H I 4'; I}: +qu I l I W! ‘l I J” 1 W b l I ’ I Ill 1 MERMiTHID HEMATGDE RAEJQSEESN: Q-F Ami guysfl‘kéus (WALKER) {DIP-TERA: CUUCl-QAE) FROM ENKSEAM CQUNE'Y, .‘MCHEGAN Thesis {03' ihe Degree of M. S. MECHiGAN STATE UNSHIERSET‘I’ Nana- K. 2. Eniagan m . LIBRARY 3 Michigan Sta cc University THESIS p F; ”fl 1*. i H fl 1;“ --'" 3 M Y .-_~ 52:: ”x * "'1": \mei VL. UAL. ABSTRACT MERMITHID NEMATODE PARASITISM OF AEDES STIMULANS (WALKER) (DIPTERA: CULICIDAE) FROM INGHAM COUNTY, MICHIGAN Nana K. B. Hagan A study on mermithid nematode parasitism of Aedes stim- glgng (Walker) was conducted and infestation rates were com- piled essentially through dissection of the mosquito host in all stages of growth. Observations were made of the develop- mental pattern of the nematode within its host and culturing of the nematode was attempted. Histological sections of the juvenile mermithid in the head of the fourth instar larva of the mosquito were obtained. In the prime collecting area, approximately 55% of the mosquitoes were parasitized but in individual pools the per- centage rose to 78%. The distribution of the mermithid nem- atode was found to be discontinuous and up to seven nematodes infested a single host. A minimum length of time for parasitic existence prior to emergence coincided with the life cycle of the mosquito host up to the adult stage. Emergence of the parasite in- variably caused the death of the host° Eight weeks after emergence the nematodes were still undergoing moulting. Gonads had not fully formed at this stage but the beginning of spicules could be observed in the males. Ectocommensal protozoa were noted on both parasitized and non—parasitized mosquitoes. MERMITHID NEMATODE PARASITISM OF AEDES STIMULANS (WALKER) (DIPTERA: CULICIDAE) FROM INGHAM COUNTY, MICHIGAN. By NANA K. B. HAGAN A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Entomology 1966 ACKNOWLEDGEMENTS The author would like to record a debt of gratitude and profound appreciation to Dr. R. A. HOCpingarner for his kind guidance and counsel during the course of this investiga- tion and to Dr. Gordon E. Guyer, Chairman, Department of Entomology, for sacrificing his busy time to advise on the conduct of this study and for providing funds to make this study a possibility. Finally my thanks are due to Dr. Harold E. Welch, Head of Zoology Department, The University of Manitoba, Winnipeg, Canada, for attempting to identify specimens of nematodes. ii TABLE OF CONTENTS Page INTRODUCTION ........................................ 1 REVIEW OF THE LITERATURE ............................ 2 METHODS AND MATERIALS ............................... 13 Collecting equipment and techniques ............ 13 Laboratory procedure - nematode investigations in the immature mosquitoes oooooooeoeoooooocoooo 15 Recovery of mosquito larvae from sod samples ... 15 Attempt at recovering mermithids from the soil . l7 INVESTIGATIONS IN THE 1966 SEASON ................... 18 Aedes stimulans and its bionomics .............. 18 Studies on mermithid parasitism of Aedes Stim- ulans ....0.0.0.000.........OOOOOOOOOOOCOOOOOOO. 22 Culturing of the immature nematodes ............ 31 Muspratt's sand culture technique .............. 33 RESULTS ............................................. 36 Local distribution of mosquitoes ............... 36 Bionomics of the parasite ...................... 37 Effect of the parasite on the host ............. 46 Presence of ectocommensals in the host ......... 47 Effect of host on the nematode ................. #7 Incidence of parasitism ........................ #7 Attempt at identifying the nematode ............ 51 DISCUSSION .......................................... 52 Survival of the mermithid parasite ............. 53 Considerations on the mode of infestation and growth pattern of the parasite ................. 55 SUMMARY ......OOOOOOO......OO'OOOOOOOOOOOOOOOOOOOO.... 58 REFERENCES 0.00.00...0.0.......OOOOOOOOOOOOOOOOO...O. 60 iii LIST OF TABLES Table I Result of a preliminary survey to determine, II III by dissection, nematode infestation of mos- quitoes in the northwestern part of Ingham County, Michigan. Summer, 1965 .............. Percentage parasitism of Aedes stimulans (Walker) by a mermithid nematode in three areas of Ingham County, Michigan ............. Frequency distribution of mermithid nematode parasites in Aedes stimulans (Walker) from one location - Willoughby No. I, Ingham county. Michigan 0.0.0000...OOOOOOOOOOOOOOOOOO iv Page 38 48 50 Figure l. 2. 3. 4. 9. 10. 11. 12. 13. 14. 15. LIST OF FIGURES Habitat Of Aedes Stimulans OOOOOOOOOOOOOOOOOOOO. Fourth instar larva of A; stimulans ............ Adult OfA-Q-Stimulans ......OOOOOOOCOOOOOOOO.... Sagittal section of the head of a fourth instar larva of A; stimulans .......................... Sagittal section through the brain of a fourth inStar larva Of A: Stimulans 0.0000000000000000. Sagittal section of the head of a fourth instar larva of A; stimulans to show multiple para- Sitism ....OOOOOOOOO......OCOOOOOOOOOOOOOOOO.... Sagittal section of a Juvenile mermithid nema- tode within the left brain lobe of a fourth instar larva of A; stimulans ................... Sagittal section of a Juvenile mermithid nem- atode within the brain lobe of a fourth instar larva 0f_A_:_St1mulanS ....OOOOOOOOOOOOOOOO0.0... Sagittal section through the head (posterior) half) of a fourth instar larva of A; stimulans . A soil culture of mermithid nematodes .......... Post-parasitic, Juvenile mermithids ............ A female Juvenile mermithid emerging from the posterior region of the abdomen of adult A. Stimulans .........OOOOOOOOO......OOOOOOOCOOQOOO A male Juvenile mermithid emerging from inter- segmental fold at the posterior region of the abdomen of a male A; stimulans ................. Adult A; stimulans with distended abdomen show- ing the position of irregularly coiled mermith-_ id par881tel-r181tuo .....OOOOOOOOOOOO0.0.0....0 Relative sizes of a mermithid nematode parasite and its mosquito hOSt 0.090000000000000...oooooo Page 19 21 23 25 26 27 28 29 30 34 4O 41 42 44 INTRODUCTION In the summer of 1964, a high incidence of an unidenti- fied mermithid nematode parasite was reported in Aggg§.§time glans (Walker) from vernal ponds in Ingham County, Michigan. Additional collections were made the following spring which confirmed the 1964 report. A high percentage of mortality was observed in these parasitized mosquitoes. Dr. H. E. Welch to whom the specimens were sent for identification re- ported that the parasites were larval mermithids, which lacked characters for positive identification, but were close to larval specimens of Hydromermis sp., a mermithid reported from several species and genera of mosquitoes. These reports stimulated interest in nematode distribu» tion within the range of their host, and particularly in their role as natural agents controlling mosquito populations. The primary obJective of this investigation, therefore, was to compile information on the occurrence of mermithid nematode parasites in certain mosquitoes in Ingham County, Michigan, to follow the developmental cycle of the nematode within the mosquito and outside it, and to attempt in culturing the nematode to adulthood to determine its taxonomy. These studies started in the summer of 1965, with a survey of the potential mosquito breeding grounds in the county, followed by collection and dissection of immature mosquitoes in the area to record any incidence of nematode parasitism in them. The investigation continued until the end of summer, 1966. REVIEW OF THE LITERATURE Hyman (1951) frequently places the Mermithoidea under Enoploidea and generally regards them as related to the Dory- laimoidea. The mermithoids are smooth filiform worms often of considerable length (50 cm., but usually shorter) that are parasitic in Juvenile stages in terrestrial or fresh- water invertebrate hosts, usually insects, but also crustam ceans, spiders, and snails whereas the adults lead a free existence, often of some duration in soil or fresh water. The superfamily embraces two families: Tetradonematidae and Mere mithidae. The taxonomy of the latter rests mainly on Russian nematologists, most notably FilipJev and Schuurmans Stek- hoven (1941) but due to the difficulties of the taxonomy of these worms only a few contributions were made in the last twenty years (Welch, 1963). Polozhetsev and Artyukhovskii reviewed the genera, Hydromermis (1960) and Paramermis (1959). Artyukhovskii rem vised Pseudomermis (1960) and Amphidomermis (1963), erecting in the latter work two new genera, Spiculimermis and gglplonthimermis. Ipateva (1963) described a new species of Filipjevimermis. Kir'yanova.§t‘a;. (1959) reviewed Kirgistan mermithids and erected the genus Pologenzevimermis. Welch and Rubstov (1964) studied a species complex in‘ Gastromermis. Coman (1961) published a monograph on the mermithids of Roumania, and listed eight genera and 29 species of which two genera, Romanomermis and Quadrimermis and eleven species were new. In North America, Johnson (1963) reported Octomyomegmis, a new mermithid from chironomids. Poinar and Gyrisco (1962) named a new gexamermis, and Poinar (1964) described a new and unusual genus and species from chironomids. Welch (1960 a, b, c.) named a new Hydromermis and discussed taxonomic problems of Hydromermis contorta (Linstow, 1889) Hagmeier, 1912. Re named a new species of Gastromermis, of Isomermis, and of Mesomermis from simuliids (1962) and new Hexamermis from Australia (1963). A new Romanomermis from mosquitoes in India was also described (1964). The occurrence of mermithid nematode parasites in mosquito larvae was reviewed by Jenkins and West (1954), who reported finding high infestation by the worm (subse- quently described as Rydgpmermis chugchillensis by Welch, 1960) in larvae of Aedes communis (De Geer) in northern Canada, with light incidence in two other Species of Aedes, namely A.nearticus Dyar and A.nigripes (Zett). In 1956 Laird reviewed world records and later Welch (1960) summar— ized mermithid parasitism in North American species of mos- quitoes. Frohne (1953, 1955) also reported the presence of mermithids in Aedes communis, and added several species to the list of known mosquito hosts given by preceeding authors. The earliest record of mermithid infestation in mos— quitoes dates back to 1898 when Ross observed parasitism in Culex fatigans from India. According to MuSpratt (1945) nematodes other than Filaria, parasitic in mosquitoes have been recorded from the United States, Canada, EurOpe, Russia, India, Ceylon, Sumatra, and Africa. These mermithids were Limnomermis Daday, Paramermis Linst, Mermis DuJ. and Agamomermis Stiles. Agamormermis is an artificial collec- tive group for larvae which cannot be identified. At Leipzig, Germany, Agamomermis sp. were collected in the abdominal cavity of larvae pupae, and adults of 9312; nemoralis by Stiles (1903). Infestation was thought to have occurred in water. The infested insects were very sluggish in movement and many of them died from the effects of the parasite. The ovaries of parasitized females were under- develOped and during the years when the nematodes were most common the mosquitoes were less numerous. Smith (1904) collected in New Jersey, large numbers of female Aggg§_§gllicitans (Walker) which were infested with Agamomermis culicis Stiles (1903) from late June to late September, 1903. Up to 50 percent of the specimens collected contained worms. The peak of infestation occurred in late July and into August. The ovaries of A.sollicitans did not develOp when the female was parasitized and the nematode was considered to be a material check on the species. In French Guiana, nematodes were found in pairs, one large and one small worm in the body cavity of Aedes aegypti (L.) The larvae develOped normally but Just before pupation the worms left the host at the posterior end of the body by perforating the membrane surrounding the anus. The larger worm emerged first followed by the smaller and both died several hours later. The mosquito larvae died as a result of inJuries caused by the emerging worms. These observa- tions by Gendre (1909) also mention that no parasitized adults were found. The life cycle of mermithids that destroy mosquito larvae was studied in India by Iyengar (1927). He found that the minute pre-parasitic worms swim in water and probably pene- trate the cuticle of mosquito larvae. The smallest nema- todes seen in the mosquito larval haemocoele were 650 to 700 microns in length and 115 to 165 microns in width. The parasitized mosquito larvae were found to remain as fourth instar longer than normal larvae. The larvae died on emer- gence of the nematodes. Muspratt (1964) has shown that an undetermined mermithid, subsequently referred to as Romanomer- mis Coman, will readily parasitize mosquito larvae of the 9312; pipiens complex. Hearle (1926) reported that as many as 80 percent of Aedes vexans Meig. mosquitoes in the Fraser Valley, British Columbia in 1920, contained a nematode parasite, Paramermis canadensis Steiner (1924), but only about 20 percent in 1921. Hearle observed the effect of the parasite to be a retardation of the development of the ovaries, since no parasitized females contained well~developed eggs. His measurement of the largest worm was 1.25 in. in length, with from one to six nematodes in a single mosquito. Hearle's (1929) further observation of mermithid parasitism of mos- quitoes was that of an adult female Aedes flavescen§_(Miller) in Canada infested with a single small nematode, and a single female Aedes aldrichii D. and K. These earlier observations of parasitism of mosquitoes by mermithids have been confirmed more recently by Smith (1961) and notably by Welch (1960). The literature gives detailed account of the structure of those members of the family that have been described. According to Welch (1963) members of the Mermithoidea are generally identified by the degenerate musculature of the oesophagus, the very long oeSOphagus, the presence of numerous oeSOphageal cells along its length and the deve10pment of the intestine as a food storage organ. Hyman (1951) gives a more detailed description of the superfamily and emphasizes the absence of a buccal capsule and the direct Opening of the mouth into the pharynx. She describes the pharynx as long and tenuous, reaching half or more the body length of some mermithids, and consists of cuticular tube embedded in a thin cytoplasmic layer. Poster- iorly they are attached to a varying number of variously arranged stichocytes, four in the family Tetradonematidae and usually more than four in the Mermithidae. The didelphic female system is of the Opposite type. The males, much small- er than the females, are usually diorchic with one or two specules and with numerous genital papillae on their poster- ior ends. Reproduction is either bisexual or parthenogenic. Most authors agree on the subdivision of the Mermithoidea into two families, the Tetradonematidae and the Mermithidae. According to FilipJev §t_al. (1941), Hyman (1951) and Welch (1963, 1965) only three of the Tetradonematids have been named, though Rubstov (1963) found new unnamed species in all stages of simuliids. The Tetradonematids are generally regarded as primitive mermithoids which remain and become adults in the haemocoele of their hosts whereas the mer~ mithids emerge and become free-living adults. The life cycle of mermithid parasites varies for dif- ferent species. Welch (1963) states that it commences when the second stage Juvenile armed with an odontostyle penetrates the host cuticle and enters the body cavity. The nematodes grow, fill the host body cavity, then emerge to begin a free~ living existence. Maturation, fertilization, and oviposi- tion require varying periods according to the specific mer- mithid. According to Anderson and Defoliart (1962), in aquatic forms such as Gastromermis sp. and Isomermis sp., the above sequence of events may take several months, or take up to a year and half in terrestrial forms such as Agamermis decaudata parasitic in grasshOpers (Cobb, Steiner, and Christie 1923, Christie, 1929, 1936). Welch (1963) states the case in Mermis nigrescens DuJardin, a parasite of OrthOptera and Dermaptera, in which the life cycle is altered to passive host infection through the consumption of elaborately tasseled eggs deposited on grass blades. Certain species, however, require rather short life cycles. Muspratt (1963) refers to some species parasitizing chironomids which require only 24 hours or even less. Welch (1960) found that the subarctic mosquito parasite Hydromermis churchillensis requires 10w15 days to attain sexual maturity after emergence from the host while in two blackfly mermithids it varies from 5 to 12 days. Most records in the literature give only larval hosts, and Stabler (1952) concluded that the presence of the worm prevented pupation. Apparently the worms can carry over from larval through pupal to adult hosts. (This occurred fre- quently in the Michigan observations). According to Welch (1960) this assists, in the culicids, in the distribution of the parasite but in simuliids it also counteracts the tendency of the river current to carry the free living stages down- stream. Many records of percentage parasitism may be found in the literature. Welch (1963) advises that despite the sampling problems involved in their derivation, and the care that must be exercised in their use, they are indicative of interactions. Rates range from 0 to 100%; their magnitude usually shows an increase with host deve10pment or age, or else with the degree of suitability of the physical environment of the nematode such as the moisture content of the habitat. Correlations with host abundance are given in a few cases. Records of mermithid parasitism of mosquitoes is rather fragmentary. Most of the data is on parasitism of simuliids, grasshOppers and chironomids. Krall (1959) recorded 50 percent parasitism of adults of Chironomus plumosus L. in a lake in Estonia. Johnson (1963) reported 20 percent of the larvae of this same insect parasitized in Minnesota. The numerous data on mer- mithid parasitism of simuliids were analyzed by Weiser (1963) and Welch (1960, 1963). A detailed study by Phelps and DeFoliart (1964) on parasitism of Simulium vittatum Zetter- stedt by three mermithids in Wisconsin showed 50 percent parasitism of larvae and 37 to 63 percent of adults. The authors considered that mermithids were a limiting factor on simuliid papulations and that eradication had occurred twice. Rubstov (1963) suggested that simuliid eradica- tion by mermithids had occurred in certain Soviet Rivers. He recorded an average of 17 percent and range of 0 to 99 per— cent in the Leningrad region. Shipitsina (1963) gave parasi- tism rates of adult simuliid flies from the Krasnoyarsk region of l to 11 percent. High rates of mermithid parasitism of mosquitoes have been recorded in pools by various authors who have pointed out that their high rates were rare, as most breeding sites are without nematodes. These and his own observations led Laird (1956) to generalize that wherever mermithid parasitism was noted, its incidence was high. Welch (1960) does not sup- port this view. The parasitism given by these authors seems to correspond only to the maximums of their ranges. Welch (1960) gives more realistic figures of infestation rates of Aedes communis (DeG.) by Hydromermis churchillensis n.sp. in three areas at Churchill, Manitoba. The average infestation per pool for each area where infestation occurred was almost the same each year and was in the order of 5.1, 0.2 - 1.4, and 1.1 - 9.8 percent. The corresponding range of infesta- tion for the three areas were 0 - 32, 0 - 20, and 0 - 82 10 percent, respectively. Welch (1965) states that percentages of parasitism given in the literature are true measures of insect mortality re- sulting from mermithid infection as mermithids kill their hosts upon emergence. Many authors agree that mermithids dam- age their hosts only during the later stages of their parasi- tic development when their growth is rapid, and their rela- tively large size compared to that of the host. Instances of multiple parasitism are often recorded. Welch (1959, 1960) and other authors have analyzed frequency distribution of the number of parasites per host and showed that departures from a chance distribution were involved in all the reported cases. This, Welch (1963, 1965) suggests, shows a discontinuous or contagious distribution of nematodes within the range of their host. Such discontinuous parasite distributions are apparent not only within, but also between populations of the same host. Gendre's (1909) observation of high but localized parasitism of mosquito larvae by mermithids was duplicated in Canada by Welch (1960) who found neighbor- ing pools to have infested and non-infested populations of larvae. In a number of observed cases high parasite numbers per host usually results in the emergence of males; and low numbers, in female mermithids. There are few observations on the factors that cause emergence from the host. According to Welch (1960) emergence of mermithids is correlated with special deve10pmental events, such as pupation or adult emergence of the host. Very little 11 is known about the causative factor of mermithid emergence from the adult host. Insects generally show little outward evidence of the presence of nematodes and dissections are usually necessary to reveal nematode parasites. The literature gives an account of anomalies due to external effects of mermithid parasitism of insects other than mosquitoes. These involve the shorten- ing of wing in ants and locusts, and the malformation of elytra in weevils (Poinar and Gyrisco, 1962). Various authors have reported formation of intercastes and intersexes in ants, chironomids, culicoides and simuliids. Discoloration and cuticular transparency have also been noted in mermithid infested weevil larvae. Internal effects of mermithid parasitism of Aedes communis (DeG.) larvae was reported by Welch (1960) who noted a reduction in fat body tissue and also in thoracic musculature. Other authors agree that probably because of the large size of mermithids, their damage to genital and intestinal organs is greater than that caused by other nema- todes. Host reaction of mosquito larvae to nematode infesta- tion by means of melanization and encapsulation was probably first recorded by Welch (1960) from observations in first and second instar larvae. Brug (1932) summarized the records in adult mosquitoes. Welch and Bronskill (1962) discovered the melanization and encapsulation of a Neoaplectanid nematode by larvae of several species of mosquitoes. Agdgfi,atimnlans. 12 is reported to react similarly to an unidentified rhabditoid nematode. The culture of nematodes has been reviewed in consider- able detail by Daugherty (1960). The literature cites in- stances of many entomOphilic nematodes that were cultured xenically. A sand culture of a mermithid nematode of mos- quitoes by Muspratt (1964) illustrates a most convenient way of culturing this group of nematodes. METHODS AND MATERIALS Studies and observations were commenced in the summer of 1965. The season proved to be rather dry, especially in the first half, and consequently many of the potential breeding grounds for mosquitoes were virtually dry, particularly puddles and vernal ponds which serve as good sources for mos- quito larvae. A survey, covering nearly 144 sq. miles of the north western portion of Ingham County, was conducted to map out the potential mosquito breeding habitats. This included essentially four townships of the county, namely, Lansing, Meridian, Delhi and Alaiedon. afllecting Equipment and Techniques. Much of the equipment and procedure used in searching for the immature stages of mosquitoes are not completely original. Some of these have been mentioned in previous publications and are considered to be standard methods of collecting mosquitoes. The basic equipment comprised metal laddles, white enamel trays and large glass containers with fine mesh wire or cotton lids which were used in transporting the mosquito infested pond water to the laboratory. In the absence of dippers large metal beakers served as good substitutes. The collecting of immature stages rather than adults was preferred in view of the ease of capture. Where larvae or pupae were present in ponds or streams there was no diffi- culty in locating them and effort was made to examine every 13 14 available water holding in the survey area. A maJority of the ponds that were examined yielded no immature mosquitoes and it was not until mid-July that mosquitoes were encountered in the field as a result of a few scattered rains. In addition, some tree holes in the area were examined for mosquitoes. The problems involved in collecting immature stages of tree hole breeding mosquitoes include the dark color of most tree hole water, the small amount of water involved, and the fact that most tree holes are less accessible than the breeding habitats of most other species. Where tree holes were found to contain water it was difficult or impossible to determine under field conditions whether or not specimens were present in a given collection. Generally tree hole water was poured into white enamel trays for examination in the field, but young instars or eggs might be present and not discovered. All water in a tree hole was routinely taken and the hole flushed with pond water. When collecting with specimens in the car, care was taken to have good ventilation, and if possible to park in the shade to keep the specimens cool. This account so far applies primarily to routine collecting when rainfall was sufficient to deposit water in many dry ponds and tree holes. At the end of the days collecting, each collection was labelled giving its location and date and taken to the laboratory for further examination. 15 Laboratory Procedure - (Nematode investigations in the im- mature mosquitoes.) In the 1aboratory,collections of larvae in the large glass transporting Jar were transferred into white enamel pans from which they were easily picked and fixed. First to third instar larvae were generally left un- til they reached fourth instar stage when they showed suf- ficient characters for positive identification. The fourth instar larvae were quickly killed and fixed in FAA solution (made up of 15-20 ml. of ethyl alcohol, 1 ml. of acetic acid, 6 m1. formalin and 40 ml. distilled water). This fixative has the prOperty of keeping nematodes in a fairly good state, free from being brittle, and has a clearing effect on the en- tire mosquito larva. Following fixation larvae were identified to species under low power stereoscOpic microsc0pe after which they were dissected without much delay to look for mermithid nematodes. Recovery of mosquito larvae from sod samples. In view of the existing drought conditions of the summer of 1965, with its attendant drying of local ponds and the consequent reduction of mosquito breeding in the field, attempt was made to re- cover mosquito larvae from the potential breeding grounds by flooding sod samples from these areas. Sod samples from a previously recorded Aedes breeding habitat were collected in plastic bags and taken to the laboratory for flooding. This selected area located in Delhi township of Ingham County, is adJacent to the Sycamore Creek on the east bank. (This is 16 the primary collecting area referred to elsewhere in this paper as Willoughby No. l). The area is essentially level and wooded and is the site of vernal ponds which appear Just after the annual spring thaw. Beginning in August 3, 1965, 32 sod samples of 1 ft. square each were cut to a depth of 2-4 inches from eight' different sites, with four samples from each site. Each sample was put into a plastic bag and kept in the shade un- til ready for transfer to the laboratory, For flooding, a sample was transferred to an enamel or plastic pan and sufficient water was used to fully cover the sod and any plant material. Four sod samples were flooded each day for 12 days. After a flooding period, the water was poured off carefully to look for larvae. In these flooding exercises some attention was given to some suitable egg hatching stimulus. The literature on egg hatching stimuli has been reviewed by Burgess (1959 after Shannon and Putnam, 1934; Garnet and Haynes, 1944; GJullin gt; gig, 1941; Borg and Horsfall, 1953; Horsfall, 1956; Horsfall 92; al;, 1958; Barr and Al-Azawi, 1958). The hatch-promoting stimuli have been found to be essentially distilled water and distilled water with low oxygen tension. Both types of distilled water were used for flooding. Diem solved oxygen was removed from distilled water by boiling for thirty minutes, then cooling it to 28-2900. for 30 minutes in the absence of air. This has been proven to reduce the amount of dissolved oxygen in the distilled water from its 17 normal value of 7-8 p.p.m. to less than 2 p.p.m. by weight. (After Burgess, 1959). The results from flooding sod samples of eight different sides in four replications yielded only 25 larvae of Aedes vexans (Meigen) from one sample. The possible reasons for this astonishingly low yield will be discussed later. These larvae were free from any nematode parasites (Table 1.). Attempt at recovering mermithids from the soil. The 1965 season investigations embraced processing of soil from habi- tats where mermithid nematode and mosquito relationships had been previously reported. Soil samples from "Willoughby 1" were obtained as previously described and passed through 25, 60, 100 and 200 mesh sieves and finally centrifuged in sugar solution. This technique leaves all nematodes in every stage of growth in the supernatant fluid. This method of investigation yielded some plant parasitic nematodes, but no mermithids. INVESTIGATIONS IN THE 1966 SEASON With the onset of the spring season, the search for Aedes mosquitoes was most actively resumed. Attention was paid to previously recorded Aedes breeding places in the four townships of the county previously mentioned. Three areas were selected for collecting and were designated Willoughby No.I, Willoughby No.II, and Holt. Willoughby Nos. I and II are situated on the east bank of the Sycamore Creek and might be regarded as similar habitats except that the two are separated by a road. The third location in Holt, about three miles away from the first two, was chosen to see any differences due to distance that might exist in the in- cidence of parasitism of the mosquito papulations. When the winter snow had sufficiently melted for ground pools to form, the search for mosquito larvae was commenced. Larvae were collected from the three sites and taken to the laboratory every five days starting on April 21, 1966. The regularity of sampling was broken owing to the unusually cold spells that occurred intermittently during the spring season. Collecting was therefore limited to good weather conditions as well as the unavailability of working material in the laboratory. The woodland nature of the collecting sites is shown in Figure l. Aedes Stimulans and Its Bionomics. The species that was found to occur in tremendous numbers in woodland pools and 18 l9 HABITAT OF AEDES STIMULANS Woodland pool, near the Sycamore Creek in Ingham County, Michigan. This spring pool contains an enormous pOpulation of Aedes stimulans. It dries up in summer. 20 stump holes in all the three sites throughout the spring is Aedes stimulans (Walker). Ross (1947) reports that the larvae of this species (Fig. 2) occur in cold water of wood- land ground pools flooded by melted snows, develOp slowly, but are usually fully grown by the time the first warm weather of May occurs. Horsfall stated that a survey of 50 snow pools showed 58 percent of the larvae to be this species in Canada (Horsfall, 1955 after Twinn, 1931). The range of Aedes stimulans is widespread throughout the northeastern states, across the north central states, through Canada, and into the Yukon (Ross, 1947). The species is reported throughout the state of Michigan. It has one generation per year (Horsfall, 1955 after Twinn, 1931; Owen, 1937). The pupal stage requires 3-4 days when the temperature range is 20°~25° (Horsfall, 1955 after Jordan, 1902). The females seem to stay near their larval sites, where they attack all sorts of animals that come near. This is evidenced by the ferocity with which they attacked to bite during the collecting period. Horsfall (1955) reportscthat the males feed on the flowers of smilax. A.stimulans in captivity is found to be reluctant to oviposit on moist cellucotton where conditions are favorable for obtaining eggs of other species like A.vexans or ngrg- phgrggp Like A.trivittatus, most eggs have been obtained only when caged females were trapped on the surface of the water, and eggs were extruded before they died. (Horsfall,l955). 21 ans a: meaotoacasmhz m>th 0:» mo one: on» Ca mhdooo spammhwa oUOmems UHapHsaos a mz¢ADSHBm .< mo <>mmda mapmzH mbmbom 4 SO Qflmm mm tuaawwoa h.cuoahm osv mason can“ Comet; cue mzosu QHmAM exp m0 c£OH HmhfluhmochHQ pm \.\ ants In Ceapoc.ndmwm MW m_%HQHH®L ll §6W+m .J..¢ . 4 \. .hgm,la miss .:e A y. r r I u q ;macd tap ma uquH madam on» sowom .pcoecpos uaanana on we p~3.oa w mm pcmn .cUOCKLus mfimpfisnca a mo tto J .la Jr. .L .uél. ‘34 mo ZOHBUmm Qsm can no Goduwwoc o>nuflramo© one mZan.HBm mmmmi mo r>mma «dtmzH mBmDom 4 mo 2H¢mm mmH mwboflma ZOHhomm HEBBHUmDfi a mo ZOHeomm nflBBHomsq mdBWZH menuom so Abh 4 m0 ZOHEU ZHsmm mmB ZHEHH. THESIS 30 as \Q m I scapucflwfihdoa Anoapmofidwoos m.cumaasmodv :Hspm caaflap m.>aoamsa II cflmun item magmatic mam tampons one. we was: Mozpo 93 CH maomonaoeap memo. ,wo uHHco and: usmtm awHHcsn one one hams oso 2H mHHoo oumoogofipm weasampm hasten Homaoa are .Nsaogp pscomnom o£+ swapfis psosaoao>oo so owupm cowpfimcmap msp mcooocaa push pcosaoaoeoe go mmmum mane .emmx m.pmos o£s mo cam Lofiaopmoa mg» no zoom ma soonest: maficmaah .mzeaaaHem mmome mo <>mDb .OHBHm¢m¢mIBmom Ha oaomaa 41 N on II coHpmonfimez mz¢AD£HBm .fi BQDQ< mo zmzomm< mmB mo onomm mOHmmBmom mmB 20mm ozHommzm QHmBHzmmz mQHzm>ph mq¢zmm ¢ NH enemas 42 ama .. eoaoooaeaamez .mzDh mamz ¢ .,t a».« “ Q 0 ma oaswam 43 the siphon as reported in the parasitism of Aedes communis (De.G.) by Hydromermis churchillensis (Welch, 1960). Growth of the nematode within the mosquito host as revealed by dissection of the various stages of the mosquito, showed a distinctive pattern. With growth and maturation of the mosquito, the nematode showed corresponding growth, migrating from the head where it was first seen in the larval instars to the abdominal haemocoele where it was seen irregu- larly coiled or doubled back several times (Fig. 14). After emergence the worm was seen to be definitely longer than the adult mosquito it had parasitized (Fig. 15). Evidence in support of this migratory tendency of the parasite was afforded by the observation of nematodes within the thorax of fourth instar larval mosquito, in two dissections. In all other cases of nematode infestation in the larva, it was always seen in the head, and when it occurred in the pupa, it was seen mostly in the abdomen or both the abdomen and the posterior part of the cephalothorax. Nematode infestation of the adult mosquito was invariably within the abdominal haemocoele. This pattern of growth also showed a marked departure from that of most reported cases where infestation was never seen beyond the larval stage of the mosquito, and usually resulted in preventing pupation, and the killing of the host larva after emergence of the worm. The life cycle of this nematode has not been fully worked out. Postparasitic stages that emerged from adult mosquitoes have been cultured for nearly three months. After six weeks 44 me In Cofipmonacmmx .ommum oHanpsm w ma 5903 ZH MBHmHE