, however, and the dog appears to be the primary reservoir host. JD. immitis has a world-wide distribution and is known from 34 of the 48 continental United States. Renewed interest in this disease has been stimulated by an Increased number of severe clinical cases being reported and a rapid northern spread of the Infection (Otto, 1974). Michigan, the reported incidence of dog heartworm has increased at an alarming rate. In OBJECTIVES Numerous researchers have studied the development of I), immitis in the laboratory or have Isolated suspect larvae from some 80 species of field-captured mosquitoes. Of theaet 2A are known to occur in Michigan. Because of the blo-ecological characteristics of individual mosquito species, the vectors of D. immitis must be studied on a local basis. Michigan does not have a state-wide mosquito control program and there is a threat of infection to Michigan dogs and humans. No field studies concerning dog heartworm transmission in Michigan have been reported and little is known about the natural maintenance of this parasite in the state. The lack of this basic knowledge and the increased Interest in this problem were reasons to undertake this project. The objectives of this study were to: 1) Determine which species of mosquitoes in Michigan are attracted to dogs and are present in sufficient numbers to make them suspect as potential vectors of D. immitis; 2) Determine by examining field-captured mosquitoes for the presence of Infective larvae, which species may carry the parasite under natural conditions; 3) Determine if D. immitis develops to the infective stage in species of mosquitoes found to be the best potential natural vectors; and A) Transmit J). immitis from dog to dog to prove that Michigan strains of suspect mosquito species are capable of transmitting dog heartworm. LITERATURE REVIEW Classification and Evolution Dog heartworm, Dlrofliarla immitis, was first found in the blood of dogs by Panthot in 1679 (Neumann and Maqueen, 1905). In 1850 Leidy (1850) described this parasite and in 1856* he named it Filaria immitis (Leidy* 1856). In 1911 Ralllet and Henry (1911) created the genus Dirofilaria and I), immitis became the type species. Chitwood (1969) placed this parasite in the phylum Nematoda and although the ranking of this taxon may be disputed* most authors agree that D. immitis is correctly classified in the superfamily Filarloidea* family Dlpetalonematidae. Hawking and Worms (1961) reported that the chief attributes of filarial worms are the production of embryonated eggs or larvae by the female in the body of the vertebrate host* Ingestion of the larvae by an arthropod in which two molts occur and* entry into another vertebrate while the arthropod is feeding. Anderson (1957) contended that the Filarloidea and Splruroldea evolved from a common ancestor which lived in the gut of its host and that this postulated ancestor established Itself in the orbit where larvae were taken up by arthropods feeding on lacrymal secretions and then were transmitted to the eyes of other hosts. In the next phase of evolution Anderson suggested that adults became established in subcutaneous tissues but returned to the orbit to deposit their larvae. A 5 Eventually adults pierced the skin to deposit their larvae In lesions which were attractive to hematophagous arthropods which Ingested larvae at this site. Larvae then accumulated In the subcutaneous tissues, being accessible only to arthropods able to pierce the skin. Finally Anderson hypothesized that larvae went Into the circulatory system, allowing adults to penetrate deeper Into the host's tissues. It Is to this stage of development that the dog heartworm has evolved. Vector Determination Dog heartworm was known prior to the 1900's. It was not, however, until Manson's discovery in 1878 that Wuchereria bancrofti (Cobbold) developed In the mosquito that researchers began examining the possibility that other fllarids might develop in mosquitoes. In 1900 Grassl and Noe (1900) experimentally demonstrated the development of D. immitis in the mosquito and this observation was further substantiated by Bancroft (1904). Since 1900 it has been suggested that fleas may also be vectors of dog heartworm (Breinl, 1920; Brown and Sheldon, 1940; Summers, 1943; Stueben, 1954) but in 1956 and 1957 the experimental evidence of Newton and Wright (1956, 1957) proved the flea to be the vector of another filarid, Dipetalonema reconditum (Grassl). Since the time of these publications the mosquito has been considered the sole vector of Dirofilaria immitis and the complete life cycle is now well understood. The Dog as a Host Adult male and female D. immitis live in the heart and pulmonary artery (Kume and Itagaki, 1955; Otto and Bauman, 1959) where they feed 6 on blood (Blcknell et al., 1956). Otto <1974) ravlawad the literature on heartworm In abnonal locations In the dog. These Include the posterior vena cava, hepatic vein, liver trachea, esophagus, stomach, and (encysted in) the subcutaneous or lntermuscular connective tissue. Generally only one worn was found In these unusual locations and rarely did these aberrant worms produce circulating microfilariae. Various authors (Bicknell et al., 1956; Crans, 1963; Otto and Jackson, 1969) have written about the affect of the parasite on the dog. Symptoms M y be absent In light infections or may Include coughing and quick loss of energy in moderate infections. In severe cases, dogs may be subject to dyspnoea, collapse, weight loss, ataxia, anaemia, e d e m a of the lower limbs, enlarged heart, congestion of the lungs and liver, endocarditis, ascites, nephritis and sudden death. Adult females M y produce over 1000 microfilariae, or young embryos, each day and these circulate in the blood stream of the host. Underwood and Harwood (1939) transfused blood containing microfilariae from an infected dog to a 4—month old noninfected dog. These survived for over two years in the animal in the absence of any adult _D. immitis infections. Kartman (1953c) found that transfused microfilariae were infective to Anopheles quadriMculatus Say for a period of only 3 months after transfusion. Afterward the microfilariae seemed to lose their lnfectlvlty and failed to develop to the infective stage in the mosquito. He estimated the age of the microfilariae to be between 3 and 12 months at the time of transfusion. 7 Development in the Mosquito Microfilariae, or first stage larvae, are taken into the mosquito,s midgut while the insect feeds on blood. Fewer microfilariae than expected are Ingested in the amount of blood consumed (Kershaw et al., 1955), although the number Ingested is quite variable. Gordon and Lumsden (1939) studied the filarid Foleyella dolichoptera Wehr and Causey in the frog Rana aphenocephala (Cope). They thought the variability in the amount of Ingested microfilariae resulted either from different concentrations of microfilariae In various capillaries or whether or not blood was taken directly from a capillary or from a pool of blood formed from a broken capillary. From the mosquito mldgut, the microfilariae migrate to the Malpighian tubules. Kartman (1953b) found that this migration can occur within eight hours In susceptible mosquito host, but Is mechanically inhibited by the clotting of blood In the mosquito mldgut. In his experiments, twice as many microfilariae reached the Malpighian tubules Aedes aegypti (L.) fed on blood to which an anticoagulant was added than when fed on blood without an anticoagulant. Kutz (1972) postulated that migration Into the Malpighian tubules can occur within an hour after ingestion. Kartman (1953a and b) also found that in refractory mosquito hosts dead microfilariae passed to the hindgut, presumably for excretion, 48 hours after ingestion and also observed the loss of microfilariae from the anus of Anopheles quadrimaculatus Ssy during the act of feeding. Taylor (1960) studied the development of D. Immitis in Aedes aegypti. She reported that during the first 6 or 7 days, larval 8 development occurred Inside the distal cells of the Malpighian tubules In "sausage" larvae. These larvae broke out of the cells to Inhabit the lumen of the Malpighian tubules for the next 6 days. The first molt occurred about the tenth day of development and took place in the lumen of the Malpighian tubules. be shed at this time. The first cast larval cuticle may not always Finally Taylor noted that the second molt In this species of mosquito occurred between 13 and 17 days post Infection after which Infective larvae broke out of the Malpighian tubules and moved toward the head and proboscis. Burton (1963) observed infective larvae of D. immitis emerge from the antennae and palps of Aedes taeniorhynchus (Wiedemann) and Culex pipiens quinquefasciatua Say. Fate of the Infective larvae While the infective mosquito feeds, the infective larvae escape from the proboscis and can be observed on the skin of the host. Hawking and Worms (1961) have cited several references for various fllarids which indicate that penetration is possible only through broken skin. Emergence of the infective larvae of Brugia pahang1 Buckley from Aedes togoi (Theobald) was shown to be unrelated to temperature, moisture or chemical stimuli, but appeared to be initiated by the mechanical bending of the labium (Lavolplerre and Ho, 1973). There is the possibility of spontaneous loss of infective larvae from the mosquito* Ho et al. (1974) reported significant loss of infective larvae from A. togoi deprived of a blood meal while on the other hand, Bemrlck and Bemrick (1969) found no significant loss of larvae from infective Anopheles quadrimaculatus feeding on a sugar solution. 9 Controversy has arisen concerning the exact proboscis location froa which the third stage larvae emerge. It Is likely that variation exists but that the tip of the labium and the labial sheath are the usual places. Bancroft (1904) saw larvae emerge from the tip of the labium and Lavloplerre (1958) also reported that this is the usual escape site. Grassl and Noe (1900) thought the bending of the labium ruptured the sheath, allowing the larvae to escape. More recently, McGreevy et al. (1974) observed larvae emerging from the tip of the labellae and the midportion of the labium. feeding had ended. Occasionally, larvae continued to emerge after Heavily Infected mosquitoes had trouble feeding because the labium would not bend. In addition, McGreevy noted that fluid, possibly hemolymph, always escaped from the mouthparts along with the infective larvae but never escaped while noninfected mosquitoes were feeding. Development in the Dog Once Inside the dog the larvae molt twice before becoming mature adults. Kume and Itagakl (1955) showed that these larvae develop in the submuscular membranes, subcutaneous tissue, adipose tissue subserosa and muscles. Orlhel (1961) found them In these areas during the first 80 days of development. He also noted that the first molt occurred In the dog about 9-12 days and the second molt 60-70 days after Inoculation. Worms begin moving toward the heart via the circulatory system as soon as 67 days after inoculation (Kume and Itagakl, 1955). are not produced until 8-9 months after Inoculation. Microfilariae No correlation has been found between the number of circulating microfilariae and the number of adult female worms (Hlnman, 1935; Fowler et al., 1973). 10 Newton (1968) reported that a laboratory infected dog maintained the heartworm infection for over 7*s years. Daily and Seasonal Periodicity Microfilariae circulating in the dog have an Incomplete nocturnal periodicity. They are present in the peripheral blood at any point in a 24 hour period, but occur in greatest numbers between 6:00-12:00 P. M. Bicknell et al. (1956), among others, found a second Increase in microfilaremia in the peripheral blood between 7:00-11:00 A. M. Ansari (1970) indicated that there is an active and a passive stage of periodicity. In the active stage microfilariae accumulate in the capillaries of the lungs, where oxygen is available to the larvae and conditions Insure the survival of the individuals. In the passive stage, microfilariae are evenly distributed in the circulatory system and are subject to ingestion by susceptible mosquitoes. survival of the species. This Insures Hawking (1956) demonstrated that the periodicity of I). Immitis was related to oxygen tension. In 1967 (Hawking, 1967) he reported that under conditions of low oxygen tension (30-60 mm Hg) microfilariae were stimulated to initiate undulating movements sufficient to maintain their position is vessels less than 20 um in diameter (presuisably in the lungs) . No response was given when oxygen tension was higher and microfilariae were swept through the vessels. Otto (1969) has mentioned the possibility that the spleen may be Important in the maintenance of periodicity, but Hawking (1962) has presented evidence to the contrary. A seasonal periodicity has also been demonstrated in which microfilariae occur less frequently in the peripheral blood during the colder months of the year (Eyles et al., 11 1954* Kume* 1974; Sawyer* 1974) and Hawking (1967) suggests that daylength* in conjunction with hormonal balance may be responsible for this. Alternate Vertebrate Hosts Besides dogs* D . jmmitis has been found in red foxes (Erickson* 1944; Stuht and Youatt* 1972), beavers (Foil and Orlhel* 1975), coyotes (Gler and Ameel, 1959), wolves (Hartley, 1938; Coffin* 1944; Faust et al.* 1941)* dingoes (Otto* 1969), gibbons (Johnson et al., 1970), cats (Farles et al., 1974; Sharp, 1974; Donahoe, 1975), seals (Medway* 1975) and was reported from tigers, Jaguars* and sea lions (Faust et al.» 1941). Evidence indicates that the dog is the primary host (Otto, 1969) and that these other wild animals do not represent a substantial reservoir for the parasite. Numerous human cases also have been reported (Abadle et al., 1965; Brine et al., 1971; Moorhouse et al., 1971; Feld, 1973; and Martire et al., 1975). In humans. I), inmitis tends to localise in the lungs where it becomes enclosed in a non­ calcified cyst. Lesions as large as 5 cm have been reported. Symptoms may include chest pain, fever, cough, pleural thickening* and adheslona between the chest wall and lung (Feld, 1973). attributed to D. immltis Infections in man. No deaths have been In only one case have circulating microfilariae been found and this case was further complicated because the patient also suffered from Lupus Erythematosls (Green, 1974). Microfilariae were found only on one occasion in the patient even though more than 50 additional blood samples were examined from this patient. 12 Geographic Distribution A near world-wide distribution for dog heartworm vaa reported in 1905 (Neumann and Maqueen, 1905). More recently, Kuts (1972) reported it from the United States, Europe, India, Burma, China, Japan, Australia, and various South Pacific Islands. In the United States reports are so numerous that it is impossible to discuss them individually. The most recent reported continental survey was conducted by Young (1955) (898 of 2337 questionnaires were returned by responding veterinarians). Survey results Indicated that only 11 states, Including 3 from which no veterinarians responded, had no diagnosed cases of heartworm (Figure 1A). Young's survey was conducted prior to the work of Newton and Wright (1956, 1957) which showed that in the United States at least 2 filarids occurred in dogs. Subsequent to Young's survey reports between 1956 and 1965 show 12 states in which D. immitis was diagnosed (Figure IB) (Soltys, 1956; Currell, 1957; Durrer, 1957; Bailey, 1958; Wallenstein and Tibola, 1960; Healy and Kagan, 1961; Leash and Hanson, 1961; Crans, 1963; Thrasher et al., 1963; Groves and Koutz, 1964; Lillis, 1964; Schlotthauer, 1964; Mann and Bjotvedt, 1965). Between 1966 and 1976 heartworm was reported from 34 of the 48 continental states (Figure 1C) (Hirth et al., 1966; Marquardt and Fabian, 1966; Kravis, 1968; Thrasher et al., 1968; Butts, 1970; Joiner and Jardlne, 1970; KcGreevy et al., 1970; Zydeck et al., 1970; Mallack et al., 1971; Rabalals and Votava, 1972; Monson et al., 1973; Trltch et al., 1973; Graham, 1974; Alls et al., 1974, Jaskoskl, 1974; Graham, 1975; Georgi et al., 1975; Sengbush et al., 1975). Several publications by Dr. Gilbert Otto outline the distribution of canine heartworm disease in the United States and these summarize 13 Fig. 1. Distribution of dog heartworm in the continental United States. A) Results of Young's survey in 1955. B) States reporting cases between 1956 and 1965. C) States reporting cases between 1966 and 1976. 15 well the changing opinion on the spread of this disease. In 1949 (Otto, 1949) he wrote that the disease occurred on the Atlantic seaboard from New Jersey to Florida and around the coast to Texas but that the incidence of disease was markedly reduced inland, especially in the north. He considered the disease to be serious only in these coastal regions and said that the inland spread of the disease has not been demonstrated. In 1972 Otto (1972) reemphasized the Importance of the disease along the east coast where reported Infection rates were as high as 63%. In the midwest reports at that time Indicated lower rates of infection but that heartworm was widespread. In 1974 (Otto, 1974) he wrote that the Infection was recognized with increasing frequency in the middle Atlantic states and interest in the disease was stimulated by the increased number of severe clinical cases being reported in the northern states and the rapid northern movement of this infection which was once considered to have mainly a tropical and subtropical distribution. Importance in Michigan Like so many other states, Michigan has had a rapid increase in the number of reported cases of canine heartworm disease during the past 25 years. An unpublished report by Newson and Stuht (1972, H. D. Newson, Michigan State University, personal communication) indicated that dog heartworm was present in 54 of 83 counties in Michigan. In total, from 1951 to May of 1972, 14,525 cases were reported by responding veterinarians. Forth-three and one half percent of these cases were reported from 1970 to mld-1972. Leash et_ aj. (1961) screened 192 dogs at the Michigan State University Veterinary Clinic from mid-April to early August 1960. An infection rate of 2% was found. Worley (1964) 16 reported 5.7Z of 123 dogs Infected with I), immitis In southeastern Michigan. Zydeck et al. (1970) found 1.67Z of 248 dogs with heartworm in Detroit, Michigan. Prouty (1972) reported infection rates of 22Z of 880, 6% of 399, and 6Z of 698 dogs in Belleville, Detroit, and Farmington, Michigan, respectively. An incomplete survey of veterinarians in the Lansing, Michigan area detected over 30 cases in the spring of 1974. A subsequent follow-up survey revealed an additional 83 cases reported during the same summer. In addition to cases reported in Michigan dogs, Sharp (1974) reported that mortality in a cat was due to 7 worms in the pulmonary artery and right atrium. Stuht and Youatt (1972) found 11 of 39 red foxes examined harbored adult heartworms. These foxes were taken from the Shiawassee River State Game Area in Saginaw County. Daahlell (1961) reported a resident of Detroit, Michigan had been found, through x-ray examination, to have a nodular lesion caused by a nematode of the genus Dlrofilaria. Its morphology and location in the lung suggested that it was D . immitis. The patient had visited South Carolina prior to diagnosis so it could not be proven that the infection was incurred in Michigan. MATERIALS AMD METHODS Site Selection Mosquitoes were collected at seven sites (Figure 2) in the Lansing, Michigan area in 1974, 1975, and 1976. Adult females were collected at Sites 1-5 in 1974 and 1975 and brought to the laboratory for identification and examination for the presence of infective I), immitis larvae. These sites (1-5) were selected because dogs at each of these private residences had been infected with D. immitis in the recent past and the chances of finding infective larvae in field-captured mosquitoes would presumably be Increased if carrier dogs were still present in these particular areas. Collecting was done at Site 1 in 1974. Sites 2-5 were used for study in 1975 and dogs at each of these households were treated for D. immitis infections in 1974. Dogs at Sites 3 and 4 died from heartworm in 1974 and one dog at Site 2 was considered cured of heartworm in 1974 but in 1975 again developed a low microfilaremia. Because of his age and the severity of symptoms he was euthanized. In 1976, adult female and/or larval mosquitoes were collected at Sites 2, 5, 6, and 7 for use in laboratory studies. These sites were selected as the best local source of adult Aedes vexans (Meigen) (Site 2); A. vexans larvae and adult Mansonla perturbans (Walker) and Anopheles quadrimaculatus (Site 5); Aedes stimulans (Walker) larvae (Site 6); and Culex pipiens larvae (Site 7). 18 Fig. 2. Seven sites In the Lansing, Michigan area where adult and larval mosquitoes were collected in 1974, 1975 and 1976. Stoll Rd Stato Rd in* Lake Lansing Lansing VO ss&i spas "’;i$ $ $ & I•A ViJltfi* ,vy.*»••<«■ « a » p Jo y Rd K i i i s V:*Ay..y.>Vv.' •joSSfjS'V.*1. 20 Description of the Sites Site 1 wee a kennel where 25-30 doge were maintained* The immediate surrounding area was used for farming but nearby therealso were several permanent ponds* marshy areas* a drain canal* and a large wood lot. Site 2 was a more populated area where seven dogs were maintained. Several cases of heartworm were reported within a half mile radius of the site during 1975 and 1976. Within this area were large fallow fields and at least one woodlot. Close by was a large marshy area into which 2 drainage canals emptied. Site 3 was mainly pastureland and included the Michigan State University horse barns. On the eastern side of the site was a pine woodlot behind which Herron Creek flowed through a marshy area. In the immediate area around Site 4 * at least 3 dogs were diagnosed with heartworm in 1974. and east of a large pond. This site was located west of the Grand River A dense woodlot between these waters flooded each spring and was subject to flooding after heavy rains. Site 5 was located at the from the lake. north tip of Lake Lansing about 250 feet The surrounding area Is marshy and at the collecting site itself* a small woodlot yielded as many as 15 different species of mosquitoes In a single night. This woodlot became flooded after heavy rains. Site 6 was a low-lying woodlot flooded each spring by melting snow and overflow from the Mud Lake Drain. of early season snowpool mosquitoes. It provided an excellent source 21 Located at Site 7 were 4 sewage lagoons. Both terrestrial and emergent foliage around the periphery of the #1 pond provided enough cover for Culex plplens to breed. Collection Methods Adult mosquitoes were collected weekly or biweekly In 1974 and 1975 In the following manner. Collecting was done at night during the season for an approximate 12 hour period which Included dusk and dawn. Biweekly collections were made at Sites 1, 4, and 5 (Figure 2) where both CDC miniature light traps and dog-baited traps were used. Weekly collections were made at Sites 2, and 3 (Figure 2)» where only CDC miniature light traps were utilized. In 1976 adult mosquitoes were collected in CDC miniature light traps and larvae were collected with pint dippers. These adult and larval collections for laboratory studies, were made when mosquito species became available in the field. In 1974 and 1975, at Sites 1-5 (Figure 2) 3 CDC miniature light traps, baited with CO^ (dry Ice), were operated during all collecting periods. At Site 1 in 1974, 2 dog-baited traps (Figure 4) were used. One of these traps was placed in a large woodlot and the other placed In an open field situation. This latter dog-baited trap was relocated between the July 18 and July 24 collecting periods and placed closer to the kennels located at Site 1. It was hoped that this change would Increase the catch of mosquitoes attracted to the dogs located in the immediate area. Only one dog-baited trap was used at Sites 4 and 5 in 1975. The CDC miniature light traps were modified for this study as shown in Figure 3, to increase the longevity of the captured mosquitoes. 22 Fig. 3. CDC miniature light trap. A) Standard CDC trap with gauze mesh collecting bag. B) Modified CDC trap with hardware cloth removed at (a) as suggested by Floore at al. (1971)» pint Ice cream container Inserted at (b) with bottom (c) partially cut out to form a baffle to direct air flow (d) through the stockinette (e). Mosquitoes are held In the collecting chamber (f) which is a large ice cream container. 23 \i/" M B I I b f 24 Herbert et al_. (1972) and Miller ejt al^ (1969) showed that CO^ significantly increased the number of mosquitoes trapped so CDC miniature light traps baited with CO^ were used to enhance the capture of mosquitoes needed for study in the laboratory. Although it was realized that no single trapping method Is adequate for sampling mosquito populations* the CDC miniature light trap has proven its utility as a mosquito collecting device* especially where conventional AC electric power is not available. Arcuff (1976) felt the CDC miniature light trap was one of the best methods to provide a representative sample of mosquito populations. For purposes of this study* it was felt that the CDC miniature light trap was the best single collecting method available to capture high numbers of mosquitoes needed for study and at the same time provide a representative sample of mosquito species present at the collecting sites. Dog-baited mosquito traps (Figure 4) were designed and constructed for this project. They were similar to the collapsable dog-baited trap of Vlllavasco and Steelman (1970) and although not collapsable* they were more portable. The louvers of the collecting boxes were modified as suggested by Bates (1949). The dog-baited traps were used to determine which mosquito species were attracted to dogs in the study areas. Although trapped mosquitoes were prevented from feeding on the dogs it was assumed their presence in the traps indicated the potential of these species to feed on dogs. Identification and Examination of Mosquitoes for the Presence of infective D. immitis larvae Adult mosquitoes captured in the field were brought to the laboratory in the removable end-boxes of the dog-baited traps and the 25 Fig. A. Dog-baited trap. A) Main compartment where dog is held. The wire screen la 1" hardware cloth. B) Mosquito collecting chambers which set on the platform of the dog compartment at points (a) and (b). C) Removable screened frame which is taken off to remove mosquitoes from the chamber or can be left off in the field to allow mosquitoes to feed on the experimental dog. The screen is aluminum window screen. 26 €----------- 3 2 " ---------> 24 48 " 27 collecting chambers of the light traps. These were placed In walk-In refrigeration rooms maintained at about 40° F. Mosquitoes were transferred to pint containers and kept refrigerated until they were Identified and pooled during the same day the mosquitoes were brought to the laboratory. Mosquitoes were anesthetized with CO^* generated from dry ice. and identified to species with the aid of a dissecting microscope. Each species was placed in pools of 25 or 30, (or less if too few had been captured) and kept in chilled containers. Pools were examined for the presence of infective I), immitis larvae according to the method of Crans (1971). Pooled mosquitoes were crushed between two microscope slides and their remains placed in .9% saline. This mixture was placed in a plastic, disposable beverage container from which the bottom had been removed and replaced with a fine mesh gauze. It measured 7.0 cm high, 9.4 cm across the top and 3.4 cm across the bottom. This container was placed in a 60 x 35 mm crystallization dish with saline covering the gauze. Living, third stage larvae exited the mosquito bodies and fell through the gauze into the crystallization dish. After one hour, the saline with larvae and smaller mosquito parts was placed into a 60 ml separatory funnel. Here the larvae and debris were concentrated by gravitation for 30 minutes. From this funnel 5 to 6 ml of saline were drawn off and examined for third stage larvae. This technique seemed to be selective for extracting infective larvae free in the hemocoel of the mosquitoes because few mosquito parts, especially Malpighian tubules were present in the examined debris. This method was preferable to individual dissection of mosquitoes because large numbers of mosquitoes could be examined for the presence of infective larvae in relatively short time. 28 Problems in Mosquito Identification During Che course of study* quick identification of living mosquito species was required. Members of two Aedes complexes were not always differentiated because the adults are* for practical purposes* indistinguishable. Aedes fitchii and _A. stimulans* members of the Aedes stimulans complex (Barr* 1958) were, therefore* tabulated together in the results. Similarly, Aedes cinereus, although not a member of the A. communis complex (Barr* 1958), was confused with members of this group and was not positively identified in collections made before September of 1975. Because of this* A. cinereus was included with members of the _A. connunls complex in pre-September 1975 collections. A. sticticus and A. aurlfer are members of the A. communis complex but these species were positively identified and tabulated separately throughout the study period. Differentiation of D. immitis Larvae Infective larvae of Dirofilaris immitis cannot presently be differentiated from third stage larvae belonging to other species in this genus. Chalifaux and Hunt (1971) developed a hlstochemical stain which differentiated the microfilariae of Dipetalonema reconditum from Dirofllaria immitis. Larvae, presumed to be I), immitis * obtained from field-captured mosquitoes were stained by this method. These were compared with known third stage larvae of JD. immitis and infective larvae of I), tenuis Chandler treated in the same manner. Records were kept of the date* location and mosquito species from which larvae were obtained. Several other nematodes were observed in mosquitoes during the course of study and were readily distinguishable 29 from I). Immitis larvae. The criteria used to Identify larvae, assumed to be D. Immitis, were size, as Indicated by Taylor (1960) and Symes (1960), and shape and activity of the worms compared to observations of known specimens of I). Immitis. Selection of Mosquitoes for Laboratory Studies Mosquito species were selected for development and transmission studies based on 4 criteria: 1) those attracted to the dog-baited trap, 2) those most numerous In the Lansing, Michigan area based CDC miniature light trap and dog-baited trap collections, 3) those found to be harboring presumed infective I), immitis larvae in nature and, 4) those incriminated in the literature as being potential vectors of dog heartworm. Based on these 4 criteria Aedes stimulans. A. vexans, Mansonia perturbans. Anopheles quadrimaculatus and Culex pipiens were selected for laboratory study. Dlrofilaria Immitis Developmental Trials Mosquitoes selected for Btudy In the laboratory were given an Infective blood meal. They were then held in an insectary maintained at 80° F and 80% relative humidity and dissected at various times or after their death to determine the developmental progress of I), immitis larvae. Obtaining the Infective Blood Meal For D;. immitis developmental studies, mosquitoes were Infected by allowing them to feed directly on a Basset Hound known to be infected with dog heartworm, or through cow-gut membranes stretched over glass containers that contained infected dog blood. Blood was not warmed 30 during the membrane feeding trials. To obtain the infective blood meal directly from dogs or to attempt transmission, mosquito cages were placed over dogs held in a restraining chamber (Figure 5)• Determination of Microfilaremia or Dilution of Infected Blood Before mosquitoes were allowed to feed on dogs, microfilaremia was checked by the method of Seeley and Blckley (1974). A single, 3-5 ml blood sample was drawn within 1 hour prior to mosquito feeding and microfilaremia was determined within 16 hours after the time the blood sample was taken. Blood samples were refrigerated if microfilaremia determinations were delayed. Twenty jil subsamples were placed on a microscope slide, and diluted with a drop of normal saline tinted slightly with methylene blue, a 24 x 50 mm coversllp was placed on the slide and microfilariae were counted. sample were examined. Three to ten subsamples from each For membrane feeding trials, 3-5 ml of blood was drawn from the infected dog and always diluted with 12-18 ml of blood from a noninfected dog in order to reduce excessive mosquito mortality due to the high microfilaremia in the infected dog. Concentration of microfilariae was determined by the same method described above. Transmission Trials For transmission studies, Aedes stimulans. A. vexans and Anopheles quadrimaculatus were allowed to feed on laboratory reared, parasite-free, purebred Beagle dogs obtained from a commercial supplier. A different dog was used as a recipient host for each mosquito species studied, for which transmission was attempted. Transmission attempts were made at least once dally until all mosquitoes died. Dogs were maintained for an 31 Fig. 5. Apparatus used to restrain dogs during mosquito feedings. Dog was strapped to canvas sling (b) with feet through holes (a) and supported by wooden dowels (c) resting on frame (d). 32 22 30 24 99. 33 appropriate amount of time after exposure to the bite of Infected mosquitoes and then examined at necropsy for the presence of adult heartworms. RESULTS AND DISCUSSION Field CollectIona A complete tabulation of dog-baited and CDC miniature light trap collections, Is given In Appendices A-I. Data on light trap collections made at Site 1 are not presented because all mosquitoes from those collections were not identified and pooled. It was felt that those examined were not selected randomly and tabulation of these data could not be made without bias. Pertinent weather data are recorded in Appendices J-0. The most likely vectors of dog heartworm were chosen from the dogbaited trap collections listed In Table 1. Host preferences for the mosquito species, except Culiseta impatiens (Walker), collected In these traps were reviewed by Edman (1971, 1974) and Tempells (1975). Aedes and Anopheles prefer manmallan hosts, while Mansonla prefers mamma1s but readily feeds on birds. Culex plpiena L. prefers avian hosts and C. territans Walker prefers amphibians. Determination of host preferences in a given area is difficult for any particular species. Besides considering a mosquito's usual blood meal source, host availability must be considered. To Illustrate, Tempells et. a J L . (1970) studied a population of Culex pipiens quinquefasciatus. primarily a blrdfeeder, in Hawaii from which 31Z had fed on dogs and large bovlnes when these mammals were the most abundant blood meal source in the area. 34 35 Table 1 1974 and 1975 Dog-baited Trap Collection Totals Site Species Open Area Woodland Aedes canadensis cinereus communis complex fitchii-stlmulans stlctlcus triaeriatus trivlttatus vexans Anopheles quadrimaculatus walker1 Culex plplens terrltans 1 46 4 9 7 51 2 19 11 3 149 34 92 112 5 1 51 131 168 184 9 31 461 423 59 1,016 717 11 3 9 4 7 74 Culiseta lmpatlens Mansonla perturbans TOTAL 19 Including Aedes cinereus and excluding A. aurifer and A. stlctlcus. 436 36 Because availability Is so Important, thoae mosquito species captured In the dog-baited traps were highly suspect as vectors of dog heartworm, especially those species captured most frequently. Only £. territans was not Initially suspect as a dog heartworm vector because Tempells (1975) wrote that this species feeds almost exclusively on amphibians. Site 5. C. territans was only captured In the dog-baited trap at During the season, many frogs were seen In the iassediate collecting area and several times, frogs were found in the dog—baited trap. The preferred host of £. territans was abundant in the area. spite of its presence in the dog-baited trap In territans. most probably does not readily feed on dogs and is not a likely vector of D. lsmiitls. Table 1 shows that Aedes fitchii, A. stimulans. A. vexana. A. triseriatus (Say), Anopheles quadrimaculatus. A. walker! Theobald, Culex pipiens and Mansonia perturbans were srest abundant in dog-baited trap collections. These Basie species were also most abundant in CDC light trap collections (Table 2). Additionally, Aedes cinereus Melgen, A. sticticus (Meigen), and A. trivittatus Coquillett were abundant in light trap collections, and these species were also collected in the dogbaited trap at Site 5. These 11 siosquito species, because of their presence in dog—baited traps, and their abundance, indicated by CDC light trap collections, are Initially the most suspect vectors of dog heartworm in the Lansing, Michigan area. Univoltine Mosquitoes In Michigan, A. fitchii. A. stlmulans and Mansonia perturbans are thought to be unlvoltlne as they are in Minnesota (Barr, 1958). Aedes fitchii and A. stimulans were collected until mid-August (Figure 6E) 37 Table 2 1975 CDC Miniature Light Trap Collection Totals Site Species Aedes aurifer canadensis clnereus communis complex dorsalis fitchii-stimulans flavescens solllcltans stictlcus trlserlatus trlvlttatus vexans Anopheles earlei punctipennls quadrlmaculatus walker! Culex erratlcus pipiens restuans salinarlus tarsalls territans Culiseta mors1tans lmpatiens inornata Mansonia perturbana Orthopodomy1a spp. Psorophora cillata ferox Uranotaenia sappharina 2 - 1 - 37 - 44 1 - 192 1 1,102 19,770 - 34 45 55 - 227 - 8 1 3 - A - 2 32 18 11 88 49 1 96 2 327 12,705 - 18 19 300 - 293 - 15 3 - B - 3 38 7 12 214 1 - 46 2 344 10,476 - 36 8 55 - 180 1 4 4 5 3 108 39 50 3 593 10 36 2,040 381 4 628 - _ _ — 117 236 801 6,044 1,231 22 787 20,437 - 65 396 134 - 110 1 1 27 37 2,282 5,391 9 691 1 19 - - - - — — 2 — 19 - — - - 21 - 1 - — — — — 1 375 87 - 53 137 1 — — — 2 - - - - - - - - - - 14 £ Includes Aedes clnereus and excludes A. aurifer and A. stictlcus. 1,024 2 1 1 3 38 Fig. 6. Seasonal Incidence of Aedes fitchii-stimulans at several locations in the Lansing, Michigan area during June through early October, 1975. Collections made with CDC miniature light traps. A) Rainfall in inches during the collecting period. B) Daily maximum and minimum temperatures during the collecting period. C-G) Collections at Sites 2, 3-A, 3-B, 4, and 5 respectively. No. Aodos fitchii •stimulant Trapped T 0 u fr— — 0 o 0 w I 6 ^ 0 w 0 O X ol * 8' 8* 5- \ S51 \ y ( / -Si Si O' >o' S' o' (•55* Sm 4.0 * 0 150 £ O g t 0 0 0 40 indicating a potential life span of about 2 months or more under natural conditions. Collection data for these two species show them to be more abundant in their woodland habitat (Figures 6E, F, G; 8C) than in open areas (Figures 6C, D; 7C; 9C). If they are unwilling to fly out of a woodland situation their potential to transmit heartworm would be less In areas outside their preferred woodland habitat. From collection data shown in Figures 10C-G it would appear that Mansonia perturbans adults are also long-lived. They were captured from June 10 to September 10. Barr (1958), however, noted that in Minnesota, adulta emerged over an extended period of time and this fact makes their longevity Impossible to determine based only by their presence in trap collections. Almost equal numbers (slightly more in the open area) of M. perturbans were captured in the dog-baited traps In the open area and the woodlot at Site 1. This might Indicate a willingness of this species to feed in either habitat, however, Figure 7H shows that in the open area, after peak emergence about July 18, incidence in this location falls sharply while in the woodlot trap (Figure 8H) this species was frequently captured after July 18. locations. Collecting was done the same night at both This might indicate a migration of M. perturbans into the woodlot and possibly a higher potential of heartworm transmission by this species in a woodland habitat. No experimentation was done, however, to prove that migration occurred or to determine another cause for this difference in abundance through time. Multivoltine mosquitoes breeding in temporary or fluctuating water situations Aedes clnereus. A. stictlcus, A. trlserlatus, A. trivlttatus and A. vexans may produce more than one generation per year and often 41 Fig. 7. Important mosquito species collected In a dog-baited trap located In an open situation at Site 1 during June through August, 1974. A) Rainfall In Inches during the collecting period. B) Dally maximum and minimum temperatures during the collecting period. C-H) Seasonal Incidence of Aedes f1tchli-stimulana. A. vexans. Anopheles quadrimaculatus. A. walker!» Culex pipiens and Mansonia perturbans. Rainfall (Inches) 42 3.0 3.0 1 .0 max Tamp. ( #F) m ln . 60' 40 20 10 100 Aedes fitc h ii * s tim u lan s Aadaa vaxans 50H 0 50- 100i Anophalas " tor 1 * ■ i quadrim aculatua i ■ /N_._ f 1■ “ l Anopheles w alksri SO* No. Trapped ol ■ "i 50 ISO* Q Culex p ip io n s Mansonia p o rtu rb a n s H 100* 50 0 10 20 M 30 l 10 1 I 20 30 J ■ 10 » 20 30 J ■ 10 I 20 A ■ 30 A3 Fig. 8. Important mosquito species collected in a dog-baited trap located in a woodland situation at Site 1 during June through August, 197A. A) Rainfall in Inches during the collecting period. B) Dally maximum and minimum temperatures during the collecting period. C-H) Seasonal Incidence of Aedes fitchii-stimulans, A. vexans, Anopheles quadrimaculatus, A. walkeri, Culex piplens and Mansonia perturbans. Rainfall (In ch at) 44 2.0 1.0 max Tamp. ( F ) 100i m ln . 60> 40 20 25 A adaa f it c h ii • atim ulana 25 Aadaa vaxana O1 10 ■ v v Anophalaa q u ad rim acu latu s Trapped O1 10 Anophalaa w alker! 01 50 I t "V ' '!'■■■■ v C ulex piplana 1 O ioo* Manaonla p a rtu rb an a 50* O ■ 10 i i 20 30 ./'V— \ _ / u i i i ■ i lO 20 30 10 20 30 45 Fig. 9. Important mosquito species collected In a dog-baited trap at Site 5 during June through early October* 1975. A) Rainfall In Inches during the collecting period. B) Dally maximum and minimum temperatures during the collecting period. C-H) Seasonal Incidence of Aedes fItchii-stimulans. A. vexans. Anopheles quadrimaculatus, A. walker!. Culex piplens and Mansonia perturbans. Rainfall (In ch ei) 46 4 .0 < 3.02* 0 * 1.0 ' J ■ J i Ji I U ' I . 1 .1 k. 0 m ax. m ln . 100 Tamp. C#F ) • 80 * ~*V-**%• yi • /***.£:*• •■V t V *:a 60 40 20 10m A « 4 n f it c h ii * s tim u la n t o-L— .---.--- I--- 1---.----------V* 50i L A s d s i vaxans 25* I Trapped 25 I I ■ “ ■I ■ Anophalas q u a d rim a c u la tu s 01 50i Anophalas w a lk a ri i 25- /\ Vy---, 10 T" Q Cutsx plplans o1 25 M ansonia p a rtu rb a n s H 01 10 20 30 M 10 20 J 30 10 20 J 30 10 I 20 I 30 ”T" — I- ■T" T 10 20 30 10 47 Fig. 10. Seasonal Incidence of Mansonia perturbans In the Lansing, Michigan area during June through early October, 1975. Collections made with CDC miniature light traps. A) Rainfall In inches during the collecting period. B) Daily maximum and minimum temperatures during the collecting period. C-G) Collections at Sites 2, 3-A, 3-B, 4, and 5 respectively. No. Mansonlo porturbont Trapped W W O O a a i a i a a i ■ \ ■ / > ■ f \ . r i / • \ i > / #/ ' i g a f > s-r *1 ■ i a a a a « a ■ i a a a a a a « H i • I 1 1 m 4 • U ol 4.0 W o o 49 produce several broods In one season. Barr (1958) wrote that these 5 species can be expected to have an early season emergence in May or June. Subsequent emergences result from heavy rainfalls during the summer and even early fall If temperatures are favorable. Figures 11C- 15C show that In 1975 these 5 species produced at least 2 broods: The annual early season brood shown by their abundance in June and a late suimser brood In September resulting from heavy rains which occurred In mid-August. A third emergence may have occurred In early August* 1975 because Figures 11C-15C show a small peak during this time. Moderate rainfall In mid-July may have produced an additional brood but there Is no other evidence to support this conclusion. The Important point to make here Is that these 5 species, by their seasonal occurrence are most Important as potential vectors of heartworm during early summer and thereafter, following heavy rainfall. This generalization though, must be weighed carefully because A. trlserlatus, although abundant only at Site 4 (Figure 130) was collected each night that traps were set except on September 11 (This species was not collected after September 23, 1975 at that site.) Thus, when abundant, this species can be a continual pest and potential heartworm vector throughout the mosquito season. Similarly, A. trlvittatus (Figure 14C) may be collected only during peak periods of abundance as It was at Site 2, or may be continually present as It was at Site 4. Likewise A. vexans showed peak periods of abundance at all sites similar to the peaks observed at Site 2 (Figure 15C). This was the species collected most frequently In the study area and although there were periods of peak activity, significant numbers of A. vexans were present throughout the mosquito 50 Fig. 11. Seasonal incidence of Aedes cinereus and some members of the .A. communis complex. Excludes A. stictlcus and A. aurifer, which were not distinguished from A. cinereus until September, 1975. A) Rainfall in Inches during the collecting period. B) Daily maximum and minimum temperatures during the collecting period. C) Seasonal Incidence of A. cinereus collected with CDC miniature light traps at Site 5. No. Trt ppod O •or u» Tamp. (°F ) tm O M O w 10 A tdai o cinorous 20 30 + communis 10 20 30 com piox 10 20 30 *t u a,llniaa| o M laiiHnn"11"1 o ....... u o o V/*”" Rainfall (inchas) 52 Fig. 12. Seasonal Incidence of Aedes stictlcus at Site 5 in the Lansing» Michigan area during June through early October, 1975. Collections made with CDC miniature light traps. A) Rainfall in Inches during the collection period. B) Dally maximum and minimum temperatures during the collection period. C) Seasonal incidence of A. stictlcus. Rainfall CInchat I 53 4.0^ J. J i Jii ji... I , 1 ,1 max. Tomp. t°F > m ln . * i. ?• *. 10 20 / \ 30 10 500- stlcticus Trappod 200- No. AodM 400- 10O- 300- V 10 20 M 30 r " "T IO 20 yA .V 30 10 20 30 10 20 30 * s 54 Fig. 13. Seasonal Incidence of Aedes trlserlatus at Site 4 In the Lansing, Michigan area during June through early October 1975. Collections made with CDC miniature light traps. A) Rainfall In inches during the collecting period. B) Daily maximum and minimum temperatures during the collecting period. C) Seasonal Incidence of A. trlserlatus. 304 ■OP No. Aadat trlsariatui Trapped 56 Fig. 14. Seasonal incidence of Aedes trivlttatus at 2 locations In the Lansing• Michigan area during June through early October, 1975. Collections made with CDC miniature light traps. A) Rainfall In inches during the collection period. B) Dally maximum and minimum temperatures during the collection period. C) Collections at Sites 2 and 4. 57 4.0*1 max. mln. S lt« 2 ■V 100*1 1 m 90*1 ►wa 80-1 a •M 70*1 b 60J m a • < 5oJ o Z 4030* 20* 10« 0 58 Fig. 15. Seasonal Incidence of Aedea vexans in the Lansing* Michigan area during June through early October* 1975. Collections made in CDC miniature light traps. A) Rainfall in Inches during the collecting period. B) Dally maximum and minimum temperatures during the collecting period. C) Collections at Site 2. 59 4 .0 m 2 3.0 E - 2 .0 1 .0 » of max. mln. 1 00 1 -. * 80 u ~ 40 a. I20 14000 13000 1100* 1000 I 900« 800 700600 500 400> 200 100* 10 20 M 30 10 20 J 30 20 30 30 lO 60 season. Thus this species could be involved in the transmission of I), inunitis during the entire mosquito season. A final interesting point concerns A. trlserlatus. Barr (1958) contended that this species does not readily leave its woodland breeding site and is generally not attracted to light traps. Table 2 shows that in light trap collections at Site 4 it was one of the most frequently captured mosquitoes. Table 1 shows that A. trlserlatus was captured at Site 1 more often in the dog-baited trap set in the open area than in an identical trap set in a woodlot. It may be that this mosquito leaves its breeding site more readily than is generally suspected, especially in areas, such as Site 1, where dense shrubs outside the woodlot, provide sufficient cover. This remains to be experimentally proven. Multivoltine mosquitoes breeding in permanent water situations Anopheles quadrlmaculatus, A. walkeri and Culex plpiens may produce several generations per year. Collection data shown in Figures 7E, F, G; 8E, F, G; 9E, F, G; 16C-G; 17C-G; and 18C-G does not permit the determination of the number of generation produced in 1974 and 1975 because emergence of these species is likely to be continual during summer months and la not as dependent on heavy rainfall as are the multivoltine Aedes species. Heavy rainfall can, however, increase population numbers by providing additional breeding sites or enlarging existing ones. Significant rainfall in August, 1975 apparently caused population Increases in Anopheles walkeri (Figures 9F and 17G) and Culex pipiens (Figure 18C) in September, 1975. Surprisingly, similar population increases of A. quadrlmaculatus did not occur in September, 61 Fig. 16. Seasonal Incidence of Anopheles quadrlmaculatus In the Lansing, Michigan area during June through early October 1975. Collections made with CDC miniature light traps. A) Rainfall In Inches during the collecting period. B) Daily maximum and minimum temperatures during the collecting period. C-G) Collections at Sites 2, 3-A, 3—B, 4, and 5 respectively. No. Anopheles quadrimaculatu* Trapped Rainfall tinches) 4.0 § e I a Temp. t°F ) m 63 Fig. 17. Seasonal incidence of Anopheles walker! in the Lansing, Michigan area during June through early October 1975. Collections made with CDC miniature light traps. A) Rainfall in inches during the collecting period. B) Daily maximum and minimum temperatures during the collecting period. C-G) Collections at Sites 2, 3-A, 3-B, 4, and 5 respectively. Ra|nf„, (inch##) 64 4.01 3.0 3.0H 1 .0 J. J i Ji i JJ.s I . 1 .1 k- ■. I. 0 T#mp. (oF , 100 80- m^ ■ * 60 40 20 50 c O1 1* i ------ T- 75 O1 ________ Ho. Anopholot walkori Trappod 25 O1 125 " A 1 -"-A 500' 400* 100* V 10 20 M 30 10 20 J A 30 10 20 30 10 65 Fig. 18. Seasonal Incidence of Culex plpiens In the Lansing* Michigan area during June through early October* 1975. Collections made with CDC miniature light traps. A) Rainfall in inches during the collecting period. B) Daily maximum and minimum temperatures during the collecting period. C-G) Collections at Sites 2, 3-A, 3-B* A, and 5 respec tively. Rainfall (Inctias) 66 4.0 3.0 2 .0 1.0 ■j. J i Ji * 0 max. m ln. 100 Tamp. (°F ) 1 . 1 .1 k • » **• 80 • a * ■ •.* *• » • * | m ■ • • •* H• .... «l* n ’ * r 60 ••t * Ai\ Du.*\*V •.****”••*%•* ••• . ;•' •/ • V V* *• 40 20 150 100 No. Culox pipians Trappod 25 A- ”o J1- D > P 1 l 75 O1 ■ 100 . ;v_. 01 I I I A ■ a I I^ I S ■■ IOOi I I /\ /v 50 0 - 10 20 30 M 10 20 J A ■I— T ^ ■» r - 1I.. sJ'— # A Nf " I 30 10 20 30 10 20 30 I" Q \ \ 10 20 s 30 10 67 1975 (Figures 9E and 16C-G). Table 1 and Figures 7D and E and 8D and E show A. quadrlmaculatus and A. walker! present In open area dog-baited trap collections at Site 1 but nearly absent from woodland collections at the same site. These species apparently do not readily enter the woodland habitat and would not be highly suspect as vectors of heartworm In such areas. On the other hand, _C. pipiens was equally abundant In woodland and open area collections at Site 1. This species must be considered a potential vector of heartworm in both habitats. An interesting observation for which no explanation can be given is that at Site 1 in 1974* A. walker! was collected early In the season (June 4— July 18) while A. quadrlmaculatus was collected later In the season (July 16-August 27) (Figure 7E and F). During 1975 these species were collected concurrently (Figures 9E and F* 16C-G, 17C-G). The conclusion to be drawn from the collection data discussed above is that these 11 discussed species* by their presence in dog-baited traps and abundance in dog-baited and CDC miniature light traps are the most likely vectors of dog heartworm* at least in the study area. Information is lacking concerning which of these species survives long enough* In nature, to support complete development of I), immitis larvae. Kutz (1972) found that under laboratory conditions at 70° F* 21 days were required for I). Immitis to develop to the infective stage and reach the head and mouthparts in A. quadrlmaculatus and development was arrested at 60° F. Jaskowski and Blckley (1976) found infective larvae in the head and mouthparts of Aedes canadensis (Theobald)* held at 64.4° F* between 27 and 37 days after an infective blood meal. If approximately 27 days is required for complete development in an ideal 68 host, It is possible to speculate on the longevity required for a mosquito to support development of D. immitis under natural conditions. In Lansing, Michigan the average temperature (Environmental Data Service, Ashvllle, North Carolina) from 1936-1975 for May through September was 56.6, 66.2, 70.7, 68.9, and 61.8° F, respectively. Temperature may limit development of heartworm in the mosquito to June, July and August when average temperatures are around 70° F. It seems logical that the most likely vectors of dog heartworm would have to survive a minimum of 30 days, allowing time to search for blood meals. Certainly, development may not require 27 days under ideal conditions and some mosquito species may serve as vectors during warm seasons but not during cooler seasons. Assuming development is possible, the most likely vectors are those in which a good portion of females in the population have a longevity of approximately 30 days. Additionally, Crans and Feldlaufer (1974) wrote that the time of greatest danger for transmission is when population numbers are low. Obviously it is at this time when the population consists mainly of older females that have had at least one, possibly infective, blood meal. Examination of Mosquitoes for the Presence of Infective Larvae Tables 3 and 4 list the mosquito species and numbers examined for nematode larvae in 1974 and 1975. Table 5 shows the site and date on which infective larvae, possibly I), immitis, were extracted from field captured mosquitoes. These nematodes, by their size, shape, and activity were indistinguishable from infective D. immitis larvae, and for the sake of discussion, it will be assumed that they were D. immitis. 69 Table 3 Mosquitoes Pooled in 1974 from CDC Miniature Light Trap and Dog-baited Trap Collections Species # Pooled Aedes canadensis 9 communis complex3 24 fltchli-stlmulans 122 triserlatus 53 vexans 2,209 Anopheles punctipennis 31 quadrlmaculatus 422 walkeri 383 Culex plplens 463 territans 2 spp. 10 Mansonla perturbans 2,492 TOTAL a 6,220 Includes Aedes cinereus and excludes A. aurifer and A. stlctlcus. 70 Table 4 Mosquitoes Pooled in 1975 from CDC Miniature Light Trap and Dog-baited Trap Collections Species # Pooled Aedes aurifer canadensis cinereus # communis complex dorsalis fltchli-stlmulans flavescens stlctlcus trlserlatus trlvlttatus vexans 13 150 413 359 2 1,437 51 779 243 1,031 24,912 Anopheles earlil punctlpennls quadrlmaculatus walker1 33 137 2,340 3,462 Culex erraticus plplens restuans sallnarlus territans 1 828 2 14 34 Culiseta Inornata moraltana 1 7 Mansonla perturbans 1,547 Orthopodomyia spp. 1 Uranotaenla sappharlna 3 TOTAL 37,800 mlncludes Aedes cinereus and excludes A. aurifer and A . stlctlcus. 71 Tabic S Nematodes, Possibly Plrofllaria Immitis, Extracted from Mosquitoes Mosquito Site Aedes vexans Anopheles quadrlmaculatus Gulex piplens Date # Pool Size Worms 3 June 30, 1973 25 12 2 July 20, 1975 25 3 5 August 26, 1975 25 20 1 August 27, 1974 4 4 5 August 19, 1975 25 20 August 26, 1975 12 33 72 There la additional evidence that the nematodes, extracted from Aedea vexana on July 20, 1975, may have been I). Immltla larvae. After these larvae were found, dogs at Site 2 were screened for microfilariae. A single dog, thought to be cured of heartworm the previous year, again showed a low microfilaremia. An Infected dog was at Site 2 during the time mosquitoes were being collected and examined for infective larvae. Table 5 shows that larvae, possibly I).Immitis, were recovered from mosquitoes collected at4 of the 5 collection sites. These larvae were found most often In A. vexans while the highest number of larvae were found In Culex pipiens. Because of the low incidence of larvae found in mosquito pools, it is probable that nematodes emerged from a single Individual of the pools. Results of the developmental trials, to be discussed later, show £. pipiens to be a poor host for D. Immitis. Never was more than a single 1). immitis larvae found in C^. pipiens fed an infective blood meal. Thirty-three larvae were extracted from a pool collected in 1974 (Table 5). This pool of £. pipiens was collected at Site 1 in the dog-baited trap set in the woodland area. are Filarld worms commonparasites of birds (Anderson and Freeman, 1969) and birds are thepreferred host of £. pipiens. Anderson and Freeman (1969) found four genera of the family Onchocercidae in Ontario, Canada. Cardiofilarla Strom, is an especially common genus which they suggest is present throughout North America. Since this genus has been found in many different bird species, these authors suggest that the vector's feeding habits are not highly selective, and typical of a mosquito. At least one species of Cardiofilarla. from Ceylon, is known to be transmitted by mosquito. Furthermore, £. inornate (Anderson) a common 73 North American speclea la known from the woodcock, robin, olive—backed thruah, long-eared owl, aharp-aklnned hawk, marah hawk, raven and the oven-bird. Thle la not to auggeat that the nematodea laolated from Culex plplena were Cardlofliarla Inornate but to bring out the fact that bird filarIda are no doubt common In the atudy area and It appeara likely that the nematodea found In Culex plplena were not D. Immitis. Again, If we asaume, based on the low numbers of larvae extracted from fleld-captured mosquitoes, that nematodes recovered from pools originated from a single individual In the pool, we can calculate Infection rates In the population. Nematodea were obtained from A. vexana on 3 occasions from a total of 27,121 mosquitoes of that species. This indicates that 0.01Z of the population may have been carrying I). Immitia larvae. Similarly, nematodea were extracted from Anopheles quadrlmaculatus on 2 occasions from a total of 2,762 pooled individuals. In this case the infection rate would be 0.072Z. From these data it may be suggested that Anopheles quadrlmaculatus carry proportionately more presumed D. immitis Infective larvae under natural conditions and would be more Important as a vector of dog heartworm than Aedes vexans. Differentiation of D. immitis Larvae The histochemical stain developed by Challfaux and Hunt (1971) to differentiate microfilariae of I), immitis and Dipetalonema reconditum proved to be of no value in identifying Infective larvae from fieldcaptured mosquitoes. Third stage larvae of D. immitis treated according to this method appeared similar to third stage larvae of I), tenuis prepared in the same manner. Orlhel (1959) concluded that the 74 developmental stages of I), immitis and I), tenuis In the mosquito host are Indistinguishable on a morphological basis. Thus, Infective larvae isolated from field-captured mosquitoes could not be positively Identified. It Is not surprising that the hlstochemical stain, to locate areas of acid phosphatase activity, did not react differently In these 2 Dirof ilarla species. D . insultis and D. tenuis are closely related taxonomlcally and because both species have developmental stages in the Malpighian tubules of mosquitoes, their physiology Is no doubt, very similar. Dirofllaria Immitis Developmental Trials Complete tabulation of I). Immitis developmental trial results appear in Appendices P-FF. Microfilaremia of the Infected dog or dilution of blood, for membrane feedings, at the time of the infective blood meals are listed for the various trials in Appendix GG. Results of the developmental trials will be discussed from Table 6. Results with Aedes stimulans Aedes stimulans proved to be a poor host for I). Immitis larvae. Infective larvae were never observed in the head and proboscis of this mosquito. Development appeared to be retarded because second stage larvae were observed In the Malpighian tubules as late as 26 days after the infective blood meal. Microfilariae did not become established in the Malpighian tubules of 16 of 19 (84.2Z) mosquitoes dissected. Encapsulation was observed in every mosquito in which larvae did reach Table 6 Suamary of Developmental Trials Species Aedes stinulans Nansonia perturbans Aedes vexans Anopheles quadrinaculatus Culex pipiens # Mosquitoes Taking an Infective Blood Trial Meal I Days Required for Development L3 L2 M. T.a Head # Mosquito Mortality until First L^ Observed in Head I Mosquitoes Dissected I Mosquitoes Dissected with No Larvae in M. T. I Mosquitoes Dissected I Mosquitoes with some Encapsula­ Dissected tion with Ho Larvae Observed 1 57 N0b 18 ND° - 19 16 84.2 3 1 2 3 4 35 54 84 71 HD HD HD 8 ND ND ND HD ND ND ND ND • 4 4 23 31 4 1 22 31 100 25 95.7 100 0 3 1 0 1 2 3 4 45 14 210 212 10 HO 7 7 NO NO 9 10 12 NO 12 12 68.9 10 2 10 7 6 0 3 1 60 0.0 30 14.3 3 0 1 3 1 2 3 4 5 10 37 15 15 6 9 HO NO NO 9 11 10 HO NO NO 13 14 NO NO NO 70 89.2 3 5 2 2 3 0 0 0 0 0 0.0 0.0 0.0 0.0 0.0 0 0 0 0 0 1 2 3 10 84 27 NO 9 14 HO 14 NO NO NO 15 4 45 23 4 40 19 100 88.9 82.6 0 0 0 aKalpighian tubules. not occur. - — - 98.6 97.6 - - - 22.2 bStage not observed but nay have occurred. cDevelopment to this stage aost likely did 76 the Malpighian tubules. observed. No unencapsulated third stage larvae were Yen (1938) found only 21 of 170 mosquitoes of this species alive 10 days after taking an Infective blood meal. 10 (472) had developing larvae. Of these 21, only Retardation of development was also reported by Yen and Infective larvae were found In 3 mosquitoes but never In the head. Encapsulation was common but Yen did not observe encapsulated third stage larvae. Yen reported that this mosquito Is a likely potential vector of dog heartworm in Minnesota but the results of the present study Indicate that the local Michigan strain of A. stimulans Is not a natural vector of dog heartworm. Results with Mansonia perturbans Mansonla perturbans proved to be an unacceptable host for I). Immitis larvae. Second stage larvae were seen In only one Individual and Infective larvae were never seen. Microfilariae did not become established In an average of 80.22 of the individuals dissected and encapsulation of larvae was always observed In Individuals In which microfilariae did reach the Malpighian tubules. results with this species. Yen (1938) had similar He found only 1 of 8 mosquitoes taking an infective blood meal had microfilariae In the Malpighian tubules. microfilariae were dead and one was encapsulated. All Results indicate that the local Michigan strain of M. perturbans Is not a vector of dog heart­ worm under natural conditions. Results with Aedes vexens Aedes vexana proved to be an acceptable hoot for D . Immitis larvae. Under laboratory conditions infective larvae reached the head and 77 mouthparts of Infected mosquitoes In as little as 12 days. Mortality of infected mosquitoes was very high; only 22 of 481 individuals (4.6Z) in 4 trials, lived long enough (12 days) for infective larvae to reach the labium. Additionally, microfilariae did not become established in an average of 34% of the mosquitoes of this species taking an Infective blood meal and encapsulation of larvae was common although not every larva was encapsulated in any individual mosquito. Hu (1931) wrote that 80.3% of A. vexans taking an Infective blood meal became Infected. He felt that relatively few larvae (10.6 larvae/mosqulto) became established although one mosquito had 33 developing larvae. Similarly, Jankowski and Blckley (1976) reported 78.9% of 78 individuals (held at 80° F) became infected after an Infective blood meal with an Infection rate of 10.6 larvae/infected individual. Yen (1938) found that 25 of 129 (19.3%) lived long enough for D,. lmmltla to complete development to the infective stage and all of these 25 mosquitoes harbored Infective larvae. Infective larvae reached the labium in as little as 13 days and one specimen contained 76 infective larvae. common. Encapsulation of larvae was Yen considered this mosquito to be a likely vector. Bemrlck and Sandholm (1966) found 72.4% of A. vexans Infected after taking an infective blood meal and 84.6% of these 113 mosquitoes harbored larvae sfter 16-18 days. Over half of these were carrying infective larvae in the head and body cavity. these previous studies. The present study confirmed the results of A. vexans Is a suitable host for JD. immitis larvae and is most likely involved in the natural maintenance of this parasite. 78 Results with Anopheles quadrlmaculatus Laboratory experiments during the present study shoved Anopheles quadrlmaculatus to be an excellent host for dog heartworm larvae. Individuals taking an Infective blood meal became Infected. All Infective larvae reached the head and mouthparts of this mosquito In 13 post­ prandial days. Mortality up to this point of development for 5 trials (78/83 mosquitoes) was 94% indicating that A. quadrlmaculatus Is a better host for I). Immitis larvae than Aedes vexans» especially considering that Anopheles quadrlmaculatus was fed directly on an Infected dog and thus carried a heavier parasite load. Kutz (1972) noted a mortality of 75.3% after 13 postprandial days in a laboratory colony maintained under similar conditions. Phillips (1939) reported 100% of 90 mosquitoes Infected after taking an Infective blood meal. found In 72 of these 11-18 days later. sacrificed. Infective larvae were The remaining 18 died or were He found an average of 40 larvae per mosquito. Kartman (1953b) found all of 210 A. quadrImaculatus infected after taking an Infective blood meal. Infective larvae reached the labium In as little as 14 postprandial days. Kartman did find a negligible amount of encapsulated larvae (Microfilariae, 0.09%; first stage larvae, 0.2%) and some degenerate first and second stage larvae but A. quadrlmaculatus still proved to a very acceptable Intermediate host for I). 1mn1tls. Similarly, Keegan et, al. (1968) reported successful development of D. Immitis in A. quadrlmaculatus. They worked with fewer Individuals and found 2 out of 11 Individuals to harbor infective larvae after 12-18 days. Assuming that A. quadrimacula tus is not limited by Its longevity, this species appears to be an efficient host for D. immitis and Is most 79 likely the species most important in the natural maintenance of this infection in the Lansing, Michigan area. Results with Culex pipiens Laboratory studies showed that Culex pipiens is a possible vector of dog heartworm in nature. Development of larvae in this mosquito did not appear to be retarded and no encapsulation was observed. It is not an efficient host, however, because microfilariae did not become established in the Malpighian tubules in 87.5X of 72 mosquitoes dissected. Never was more than a single larva, at any stage, observed in any individual. Only one Infective larva was seen in the proboscis of one mosquito during all 3 of the developmental trials. Similarly, Hu (1931) found only 27.42 of 182 mosquitoes infected after taking an Infective blood-meal but found larvae able to complete development to the infective stage. Kartman's (1953b) infectlvlty results were similar to these but he found Infective larvae in the proboscis of <2 . pipiens in as little as 10 days after the infective blood meal. Because £. pipiens is not an efficient host and because it normally feeds on birds, it is not likely that this species plays an Important role in the natural maintenance of D_. Immitis Infections. Apparently however, assuming its longevity is adequate, it may serve as a vector of dog heartworm under natural conditions. During the course of this investigation, two phenomena occurred which have a bearing on the suitability of a mosquito as a vector of heartworm: encapsulation of larvae and elimination of microfilariae before they become established in the Malpighian tubules. Encapsulation was observed in Aedes stimulans. A. vexans and Manaonla perturbans. 80 Kartman (1953b) found conslatant encapsulation of I), immitis microfilariae in Aedea aegypti but felt this had little bearing on the ability of this mosquito to act as a vector of dog heartworm because only 12Z of the microfilariae were encapsulated. In the present study• encapsulation prevented Aedes stimulans, A. vexans and Mansonia perturbans from acting as an efficient host for D. immitis larvae. Complete loss of larvae from mosquitoes which took an infective blood meal, was noted in Aedes stimulans, A. vexans and Mansonia perturbans and Culex pipiens in which this was observed in an average of 84.2, 34.8, 80.2, and 90.5% of these species, respectively. Kartman (1953b) observed this phenomena in Culex pipiens and £. quinquefasciatus and Yen (1938) reported it in Aedes trivittatua. It is not known if digestive enzymes work against dead microfilariae killed by another substance or whether digestive enzymes act directly on living microfilariae. In the present study, inability of larvae to reach the Malpighian tubules proved to be an important factor in preventing Aedes stimulans and Mansonia perturbans and limiting the ability of Culex pipiens to act as an efficient host of dog heartworm. Kartman (1953b), among others have shown that, different geographical strains of a mosquito species may show variation in their efficiency to act as a host for I), immitis larvae. In the present study development of D. immitis larvae in Michigan strains of mosquitoes were compared with the results reported with other strains in the United States. Because the results of the present study so closely parallel the results of these previous studies, it might be suggested that, at least in the continental United States, a species shows little geographic variation in its ability to support development of D. itis larvae. 81 It must be understood* however* that these comparisons are difficult to make. In the past* some authors have considered a species a suitable host If development appeared normal or If Infective larvae were observed. Realistically* development cannot be considered complete until Infective larvae migrate to the labium of the mosquito. Additionally* techniques among authors vary. One Important difference among various studies is the parasite load received by the mosquito at the time of the Infective blood meal. Vlllavaso and Steelman (1970) have shown that there is a direct correlation between mortality and parasite load. Seeley and Blckley (1974) reported development of D* immitis larvae was complete only In one of three United States strains of Culex sallnariua. It remains then* that host efficiency must be determined locally to determine the vector potential of any mosquito species. Transmission Trials Complete observations of the transmission trials are given In Appendices HH-JJ. Aedes stimulans was allowed to feed on experimental dog MW 75. Of 57 mosquitoes known to have taken an Infective blood meal 15 survived 16 postprandial days when transmission attempts began. All 15 mosquitoes fed on the clean dog during the first two transmission attempts. 248 days after the final transmission attempt a necropsy was performed. This dog was not Infected with I), immitis. These results are to be expected because developmental trials with a Michigan strain of 82 A. stimulans Indicated that thin speclea la not a aultable host for dog heartworm larvae. Aedea vexana was allowed to feed on experimental dog ER 54. Eighteen of 481 mosquitoes known to have taken an Infective blood meal survived at least 12 postprandial days when transmission attempts began. Only one of these mosquitoes Is known to have fed on the clean dog. This mosquito died several days later. Dissection Indicated that this Individual was not infected with 1). immitis. A necropsy was performed on dog ER 54 173 days after the final transmission attempt. was not Infected with dog heartworm. This dog Although A_. vexans appears to be a suitable host for I). Immitis, this species Is difficult to work with In the laboratory. While In the confines of the cages used in this study it did not readily feed on dogs. breed In small cages. Likewise, A. vexans does not readily It may be that the behavior of this mosquito Is altered In typical mosquito rearing cages. It cannot be concluded that A. vexans is an unsuitable vector of D. immitis. Further experimental evidence is required to access the importance of this mosquito as a vector of D. Immitis In Michigan. Anopheles quadrlmaculatus was allowed to feed on experimental dog HT 05. Seven of 83 mosquitoes, known to have taken an Infective blood meal, survived 13 postprandial days when transmission attempts began. two occasions, several mosquitoes landed on the experimental dog and engaged In probing activity. taken any blood. These mosquitoes did not appear to have Developmental trials Indicated that all A. quadrlmaculatus taking an Infective blood meal became Infected. McGreevy et^ al^ (1974) noted that heavily Infected mosquitoes had trouble feeding because the labium, filled with Infective larvae, would On 83 not bend. Similarly, during Newton's transmission experiments, mosquitoes had trouble feeding and only 8 of SS were known to have taken any blood, yet transmission was accomplished. In spite of the failure of these transmission attempts, A^. quadrlmaculatus must be considered an excellent potential vector of I). Immitis in Michigan. GENERAL DISCUSSION Barnett (1960) has outlined 4 criteria for the incrimination of an arthropod as a vector of disease. These were generalized by James and Harwood (1969) as follows: "1) Demonstration of feeding or other effective contact with the host under natural conditions. 2) A convincing biological association In time and/or space of the suspected arthropod species and occurrence of clinical or subclinical infection In the host. 3) Repeated demonstration that the arthropod under natural conditions, harbors the Infectious agent in the Infective stage. 4) Transmission of the agent under controlled conditions." The present study attempted to meet 3 of these criteria. Criterion 2 Is very difficult to dastonatrate with a disease such as dog heartworm primarily because of the present method of screening for the disease which Is through various kinds of examination for microfilaremia. Generally, 8-9 months pass before microfilariae are produced and It may not always be possible for a dog owner to recall where a dog has been over an extended period of time. Second, unless annual or biannual blood screening Is performed, an asymptomatic Infection may go undetected for several years and It may be impossible to estimate the 84 85 time the infection wee contracted* Third, unless mosquito surveys were ongoing during the time of suspected transmission, one can only speculate on which mosquito species may have been present at the time of transmission. As mentioned earlier, 24 suspected mosquito vectors of I). Immitis are known to occur in Michigan. Their importance will be discussed in relation to 3 of Barnett's criteria. Aedes Aedes spp* typically prefer mammalian hosts (Tempells, 1975). It will be assumed that all Aedes discussed here would take a blood-meal from an available dog and satisfy Barnett's criterion 1. Aedes atropalpus Coquillett was studied by Keegan et al^. (1968). In their report this species readily fed on dogs and 74.3X of 31 mosquitoes examined harbored infective larvae. Infective larvae have not been Isolated from fleld-captured specimens nor are their any reports of transmission of heartworm involving this species. It was not collected in the Lansing, Michigan area and for this reason It is not likely to be a vector In this area. Figure 19 shows the reported distribution of this mosquito in Michigan. Because it has been shown to be capable of supporting complete development of ID. immitis it may have some importance as a vector in the upper and northern half of the lower pennlnsula of Michigan. Aedes canadensis (Theobald) was seldom trapped at Sites 1-5 (Table 1 acid 2). Crans (personal communication) using CDC miniature light traps found this species to be very abundant in New Jersey. Because of the high incidence (about 2X) of these mosquitoes harboring presumed, 86 Figs. 19-24. Reported distribution of Aedes atropalpus, A. canadensis, A. cinereus. A. fitchii, A. excrucians and A. junctor, respectively, in Michigan®. £ Distribution of mosquitoes illustrated in Figures 19-42 was compiled from the following reports: Irwin (1941, 1942), Newson and McGroarty (personal communication), Obrecht (1949), Sabrosky (1946), and Zavortlnk (1973). 87 trh^rawf--i “V "!_• L -1TJ-rJ*J-ciLr— 1 , ^ 1 pnrt f" *7. >— y + '*2* |i I .?r^i-vL* |4( |w4U| .'«t | « 1* L*. I.•_I• ;_ S*7 ri*4 T - l ^ - s y . rlrL^rl L7 *_iT-_ji-r’ •_!-V-rl"p^ • *• ■• i• >• V _ !• • i i*t •• : |L x i |— • |— m iv^-vixrcv r *5* i _ t- f?*" , ’•■ --*tti . « . jZIzjzjx&s- srirteLTr-; rrKLTrr- irnTir-riTi* mmm . im I t i«« i * ,,-i*y,*■--ji ••■*“ -# 3-Ti-Eair±^ [ i. • i • _ ! _ • > • i f i 88 D. immitis. Infective larvae he considered this species to be the primary vector of JD. JLmmitis in this state. Hu (1931) and Yen (1938) both showed that A. canadensis was able to support the development of I), immitis to the Infective stage. Yen found infective larvae in the proboscis of this species 13 days after the infective blood-meal. Jankowski and Bickley (1976) found this species to feed readily on dogs during the course of their experiments. Additionally* Morris and De Foliart (1971) estimated that 19.12 of female A. canadensis searched for more than one blood meal Indicating that their life-span may be adequate to allow this species to be a natural vector of D. immitis. In Michigan* this species has been reported from nearly every county (Figure 20). A. canadensis must be considered a good potential vector of dog heartworm in Michigan, primarily in Its woodland habitat where females generally remain (Jankowaki and Bickley, 1976). Aedea cinereua Melgen was not positively identified from trap collections until September, 1975. The increased incidence of this mosquito in the field at this time (Figure 17) demonstrate its potential to occur in largenumbers and Morris and De Foliart found 36.82 of a Wisconsin population to seek more than one blood*-meal indicating some potential for an extended life-span. Phillips (1939), under laboratory conditions, found D. immitis developed to the infective stage in this mosquito in 73 of 120 mosquitoes and infective larvae were able to reach the proboscis 12 days after the Infective blood meal. Yen (1938) worked with fewer mosquitoes and observed some encapsulation of developing larvae. Maturation to the infective stage did occur and he considered A. cinereua to be highly susceptible to D. immitis Infections. A. cinereua. because of its widespread distribution and apparent 89 suitability ss sn Intermadlate host In Michigan (Figure 21), must be considered a potential vector of dog heartworm, in this state. Aedea excrucians (Walker) was not collected in the Lansing, Michigan area during this study. Phillips (1939) found Infective larvae of I), immitis in the labium of this mosquito 15 days after it took an infective blood-meal. Because of its widespread distribution (Figure 23) A. excrucians has potential to be a vector of dog heartworm in Michigan. However its importance as a vector is probably very minor, at least in the Lansing area where its density is apparently very low. Aedes fitchii (Felt and Young) as shown in Figure 6, was collected from early June through Mid-August. dogs during this study. It was readily attracted to the Carpenter and Nielson (1965) found A. fitchii to go through as many as 4 gonotrophic cycles and live as long as 53 days in nature. Its longevity would make this species an ideal host for £* immitis. Bemrlck and Sandholm (1966) found A. fitchii to support larval development of dog heartworm to the infective stage but these larvae were never found outside the Malpighian tubules. consider development complete in this species. They did not Until further evidence is obtained, A. fitchii should not be considered an important vector of dog heartworm in Michigan, in spite of its widespread distribution (Figure 22). Aedes punctor (Kirby) was not collected in the Lansing, Michigan area. In Europe, Roubaud 2nd Collas-Belcour (1937) found Infective larvae of D . *—■»•*«•*» were able to migrate to the labium of this mosquito. No further information has been reported about this mosquito and therefore it must be considered a potential vector of dog heartworm where it occurs in Michigan (Figure 24). 90 Only one specimen of Aedes aollicitana (Walker) was collected during the course of this project but It has now been collected In 5 Michigan counties (Figure 25). Hu (1931) found this salt marsh mosquito able to support the development of I). Immitis to the Infective stage. This was confirmed by Summers (1943) and Keegan et^ al. (1968) who observed third stage larvae In the head of this mosquito. It seems unlikely that this mosquito has much Importance In the Lansing area, but it has become quite a nuisance In Marysville, Michigan (H. D. Newson and D. L. McGroarty, Michigan State University, personal communication) and it may be an Important vector of dog heartworm In this and other areas where populations of this species are abundant. Aedes stlcticus (Melgen) which Is distributed throughout Michigan (Figure 26). It may occur locally In large numbers (Figure 12). Bemrlck and Sandholm (1966) did not consider larval development of D.. immitis to be complete in this species because infective larvae were not observed outside the Malpighian tubules. It appears unlikely then that this species Is a vector of dog heartworm in Michigan. Aedes stlmulans (Walker) has a widespread distribution In Michigan (Figure 27). It does not appear to be a vector of dog heartworm because In the present study microfilariae did not reach the Malpighian tubules of all individuals taking an infective blood meal. Furthermore those larvae which did reach the Malpighian tubules were subject to encapsulation and retardation of development In this species. Phillips (1939) first experimented with the tree-hole breeding mosquito Aedes trlserlatus (Say). His data Indicates that this species Is an excellent host for JD. immitis. Infective larvae migrated to the proboscis of this species In as little as 9 days after the Infective 91 Figs. 25-30. Reported distribution of Aedes sollicitans, A. stlctlcus. A. stlmulsns. A. triseriatus. A. trivlttatus, and vexans, respectively, in Michigan. lAKt » m - 10 3 i 93 blood meal and with remarkably low mortality to the mosquitoes. Keegan et si. (1968) found this species ideal for laboratory studies of heartworm especially since it readily fed on dogs. Intermlll (1973) also found that A. triseriatus readily fed on dogs but noted that this mosquito was not generally found in large numbers in the field. He reported no histological damage to the Malpighian tubules from the migration of larvae but did observe some encapsulation in those excretory organs. Infective larvae were recovered from the labium in as little as 13 days after the Infective blood meal. Similarly Kaska (personal comsunlcation) obtained Infective larvae in the labium of a Michigan strain of A. triseriatus in only 12 days development time and also noted low mosquito mortality. At least under laboratory conditions A. triseriatus appears to be an ideal vector of D. immitis. Field studies by Morris and DeFollart (1971) Indicate that 35.4Z of the females of this species seek more than one blood meal. It may be that this species has adequate longevity to support development of I), immitis under natural conditions. Trap collections indicate that this species is only locally abundant (Tables 1 and 2) although it is widely distributed in Michigan (Figure 28). At Site 5 (Table 2) this species was the fifth most abundant mosquito collected in CDC miniature light traps and was collected consistently throughout the season (Figure 13). A. triseriatus appears to be an excellent host and potential vector of D,. immitis. Because of its local distribution it may have only secondary importance in the natural maintenance of this disease. As shown in Figure 14, Aedea trivittatus (Coqulllett) can, at times, be an abundant peat. Occasionally it was collected in the dog- baited traps used in this study (Table 1). Morris and DeFollart (1971) 94 eat1m ted that 39.9% of the fesmles In e Wisconsin population sought more then one blood seal. This M y Indicate sufficient longevity to support developMnt of heartworm under natural conditions. Ten (1938) found that none of 16 A. trlvittatue feeding on an Infected dog harbored infective larvae and concluded that this species was entirely refractory as a host for I). 1— itls. On the other hand, Christensen and Andrews (1976) concluded that A. trlvittatue is the principal vector of D. i l l tie In central Iowa. on too little data. Yen (1938) M y have based his conclusions Although Crlstensen and Andrews (1976) collected for only a two week period, their finding of Infective larvae, possibly D . i— itis, Indicates that this mosquito must be considered to have at least secondary Importance as a potential vector of dog heartworm In Michigan. It has been reported from M n y parts of the state (Figure 29). Aedes vexans (Melgen) was, by far, the mosquito collected most frequently during this study. Larvae, presuMbly D. immitis, were extracted from pooled mosquitoes on 3 occasions. In Maryland, Bickley et al. (1976) also Isolated possible D. Immitis larvae from fieldcaptured specimens and Bemrlck and Sandholm (1966) reported 5 isolations of Dirofliarla larvae from field-captured A. vexans. As discussed earlier, laboratory studies indicate that this species is a good host lor I), immitis larvae. Michigan (Figure 30). A. vexans Is abundant and present throughout It is among the best potential vectors of dog heartworm in this state. 95 Anopheles Anophelw spp. typically prefer m e n u 11an hosts (Tempelis, 1975). It will be assumed that all Anopheles species discussed would take a blood-meal from an available dog and satisfy Barnett's criterion 1. Anopheles earlei Vargas was captured only at Site 5 (Table 2) during this study and is reported from only 4 Michigan counties (Figure 31). Although Bemrlck and Sandholm reported third stage larvae of D . immitis in this mosquito, migration to the labium has not yet been demonstrated. For this reason A. earlei must be considered, at best, to have only minor Importance as a potential vector of dog heartworm and Its ability to support complete development of I), immitis larvae must still be proven. Anopheles punctipennis (Say) was captured In low numbers (Table 2) at all sites In the Lansing, Michigan area. This species, however, is known from nearly every county in Michigan (Figure 32). Hu (1931) found this species to tolerate high numbers of developing larvae. Yen (1938) agreed with Hu in finding 100Z infectivity of mosquitoes taking an Infective blood meal. postprandial days. Yen found larvae in the labium in as little as 12 Phillips (1939) likewise found this species an excellent host for I), immitis although he reported some encapsulation. Bickley at al. (1976) have Isolated fllarld larvae from field-captured specimens of A. punctipennis. This species must be considered a potential vector of dog heartworm in Michigan but because of its low incidence, at least in the Lansing, Michigan area, probably has only minor importance. Anopheles quadrimaculatus Say was collected at all sites In the study area and was commonly collected in the dog-baited traps (Tables 1 96 Figa. 31-36. Reported distribution of Anopheles earlei. A. punctipennis. A. quadrimaculatua. A. walker1, Culex piplens. and C. restuans. respectively, In Michigan. 97 ® rr; ;•/ i v^rrFrj hi 3-rit^oVr^ -iTjrTrfev 98 and 2). At Site 1 fully engorged females were often observed on the walls of the kennels. This species is readily attracted to dogs and is willing to enter buildings to obtain a blood meal. Laboratory studies as well as the finding of fllarld larvae in field-captured specimens have already been discussed. These findings along with the widespread distribution of this species in Michigan (Figure 33) supports the hypothesis that this species plays an Important role in the natural maintenance of dog heartworm infections. Anopheles walker! Theobald was collected at all sites in the Lansing* Michigan area (Tables 1 and 2). This species was collected more frequently than A. quadrimaculatus in one of the dog-baited traps at Site 1, the dog-baited trap at Site 5 and in the CDC miniature light traps set at all of the sites. field-captured specimens. No I), immitis larvae were isolated from Several unsuccessful attempts were made to colonize this mosquito in the laboratory. Bemrlck and Sandholm (1966) showed A. walker! capable of supporting complete development of D. immitis larvae in the laboratory. abundant in the Lansing area. Michigan (Figure 34). This mosquito appears to be very It is known from nearly every county in A. walker! should be considered a primary potential vector of dog heartworm in Michigan. Culex The feeding habits of Culex species are more varied and will be discussed individually. Culex pipiens Linnaeus, present throughout Michigan (Figure 35) is generally very abundant. Laboratory studies discussed earlier have shown it to be* an inefficient host for D, immitis larvae. Additionally, 99 Tempelis (1975) reported that It feeds mainly on birds. Thus* (I. plplens Is not likely to be an Important vector of dog heartworm In Michigan but it must still be considered a potential vector of I). immitis. Culex restuans Theobald occurs throughout Michigan (Figure 36) but was rarely collected in the study area (Table 2). Bemrlck and Sandholm (1966) concluded that this species was a poor host for dog heartworm but they did observe infective larvae in the head of one mosquito. Because complete development is possible, C. restuans must be considered a potential vector of I), immitis even though this mosquito feeds primarily on birds (Tempelis, 1975). Likewise, Culex salinarius, known from scattered areas in Michigan (Figure 37) was rarely collected during this study. Reports by Crans (1973) and Tempelis (1975) Indicate that this species has a wide variety of hosts. Hu (1931) observed only partial development of D. immitis larvae in this species and Summers (1943) did not observe development beyond the sausage stage and reported some encapsulation of larvae. Seeley and Bickley (1974), however, reported complete development of D. immitis in one of three United States* strains of C,. salinarius. Bickley et al. (1976) found filarid nematodes believed to be I), immitis in field-captured £. salinarius. Until further information is obtained, this mosquito must be considered to have at least minor importance as a potential vector of dog heartworm in Michigan. Culex tarsalis Coqulllett was collected on only one occasion at Site 2 (Table 2) and to date has been reported from only a few counties in Michigan (Figure 38) • Yen (1938) reported that most I), immitis 100 Figs. 37-42. Reported distribution of Culex salinarius. jC. tarsalia. <2. terrltans. Culiseta inornata, Mansonia per turbans, and Psorophora ferox. respectively, in Michigan. 101 M 1t j~-_r~rj“ i— -t—-1~*—?L-,r—;j |« m Zt ^ufM ! ^ — J— rJ h-— r — 1 r ^ L j r g L>i — j--t^±^hz “— .1“ ] (^T* *-4- K-r I J _l < _1Jr*L *r1 — »i“i ~ r1 ■ i — Xl r*—“* ! ““ ; ~-r 1 •—•■•r -<* I |— —t ‘ t !.Ji*_}_•_,L^rr?,^'3' *» 1 !•« M | IH M |MI J t1 .m«ij **•« i i m I i grraiyr?:? • I.TTS7 “ .— 102 larvae did not become established In the Malpighian tubules of this mosquito. Never was more than one Infective larvae seen In the labium of a mosquito at any one time. similar to Yen. Bemrlck and Sandholm (1966) had results In their study 9 of 94 mosquitoes feeding on an infected dog retained I). Immitis larvae and all of these were found to have infective larvae in their heads. Tempelis (1965) found that £. tarsalis fed primarily on birds but more readily fed on mammals during the summer. C.. tarsalis must be considered a minor potential vector of dog heartworm In Michigan. Culex territans Walker was trapped on only one occasion at Site 3-B (Table 2) and more commonly at Site 5 (Table 1 and 2). Although Summers (1943) noted some encapsulation of developing larvae in this species, Hu (1931) and Yen (1938) as well as Summers did report complete development of D. inmltis to the infective stage. £. territans Is known from scattered areas of Michigan (Figure 39) and feeds primarily on amphibians (Crans, 1970). It must be considered at least a minor potential vector of dog heartworm in this state. Culiseta Culiseta inornate (Wllllston) was captured only at Site 3-A Table 2) and is known only from a few counties In Michigan (Figure 40). This species Is thought to prefer larger mammals as a blood meal source (Edman et_ al., 1972). Yen (1938) noted encapsulation and arrested development of D. Immitis larvae In this mosquito. Keegan et. aT. (1968) reported that D. immitis larvae did not develop in _C. inornate. It 103 seems very doubtful that this mosquito Is a vector of dog heartworm In Michigan. Mansonla Mansonla perturbans (Walker) was collected very frequently In the study area (Tables 1 and 2) and has been reported throughout Michigan (Figure 41). It was captured more than any other mosquito In dog-baited traps at Site 1 (Table 1). laboratory studies. For these reasons It was selected for Results of this study (discussed earlier), In agreement with Yen (1938) have shown that this species is an unacceptable host for I), immitis larvae. It cannot be considered a vector of dog heartworm In Michigan. Psorophora Psorophora ferox (Humboldt) was captured only at Site 5 and then only one specimen was trapped (Table 2). This species has been reported from only three Michigan counties (Figure 42) Edman (1971) reports that It prefers mammalian hosts as a blood-meal source. In 1954 Steuben (Steuben* 1954) reported the recovery of Infective larvae from this mosquito but details of his findings are Incomplete. Because of this report P,. ferox must be considered a potential vector of dog heartworm In Michigan but its apparent low density Indicates that at best it has only minor Importance as a potential vector In this state. SUMMARY AND CONCLUSIONS Mosquitoes were collected In dog-baited and CDC miniature light traps at 7 sites in the Lansing, Michigan area. These traps worked well together to determine which mosquitoes were attracted to dogs in the study area and to indicate their local abundance. Trapping results showed that Aedes cinereus, A. fitchii, A. stictlcus, A. stlmulans. A. triseriatus. A. vexans. Anopheles quadrimaculatus. A. walker!, Culex piplens. and Mansonla perturbans were the most abundant species attracted to dogs in the study area. Mosquitoes were brought to the laboratory to be identified and a pooling technique was used to isolate filarid larvae from fieldcaptured mosquitoes. Suspected D. immitis larvae were extracted from field-captured specimens of Aedes vexans, Anopheles quadrimaculatus and Culex pipiens. A hlstochemical stain developed by Chalifaux and Hunt (1971) was used to try to differentiate Dirofilaria immitis and I), tenuis. The results of the hlstochemical stain showed that D. immitis and I), tenuis could not be differentiated after being treated by this method. Thus, filarid larvae Isolated from field-captured mosquitoes could not be positively identified. Based on field collection results, Aedes stimulans. A. vexans. Anopheles quadrimaculatus, Culex pipiens, and Mansonla perturbans 105 were selected for £>. Immitis developmental studies in the laboratory. Aedes vexans, A. stimulans and Anopheles quadrimaculatus also were used in transmission attempts. 8. It was postulated that the ideal vector of dog heartworm, at least in the Lansing, Michigan area, should have a life-span of a minimum of 30 days under natural conditions. 9. Developmental studies showed Anopheles quadrimaculatus to be an excellent host for D. immitis larvae. All specimens taking an infective blood meal became infected and there was no observed encapsulation of developing larvae. Additionally, A_. quadrimaculatus seemed to tolerate a heavier parasite load than Aedes vexans. Aedes vexans was also an efficient host but some encapsulation was observed and not all individuals taking an Infective blood meal became infected. Culex pipiens supported development of I), immitis larvae but is an extremely inefficient host. Aedes stimulans and Manaonia perturbans did not support complete development of D. immitis larvae. 10. I), immitis developmental studies Indicate that the larvae Isolated from field-captured Culex pipiens were not D. immitis larvae. 11. Efficiency of a mosquito as a host for D . immitis larvae must be determined locally. 12. Twenty four potential mosquito vectors are known to occur in Michigan. Their suspected Importance as vectors of dog heartworm in this state are given in Table 7. Anopheles quadrimaculatus and Aedes vexans appear to be the mosquito species most likely involved in the natural maintenance of D. Immitis in Michigan. walker1 may be equally Important. Anopheles 106 Table 7 Hypothesized Importance of Various Mosquitoes as Vectors of Dog Heartworm in the Lansing. Michigan Area Species Collected During Study Importance as a Vector Primary SecondaryMinor Doubtful Aedes atropalpus canadensis cinereua fitchii excrucians punctor solllcltans sticticus stimulans triseriatus trlvlttatus vexans Anopheles earlei punctipennis quadrimaculatus walkeri + + + + + + + + + + + + + X X X X X X X X X X X X X X X X Culex pipiens restuans salinarius tarsalis territans + + + + + X X X X X Culiseta inornata + X Mansonla perturbans + X Psorphora ferox X APPENDICES APPENDIX A Mosquitoes Collected in a Dog-baited Trap at Site #1 In a Woodland Area, 1974 Species Month/Day 6/13 6/18 6/20 7/1 7/3 7/7 7/10 7/16 7/18 7/24 7/30 7/31 8/5 8/27 Aedes canadensis Total 1 a conounis couples'* 2 1 4 7 fitchii-stimulans 7 14 23 51 11 triseriatus vexans 1 16 11 34 Anopheles quadrimaculatus walker! Culex pipiens Mansonla perturbans TOTAL 8 13 12 38 29 41 40 184 1 5 17 51 22 42 44 48 21 22 66 40 42 2 423 11 36 55 55 22 53 58 51 26 36 105 78 87 44 717 Includes Aedes cinereua and excludes A. aurifer and A. sticticus. APPENDIX B Mosquitoes Collected in a Dog-baited Trap at Site #1 in an Open Area, 1974 Species Month/Day 6/4 6/18 6/20 7/1 7/3 7/10 7/16 7/18 7/24 7/30 7/31 8/5 8/26 8/27 Aedes canadensis communis complex3 2 - - 2 2 - - - - - - - - - fitchii-stimulans triseriatus 13 5 1 vexans 71 33 19 3 - - Anopheles quadrimaculatus Total - - 7 - - - - 1 - - 2 44 - - 4 1 9 - - 24 - - 8 - 10 1 - - 9 - 19 1 149 1 92 - 112 60 1 3 29 5 4 Culex pipiens - - - 5 1 22 6 18 2 20 11 16 29 28 168 Mansonla perturbans - 2 4 120 37 132 83 4 39 11 10 15 2 2 461 152 41 30 159 50 158 94 66 55 56 32 39 52 32 1,016 *Includes Aedes cinereua and excludes A. aurifer and A. stictlcus. - 11 - 4 valkeri Total 1 - - tfraroii c CDC Trtp Collections at Site It, 1975 Species *»th/Dsj 6/S 6/15 6/22 6/29 canadensis 1 lo— na1s coaplex* 6 fltefcli-stlaulans 27 7/6 7/13 7/20 7/27 8/3 8/10 8/17 8/24 8/31 9/7 9/14 9/21 9/28 10/5 1 37 3 3 44 1 flausscena atletleas 18 TOTAL 2 1 1 19 72 31 192 26 1 triseriatus trirlttstus 22 17 443 28 215 219 375 12 238 1.012 1,043 Anopheles punctipennis 2 10 10 10 uulksrl 5 5 Culex pipiens 12 quadriaaculatus 20 276 8 982 25 783 13,895 626 1 9 1,102 33 50 143 202 19,770 34 - 45 29 11 salinarius 23 127 55 21 1 - 5 227 8 7 1 tarsalis Mansonla perturbans 19 147 71 18 59 14 375 Orthopodouyls spp. 1 Psorophora clllata TOTAL 9 29 543 57 375 313 405 302 1,107 ^Includes Andes ctnsruus and excludes A. aurifer and A. stlctlcus. 1,105 231 303 807 15,133 706 53 202 216 21,896 AFTBDU D CDC Trap Collectloos at Slta #3 - A, 1973 Spaclaa 6/8 6/15 6/22 6/29 7/6 7/13 7/20 7/27 8/3 8/10 8/17 8/24 8/31 9/7 9/14 9/21 9/28 10/5 TOTAL Aadea caoadaaaia - - 2 ciaaraua - - - - - - - - - - _ - - 7 6 4 15 - 32 10 - 2 1 - - - - 3 1 - 1 - - - - - - 18 - - 1 - - - - - - - - - - 1 9 - - - 11 24 9 33 12 6 1 flaveteen* 2 4 35 8 aolllcltana - - - - - - - - - - - - 1 - - - - - 1 atictlcua 6 - 5 - - 1 1 - 1 3 - - 1 22 42 - 13 1 96 trlaerlatua - - - - - - - - 1 - 1 - - - - - - - 2 trlvlttatua - - - - 2 2 2 1 3 1 - - 8 228 42 - 38 - 327 vtxaaa 8 21 185 129 612 97 133 128 390 1.037 27 67 873 6,281 1,557 416 680 1 - - 1 1 2 1 - - 5 - - quadrlaaculatua - - - 1 3 5 2 - - 5 - walker1 - 20 23 34 22 13 - 19 3 26 1 Culax plpiana 3 2 4 7 23 57 28 14 34 7 1 coaaunla cooplas* doraalla fitcbii-atlaulaiie aallnarlua 49 3 2 1 1 1 1 - 9 37 63 3 I 63 25 9 6 - Culiaata Inornata - - 1 Manaoala parturbaaa - 1 11 54 57 302 TOTAL 88 64 12.705 1 18 - - 19 15 11 1 300 22 1 _ 1 293 _ - - - 15 1 8 33 14 3 201 702 192 170 6 3 3 171 438 1,088 *Iacludea Aadaa ciaaraua and txcludea A. ■urlfer and A. atictlcua • 1 30 79 a» _ _ 998 6,638 1,683 436 757 2 2 87 68 14.064 110 Anopbelaa puncclpcmiB 2 APPENDIX E CDC Trap Collections at Site #3 - 1, 1973 Species Month/Day 6/13 6/22 6/29 Aades caoadaaaia - i claereua - 8 coMtnls coaplex* - doraalla - - fltchll-stlaulans 6 61 7/6 7/13 7/20 7/27 8/3 8/10 8/17 8/26 8/31 - - j - — - 1 1 - - - - - - 105 27 - - - - - - - - - - - - 3 - 8 - 3 3 - - - — - 6 - - - 1 1 2 — — 1 3 5 - - - - 1 - - 2 trlaerlatua trlvlttatua - vexans 2 103 Anopheles punctlpenola 1 - 2 quadrlaaculatua — — aalkerl - 3 Culex plplena - 1 — _ _ 1 — _ 1 _ 2 _ _ 3 - - TOUL - - 3 18 - 38 - 1 - - 9 13 - - - - - - j 3 366 1 2 1 1 267 60 - 38 123 822 196 235 211 88 667 18 200 609 6,877 336 68 186 - I 1 12 1 - - — - - 6 16 - 57 28 25 — 3 3 - 1 5 3 - 1 - - 25 19 3 - — 7 - 6 1 6 3 — - 2 6 B 1 1 - - - - - - - - - - - - aallnarlus - - - I - - - - - - - 3 terrltans - - - - - - - - - - - Naasoola perturbena - TOTAL 7 - 18 10 11 265 880 260 277 3 1 232 103 ^Includes Aadea clnereua and excludes A. aurlfer and A. atictlcua. 2 668 - 21 211 216 - 6 6 12 - - 3 - - 2 2 7 - 2 2 - 1 - reatuana 188 3 - 10/3 2 - 5 - - flaveacens stlctlcus 9/7 9/16 9/21 9/28 - - - 6 1 - 1 66 137 10,676 - 36 8 3 55 6 180 1 7 - - - - - l - 2 - - - - 2 - 3 - - 53 683 7,199 629 71 258 170 11,682 APrnvu f Collect*4 U • Dot-baited Traj at Site #*, 1475 M k / m */2* */2l 7/1 7/3 caaadnata 7/1 7/10 7/13 7/17 7/2* 7/M 7/31 1/3 1/7 1/12 1/1* 1/19 0/21 t/2* 9/2 9/10 9/11 9/1* 9/10 9/23 9/25 10/2 TOEIL 1 fltchll-otl— laaa - trlaerlataa - 2 1 2 112 Ti~jt* 1Calcz flllna DartartaM TOTAL 0 0 0 2 1 2 5 9 5 CDC Trap Callaetloaa at tit* 14, 1973 Ipaclaa HuatL/Oay aarlfar caaaOaaala 4/10 «/19 4/24 4/14 7/1 7/3 7/4 7/10 7/15 7/17 7/22 7/24 7/29 7/31 4/3 4/7 4/12 4/14 4/19 4/2 - 2 1 23 20 7 4/24 4/14 9/2 9/4 9/9 9/11 9/16 9/14 9/23 9/23 10/3 10/7 TOTAL 3 13 12 14 2 - 1 4 - 4 elaacaaa 19 c o n a l i cnaplai* 4 4 2 4 5 3 3 - 2 Oaraalla - - - - - - - - - - - - - fltckli-atlaulaaa 43 129 109 119 44 44 17 4 5 7 4 - atictlcua 24 24 7 4 4 5 5 2 1 - - 3 1 trlaarlataa - 3 3 12 15 24 5 2 5 3 7 3 4 5 5 3 9 10 7 34 24 14 trlvlttataa - - 2 4 7 13 9 3 4 3 34 25 14 7 27 24 10 5 2 4 1 30 135 141 44 239 42 239 177 51 44 70 45 30 38 26 S3 50 17 40 28 114 2 - 3 12 3 3 1 - 1 1 1 1 2 - 4 1 2 4 - 15 3 - 1 Taadrlaaculatua 3 2 7 4 11 24 10 10 33 47 24 21 41 23 9 1 30 27 4 9 25 2 1 aalkarl - - - 3 4 1 1 - 1 - 113 1 1 I - Calas piplaaa 3 2 3 9 2 2 1 - 4 8 4 5 2 - 2 Aaaphalaa puactlpaaala raataaaa 6 2 - 3 - * - - * - - 1 - - 2 - - - - - - 104 2 39 - 50 - 3 393 2 4 9 1 7 1 4 11 - 3 5 7 - - - 117 9 13 - 5 5 2 - - - 234 120 1 1 0 13 54 123 73 2 44 401 275 635 454 249 27 42 423 1.431 244 7 2 4 2 4 - 4 - 64 6,044 1 65 - 2 1 - - - 394 3 1 - - - - 134 3 4 1 - 1 . 110 1 1 aaltaarlua 1 Cullaata tapatlana 1 Haaaoala parturbaaa 5 11 2 4 3 24 7 4 11 12 21 0 2 2 - - 1 1 1 4 5 Braanraaala aappharial - - 1 - - - * - - - - - - 4 - - 1 2 - - 1 231 340 211 437 174 432 234 45 123 153 274 109 105 40 143 44 43 100 43 TOTAL *lacla4aa AaOaa clatraaa aad o e M u A. — tad A. atictlcua. 3 1H 137 - _ _ * 130 471 1,975 393 294 745 404 - - 379 • » 32 1 * 14 133 4,453 113 «*— U 10 - Armon h Mosquitoes Collected In e Dog-beited Trap et Site #3, 1973 Species Month/Day 6/23 6/30 7/2 7/7 7/9 7/14 7/16 7/21 7/23 7/28 8/4 8/6 8/11 8/18 8/20 8/23 8/27 9/1 9/3 9/8 9/15 9/17 9/24 9/29 10/6 TOTAL 1 canadensis claereus - - conuals conplex* 2 - fltchl1-stlilans - stlctlcus - - - - - - - - - - trivlttatus - - - 2 3 3 - - - - - - - - 1 - - - - 6 7 16 3 2 welksrl 31 2 17 5 Culex plplens 1 terrltens - 1 - - - - - - - 1 - - - - - -30 2 - - 16 - - - 46 - - - - 11 9 - - 2 - 3 - - 1 vexsns Anopheles quadrlneculstus - 2 2 - - - 2 - - - - - - 1 - - - 2 - - - - - 5 10 1 11 - 1 1 1 - - - - 2 16 - - 1 - 8 1 - - - - - - 1 - 1 ..................... 5 0 - 1 1 5 - 1 16 - - - - - - - 4 - 7 - - 74 - - - - - - - - - - - - - si 2 - - - - - - - 11 33 - - - 131 - 2 - - - 4 - - - - - - - - - - - 3 - - - - - - - - - - - - 9 - - - 31 - 3 Cullaeta l^atlens Msssonls perturbsns TOTAL - - - 2 10 42 22 2 4 5 39 17 12 - - - - - 19 4 3 9 46 32 6 29 - - 1 0 includes Asdes claereus and excludes A. surlfer sad A. stlctlcus. 13 - - 0 - - - - 1 2 - - - - - - - - - - - - - 59 4 0 0 0 0 0 58 0 54 62 0 0 0 436 CK Tta*CallaetloaaatSlta13.1*73 IpatlM M U h r aarlfat cMtadi claaraaa cMala Mfln* 4/9 4/11 4/14 4/23 4/13 4/30 7/1 7/7 7/4 7/14 7/14 7/11 7/1) 7/14 7/30. 1 3 3 1 10 4 2 1 1 1 2 - - - - - - 1 2 42 14 4 7 1 4 u */l 9/3 */« »/13 9/17 ,/j* ,/n u/t w / 1 , miL 10 12 130 4/4 4/4 4/11 4/14 4/14 4/10 4/13 4/27 - 4 3 1 - 1 1 1 43 33 9 - 1 - 1 3 3 10 12 - 30 1,302 399 1 33 14 - - - - - 2 Oanalla fitchli-atiaalaaa 124 49 134 1)1 47 34 32 21 20 14 1 3 3 1 - 1 3 - - - - - - - - - - - 424 2 2 1 - 1 49) 240 127 - 4 4 .231 - - 1 3 2 4 47 12 7 - 2 4 14 4 - - - 2 It 43 30 4 9 trlaarlataa - - - 3 - 2 2 1 - - - - - - - - - 2 4 trlvlttAtwa - - - 2 2 2 3 1 3 - 1 - - 1 4 2) 2 12 11 14 120 112 44 2) 13 303 93 102 100 733 133 223 401 - - - 1 4 - 2 1 1 2 2 - - 2 2 1 - 3 3 - I 1 - 2 - 2 2 - - 3 3 3 2 3 4 * - - a»a4rlaacalatu 10 7 19 73 24 30 1 144 41 137 149 24 30 100 140 39 11 11 43 3 4 1 aalkarl 10 43 74 443 377 27 M l 1) 47 114 4 114 329 24 317 37 1 33 173 30 plfl«a 3 10 10 - 4 14 7 14 4 34 14 39 4 44 72 94 42 39 raataaaa - - - - - - - - - - - - - - - - - 1 2 - - - 1 2 2 4aofhalaa aarlal yacttyaaala 1U 131 30 444 331 144 U S 293 107 24 1)7 129 1.430 4 - 40 3 Ml - - M - - atlctlcaa 1 34 34 2.0 040 31) 230 323 2 I 1 - - - - 23 - 11 30 314 144 43 1 19 14 3 747 44 1,432 3,933 1,704 1,403 3,327 - 202 44 211 .417 - - - - - 1 - 1 - 2 314 1,044 * 139 11 43 - 11 27 3) 240 7 17 3 1 49 14 14 - 1 - - - - - Calaa ittittcw 37 9 27 M - - aaltaarlaa 34 - 2 4 4 - - - - - - 1 3 - - - 1 It 11 It 1 - tarrltaaa Callaata m l t M - 1 ala ■ria ) 11 M 43 40 IS) 21Q 44 104 71 14 99 1* 12 4 2 4 4 3 - 4 t I 3 4 OrtkopaOaaqrU 21 1 - 1 «W- 491 1.014 2 Paorefhora c 11 lata farm Uraaotaaala aaffharlal TOTAL 333 *Iarl«4ea Aa4aa claara— 233 494 444 394 311 403 1.403 34) 344 470 aa4 asela4aa A. aarltar aa4 atUtlcat. 79 749 2)4 1.701 1.2)1 249 421 1,122 319 44) 409 147 1.439 4,297 2,433 3,913 3,444 2 424 134 314 33,104 116 APPENDIX J Representative Study Area Weather Data - Capital City Airport Environmental Data Service Ashvllle, North Carolina May 1975 May 1974 Maximum Date Minimum Precipitation °P °F (Inches) 1 2 3 4 5 62 69 57 61 53 36 41 33 32 32 6 7 8 9 10 50 54 43 51 63 28 22 37 38 30 .09 11 12 13 14 15 66 63 50 75 62 46 45 36 48 45 16 17 18 19 20 70 68 66 70 75 46 52 46 60 43 21 22 23 24 25 84 73 72 61 63 53 62 54 46 41 26 27 28 29 30 65 62 66 73 76 39 35 42 59 60 31 73 50 Maximum °F Minimum °F Precipitation (Inches) 61 70 71 57 67 41 40 42 45 42 .01 T .04 .11 .01 63 70 72 74 77 43 39 39 40 37 T .38 .02 .02 .42 T 76 56 70 70 65 43 41 36 45 47 .03 .75 1.73 .18 .01 T 66 76 81 88 89 42 40 44 57 64 86 77 83 88 86 62 60 56 63 64 T .32 .39 77 76 77 79 79 64 52 44 57 60 .02 .81 T 69 55 T .06 .16 .29 T .12 00• T T .02 .21 117 APPENDIX K Representative Study Area Weather Data - Capital City Airport Environmental Data Service Ashvllle, North Carolina June 1974 Date Maximum °F Minimum °P June 1975 Precipitation (Inches) Maximum °F Minimum °F Precipitation (Inches) 72 70 72 74 76 45 42 42 50 59 .04 .88 .03 .07 .51 68 60 66 76 78 54 51 44 40 52 .27 .11 70 76 80 81 76 58 60 60 59 60 .50 .02 T .25 .50 49 46 48 59 54 .01 .16 .07 .31 .02 75 83 85 88 85 56 66 68 71 68 T .11 T .01 T 82 71 64 68 75 65 54 49 43 44 T 88 92 86 78 77 57 69 73 65 61 78 80 79 81 79 51 48 50 53 57 82 86 88 87 86 57 62 57 60 53 1 2 3 4 5 73 73 75 84 84 44 46 45 61 63 6 7 8 9 10 82 81 80 86 76 66 67 68 69 51 11 12 13 14 15 65 72 75 78 75 46 42 46 49 53 16 17 18 19 20 57 57 73 79 85 21 22 23 24 25 26 27 28 29 30 T .33 .25 .24 .38 .67 118 APPENDIX L Representative Study Area Weather Data - Capital City Airport Environmental Data Service Ashvllle, North Carolina July 1974 Maximum Minimum 1 2 3 4 5 86 90 92 85 76 52 67 71 64 51 6 7 8 9 83 88 93 94 82 50 54 62 67 61 80 84 94 98 84 53 43 55 72 60 81 87 91 91 78 49 55 73 63 51 23 24 25 83 68 76 84 81 38 61 54 54 65 26 27 28 29 30 87 89 92 82 80 62 54 50 58 55 31 81 52 11 12 13 14 15 16 17 18 19 20 21 22 Precipitation (Inches) Date 10 July 1975 .55 .44 .01 T T .12 T .07 .02 Maximum 'F Minimum 87 87 87 84 87 59 60 62 56 58 85 85 90 80 75 65 61 60 48 54 74 75 78 70 82 45 50 50 57 62 88 83 85 82 50 65 66 59 52 80 87 86 80 78 57 54 63 58 51 82 84 86 91 93 45 55 53 53 61 95 62 88 Precipitation (Inches) .58 T T .01 .65 .03 .78 .38 T .02 .01 .01 119 APPENDIX M Representative Study Area Weather Data - Capital City Airport Environmental Data Service Ashvllle» North Carolina August 1975 August 1974 Maximum °F °F 1 2 3 4 5 82 82 84 72 78 48 61 54 50 50 6 7 8 9 84 84 83 83 83 51 48 63 59 56 86 87 81 78 83 69 53 60 51 53 87 80 83 87 90 65 60 49 54 57 22 23 24 25 88 88 82 72 81 26 27 28 29 30 31 10 11 12 13 14 15 16 17 18 19 20 21 Precipitation (inches) Maximum °F Minimum °F Precipitation (Inches) .02 .55 T .01 97 79 84 87 74 65 68 67 59 58 .09 72 77 82 88 88 52 44 46 58 67 87 88 84 80 70 65 57 60 50 57 82 83 73 75 79 49 52 50 53 55 T .20 61 66 59 48 43 79 71 83 86 87 59 61 61 71 67 3.08 .83 .37 .14 T 92 80 74 77 81 61 56 46 47 53 60 53 54 67 64 .07 T 79 79 83 78 69 1.34 .35 73 44 .10 78 64 1.62 .03 1.22 .25 .40 CD .73 .39 • o Date Minimum T .06 T .55 T T T 120 APPENDIX N Representative Study Area Weather Data - Capital City Airport Environmental Data Service Ashvllle, North Carolina September 1975 September 1974 Date Maximum °F Minimum °F 1 2 3 4 5 70 57 66 67 72 46 45 40 35 36 6 7 8 9 10 74 75 80 82 81 38 40 50 55 61 11 12 13 14 15 87 81 69 64 72 67 68 49 38 43 16 17 18 19 20 71 80 68 64 61 35 44 42 43 42 21 22 23 24 25 64 52 59 64 67 41 31 28 45 36 26 27 28 29 30 82 78 75 65 49 38 53 62 41 41 Precipitation (Inches) Maximum °F Minimum °F Precipitation (Inches) T 75 76 69 72 64 63 55 53 55 53 71 75 65 64 77 49 44 42 37 43 77 60 59 65 68 51 36 35 31 46 69 71 67 71 65 55 45 52 51 48 49 41 37 43 45 .13 T T 58 60 64 63 53 43 35 34 36 48 T .51 .23 .76 T 57 67 70 70 70 .61 T .40 .04 T .02 .03 T .51 .48 .10 T T .01 .17 T T .25 121 APPENDIX O Representative Study Area Weather Data - Capital City Airport Environmental Data Service Ashvllle, North Carolina October 1975 Minimum °F 1 2 3 4 5 60 51 66 73 72 41 32 36 45 43 6 7 8 9 10 70 69 68 59 71 42 33 44 47 41 11 12 13 14 15 57 67 83 82 68 40 32 53 62 41 T 16 17 18 19 57 51 46 49 64 34 35 42 45 45 .08 .23 .07 23 24 25 70 73 75 78 71 43 36 55 53 33 .34 26 27 28 29 30 56 64 60 51 46 29 35 46 31 24 31 57 33 Date 20 21 22 Precipitation (inches) CM o• Maximum °F T .21 122 APPENDIX P Developmental Trial Aedea atlmulana Trial #1 Postprandial # Day Alive Observed Stage Development # Dead 0 2 57 55 2 - 5 7 32 28 23 4 L 8 10 12 25 21 20 3 4 1 L Lx - 14 16 4 - 15 16 15 14 1 1 17 18 9 6 5 3 L^i 19 5 1 - 20 21 22 23 24 25 26 5 4' 4 4 3 2 0 0 1 0 0 1 1 2 - Comments 1 mosquito with no larvae 3 mosquitoes with no larvae 1 mosquito with no larvae 1 mosquito with no larvae 4 mosquitoes with no larvae in M.T. 2 mosquitoes with no larvae; encapsulation of and all In one mosquito 1 mosquito with no larvae Encapsulation of L^ L, 1 mosquito with no larvae 1 mosquito with no larvae in M.T. 1 mosquitowith nolarvae; about 16 encapsulated L in one mosquito and 123 APPENDIX Q Developmental Trial Manaonla perturbans Trial #1 # Alive # Dead Observed Stage of Development 0 1 35 31 0 4 L1 2 23 3 4 7 22 21 16 8 1 8 14 9 13 10 11 6 4 17 0 1 5 2 1 Comments - 4 4 1 mosquito with no 7 2 4 - 2 mosquitoes with i 1 mosquito with no 124 APPENDIX R Developmental Trial Manaonia oerturbana Trial #2 Postprandial Day I Alive # Dead Obaerved Stage of Development Conmienta 0 54 3 20 34 4 16 4 Encapsulation of some Lt in 1 moaqulto, 5 2 14 Encapsulation (capsulation of oi some in 1 mosquito. 6 0 2 L 1 moaqulto with no larvae 1 moaqulto with complete encapsulation of all L^, 123 APPENDIX S Developmental Trial Mansonia perturbana Trial #3 0 3 6 # Alive # Dead 84 74 52 10 Observed Stage of Development Comments 22 4 8 1 mosquito with no larvae 5 mosquitoes with no larvae 9 48 40 31 9 7 mosquitoes with no larvae, 1 mosquito with encapsulation of L^. 10 23 11 20 8 3 12 11 9 13 14 5 0 6 5 7 8 1 mosquito with no larvae 4 mosquitoes with no larvae 2 mosquitoes with no larvae 2 mosquitoes with no larvae 126 APPENDIX T Developmental Trial Haneonia perturbans Trial #4 Postprandial Day # Alive I Dead 1 2 71 63 0 8 3 4 61 58 2 3 5 6 49 9 14 7 8 9 35 17 3 0 18 14 3 Observed Stage of Development Comments L1 L1 L1 L1 L1 L1 4- 4 L1 1 mosquito with no larvae 4 mosquitoes with no larvae 6 mosquitoes with no larvae 11 mosquitoes with no larvae 8 mosquitoes with no larvae 127 APPENDIX U Developmental Trial Aedea vexana Trial #1 Poatprandlal Day 1 # Alive # Dead AS 29 26 2A 20 0 2 3 A 5 6 7 19 19 8 9 19 19 0 10 17 2 11 12 16 1A 1 2 13 1A 11 8 3 3 15 7 1 16 A 3 17 18 19 3 2 1 0 0 2 16 3 2 A 1 Obaerved Stage of Development Comments L1 L1 L1 L1 1 moaqulto with no larvae 0 0 Ll> L2 - Ll ’ L2* L3 L2’ L3 V L3 - L3 1 moaqulto with encapaulatlon of L^, 1 moaqulto with L2 1 moaqulto with no larvae 1 mosquito with no larvae; 1 mosquito with partially encapsulated L- (2), and 3 L_ In head, 1 L. In abdomen and 8 L3 In M.T. 1 mosquito with 1 L_ in the head and 1 In the thorax; 1 mosquito with A L. in the Malpighian tubules. 1 moaqulto with 1 L, In the proboscis, 3 L. 1ft the head, 1 L. in the thorax and 1 partially encapsulated L In the Malpighian tubules. 2 mosquitoes with no larvae; 1 moaqulto with 3 L. In the head and 1 L. In the Malpighian tubules. 1 moaqulto with no larvae. 2 mosquitoes placed in cage with A. vexana from trial #3. 128 APPENDIX V Developmental Trial Aedea vexana Trial #2 Postprandial Day # Alive 0 14 # Dead Observed Stage of Development 1 12 2 Lx 2 3 4 3 L 2 1 9 1 1 5 6 1 1 0 0 7 0 1 1 Comments 129 APPENDIX W Developmental Trial Aedes vexana Trial #3 Postprandial Day # Alive # Dead 1 210 2 3 4 152 91 73 91 18 5 6 53 51 41 20 2 10 35 27 6 8 7 8 9 58 10 21 6 21/14 22/15 23/16 24/17 4 2 2 1 0 2 0 1 25/18 0 1 Observed Stage of Development Comments L1 L1 L1 L1 Ll* L2 V L3 1 mosquito with 13 L. in the Malpighian tubules 1 mosquito with 2 L. In the Malpighian tubules; 1 mosquito with 1 L. in the Malpighian tubules. 11 12 9 2 mosquitoes with no larvae; 1 * V L3 mosquito with 2 L- in the Malpighian tubulel and 2 encapsulated L^. 12 1 mosquito with no larvae; 1 3 9 L3 mosquito with 4 in the head 2 mosquitoes from Trial #1 placed in cage with the 3 mosquitoes from this trial for transmissioni trlala. 20/13 1 mosquito with encapsulated L^. 4 1 L1 V S l2 L3 1 mosquito with 1 L_ In the head and 3 L. in the thorax. 3 L2 130 APPENDIX X Developmental Trial Aedea vexana Trial #4 # Alive 1 2 3 4 212 167 149 141 # Dead Comments 45 18 8 16 L1 L1 19 13 L1 L1 6 125 106 93 7 65 28 8 9 51 35 14 16 10 20 15 11 12 8 12 5 7 13 14 15 16 4 1 2 2 0 2 0 2 5 Observed Stage of Development V L2 1 moaqulto with aome encapsulated L^. L2 Ll ’ L2 V L3 V L2 L3 L1 - 1 moaqulto with some encapsulated L^. 1 mosquito with 3 In the Malpighian tubulea. 1 moaqulto with no larvae; 1 moaqulto with 5 L_ In the head, 1 L_ In the hemocoel, and 1 In the Malpighian tubules. 1 moaqulto with encapsulated L^. 1 mosquito with no larvae but Malpighian tubulea were damaged. 131 APPENDIX Y Developmental Trial Anophelea guadrimaculatua Trial #1 # Alive # Dead 2 10 10 0 0 3 4 5 7 7 7 3 0 0 6 7 7 7 0 0 1 7 8 9 10 11 12 13 5 0 2 4 3 2 3 0 0 3 1 Observed Stage of Development Comments L1 L2 L2 L2 L3 Malpighian tubules packed with L2 and 1^. 1 mosquito with 6 L_ In the thorax and many L. In the Malpighian tubulel; 1 mosquito with 3 L_ In the proboscis, 7 L_ in the head, 11 L, In the tnorax. 132 APPENDIX Z Developmental Trial Anophelea guadrimaculatua Trial #2 1 2 3 4 5 6 # Alive f Dead 37 33 31 28 24 24 4 2 3 4 0 7 8 20 4 20 0 10 14 11 12 12 9 3 13 14 5 4 4 1 15 16 17 4 0 2 2 0 6 2 2 Obaerved Stage of Development Comments Lx L LL , L_ L^. 1 moaqulto with L. in the Malpighian tubulea. 1 mosquito with 9 in the Malpighian tubulei; 1 mosquito with L. and L. molting. 1 mosquito with L0 in the Malpighian tubules. L_ 1 mosquito with37 L_ removed from its body. 4 l . found in the proboscis, 4 L_ in the head, 15 L_ in the thprax, 2 L. in the abdomen, and 4 L_ ift the Malpighian tubules. L 1 mosquito with 4 L in the proboscis, and 5 t in the head. 3 133 APPENDIX AA Developmental Trial Anophelea quadrlmaculatiis Trial #3 Postprandial Day # Alive # Dead 1 15 0 2 4 10 8 5 2 6 1 7 7 1 0 8 9 1 1 0 0 10 0 Observed Stage of Development L Lx 1 Comments 134 APPENDIX BB Developmental Trial Anopheles guadrimaculatua Trial #4 Postprandial Day 1 2 4 6 7 8 9 10 # Alive # Dead 15 10 8 1 0 5 2 1 7 0 1 1 0 0 0 1 Observed Stage of Development L1 L1 Comments Advanced sausage larvae 135 APPENDIX CC Developmental Trial Anopheles quadrimaculatua Trial #5 Postprandial Day 0 1 2 3 4 5 # Alive # Dead 6 5 3 0 2 1 1 0 Observed Stage of Development snta 1 2 6 7 0 0 8 0 9 1 "sausage" larvae 136 APPENDIX DD Developmental Trial Culex pipiena Trial #1 Postprandial Day # Alive # Dead 1 10 0 4 6 6 4 0 6 6 0 5 6 7 8 9 10 11 6 4 4 4 Observed Stage of Development Comments 0 0 2 0 0 - All 4 mosquitoes dissected. No larvae were aeen and the Malpighian tubules were not damaged. 137 APPENDIX EE Development*1 Trial Culex pipiens Trial #2 # Alive # Dead Observed Stage of Development Comments 84 75 0 9 62 56 51 13 6 5 6 49 42 2 7 2 mosquitoes with no larvae. 7 39 3 2 mosquitoes with no larvae. 8 36 3 3 mosquitoes with no larvae. 9 11 35 35 33 1 0 2 12 30 3 13 14 29 26 1 3 Lj L 15 16 24 0 2 24 L_ 1 2 3 4 5 10 L. 1*2 6 mosquitoes with no larvae; 1 mosquito with 1 microfilaria in the Malpighian tubules. 1 mosquito with no larvae. 2 mosquitoes with no larvae. 1 mosquito with no larvae; 1 mosquito with 1 "sausage" larvae * 1 1<2 in Malpighian tubules. 1 mosquito with no larvae. 1 mosquito with no larvae. 1 1 1>2 in Malpighian tubules. mosquito with no larvae; 1 mosquito with 1 L_ in Malpighian tubules. 1 mosquito with no larvae. All mosquitoes sacrificed; 1 mosquito with 1 L_ in Malpighian tubules; 19 mosquitoes with no larvae. 138 APPENDIX FF Developmental Trial Culex pipiena Trial #3 # Alive # Dead 2 27 27 27 0 0 0 3 4 26 22 1 4 5 6 7 21 1 20 1 20 0 8 20 0 9 20 0 10 20 0 11 20 0 12 20 0 13 14 20 0 19 0 1 19 0 1 15 Observed Stage of Development L1 L 2 L. Conents 1 mosquito vith 1 early "sausage" larva, 3 mosquitoes with no larvae. All mosquitoes sacrificed; 16 mosquitoes vith no larvae; 2 mosquitoes with 1 L_ in the Malpighian tubules; 1 mosquito with 1 L_ in the proboscis. APPENDIX GG Concentration of Microfilaria at the Time of the Infective Blood Meal Aedea atinulans Sample # Microfliariae/20ul sample Trial # 1 - Feeding occurred on an Infected dog 1 2 3 4 5 6 7 8 9 10 294 210 192 135 179 163 225 216 122 270 _ X - 201.5 Coquillettidia perturbans Sample # Mlcrofilarlae/20ul sample Trial # 1 - Feeding occurred on an Infected dog 1 2 3 4 5 6 7 8 9 10 841 627 400 504 1050 855 1091 546 771 1387 __ X - 807.3 Trial # 2 - Feeding occurred on an Infected dog 1 1550 2 1111 3 4 1203 937 _ X - 1200 Trial # 3 - Feeding occurred on an infected dog 1 1171 2 1012 3 4 5 1238 1261 1204 _ X - 1177 140 APPENDIX GG CONT’D Coquillettidla perturbing Sample # 3 Microfilariae/20ul aaaple Trial # 4 - Feeding occurred through a membrane 1 262 2 239 3 233 4 260 5 263 _ X - 251.4 Aedes vexana Sample # Mlcrofilarlae/20ul sample Trial # 1 - Feeding occurred through a membrane 1 2 3 4 5 382 392 481 402 345 Trial # 2 - Feeding occurred on an Infected dog. determined. Microfilaremia was not Trial # 3 - Feeding occurred through a membrane 1 111 2 3 4 5 137 105 151 121 _ X - 125 Trial f 4 — Feeding occurred through a membrane 1 2 3 4 5 150 173 183 213 181 _ X - 180 Anopheles quadrlmaculatus Sample # Mlcrofliarlas/20ul sample Trial # 1 - Feeding occurred on an Infected dog 1 2 3 1372 1156 906 _ X - 1144.6 141 APPENDIX GG CONT'D Anopheles quadrlmeculatue Sample # Mlcrofliarlae/20ul sample Trial # 2 - Feeding occurred on an Infected dog 1 2 3 4 680 914 708 878 Trial # 3 - Feeding occurred on an Infected dog 1 888 2 880 3 881 Trial # 4 - Feeding occurred on an Infected dog 1 2 3 4 5 776 748 748 821 856 X - 795 X - 883 X - 790 Trial # 5 - Feeding occurred on an Infected dog 1 2 3 4 981 954 779 966 X - 920 Culex plplene Sample # Mlcrofllarlae/20ul sample Trial # 1 - Feeding occurred through a membrane 1 2 3 4 204 189 231 217 _ X - 210 Trial # 2 - Feeding occurred through a membrane 1 2 3 4 5 264 287 285 282 249 X - 273 142 APPENDIX GG CONT'D Sample # Culex plplens Mlcrofllarlae/20ul sample Trial # 3 - Feeding occurred through a membrane 1 2 3 4 351 307 272 289 X - 305 143 Appendix HH Transmission Trial Aadea stimulans Trial #1 Dog MW 75 Poatprandial Day 16 # Alive 17 15 14 18 19 9 6 20 21 5 22 5 4 23 24 4 4 25 26 3 2 27 0 Time Offered 5 00 P. M. 3:00 3 30 P. M. 3:15 - 3 45 P. M. 3:15 3 45 P. M. 3:15 3 45 P. M. 3:15 - 3 45 P. M. 4:00 - Moat mosquitoes fed - All remaining mosquitoes - - 3:15 3:15 3:00 3:15 Consents - - - 3 45 P. M. 3 45 P. M. 3 25 P. M. 3L45 P. M. No feeding trial - No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding 144 APPENDIX II Transmission Trial Aadas vflxana Trial #1 Dos ER 54 Postprandial Day # Alive 13 11 14 15 8 7 16 4 17 3 18 2 Time Offered 2:30 7:15 - CotMents 3:00 P. M. 7:30 P. M. 8:45 A. M. 8:30 11:15 - 11:30 3:20 - 3:35 6:15 - 6:45 8:45 - 9:15 A. M. P. M. P. M. A. M. 12:15 - 12:30 P. M. 3:15 - 3:35 P. M. 8:00 - 8:10 A. M. 5:05 - 5:15 P. M. 8:30 - 8:45 A. M. 5:45 - 5:50 P. M. 8:45 — 8:00 - 8:55 A. M. 8:15 P. M. One mosquito fed No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding No feeding 2 mosquitoes remaining alive from Trial combined with those from Trial 3 Trial 1 - 3 12/19 13/20 15/22 16/23 17/24 5 4 9:55 - 10:10 A. M. No feeding 9:45 - 10:05 A. M. 2 1 9:45 - 10:00 A. M. No feeding trial No feeding No feeding 0 Trial #4 14 15 16 2 2 0 8:25 - 8:35 A. M. No feeding trial No feeding 145 APPENDIX JJ Transmission Trials Anopheles quadrimaculatus Dog HT 05 Trial # 1 Postprandial Day # Alive 13 2 14 0 Time Of fered Comments 2:00 - 2:15 P. M. 7:45 - 8:00 P. M. No feeding No feeding Trial # 2 13 5 8:50 - 9:15 A. M. 14 4 1:50 - 2:15 P. M. 15 16 4 8:40 - 8:55 A. H. 9:15 - 9:25 A. H. 17 0 2 Several mosquitoes landed on dog and appeared to probe. No mosquitoes took a blood meal. Several sK>squitoes landed on dog and appeared to probe. No mosquitoes took a blood meal. No probing or feeding. 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