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1988 Spontaneous flight activity of mosquitoes (Diptera: Culicidae) infected with filarial nematodes William Joseph Berry Iowa State University

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Spontaneous flight activity of mosquitoes (Diptera: Culicidae) infected with filarial nematodes

Berry, William Joseph, Ph.D.

Iowa State University, 1988

UMI 300N.ZeebRd. Ann Arbor, MI 48106

Spontaneous flight activity

of mosquitoes (Diptera: Culicidae) infected with filarial nematodes

by

William Joseph Berry

A Dissertation Submitted to the Graduate Faculty in Partial Fulfillment of the

Requirements for the Degree of DOCTOR OF PHILOSOPHY

Department: Entomology

Major: Entomology (Medical Entomology)

Aooroved:

Signature was redacted for privacy. [aj or Work

Signature was redacted for privacy.

Signature was redacted for privacy.

Iowa State University Ames, Iowa 1988 ii

TABLE OF CONTENTS GENERAL INTRODUCTION 1 Explanation of Dissertation Format 12 SECTION I. INFLUENCE OF DEVELOPING BRUGIA PAHANGI ON SPONTANEOUS FLIGHT ACTIVITY OF AEDES AEGYPTI

(DIPTERA: CULICIDAE) 14

ABSTRACT 16 INTRODUCTION 17 MATERIALS AND METHODS 19

RESULTS 21 DISCUSSION 29 ACKNOWLEDGEMENTS 33 LITERATURE CITED 3 4 SECTION II. INFLUENCE OF DEVELOPING DIROFILARIA IMMITIS , ON THE SPONTANEOUS FLIGHT ACTIVITY OF AEDES AEGYPTI (DIPTERA: CULICIDAE) 36 ABSTRACT 38

TEXT 3 9 ACKNOWLEDGEMENTS 51 LITERATURE CITED 52 SECTION III. SPONTANEOUS FLIGHT ACTIVITY OF AEDES

TRIVITTATUS INFECTED WITH DIROFILARIA

IMMITIS 54 ABSTRACT 56 ill

INTRODUCTION 57 MATERIALS AND METHODS 58 RESULTS 61 DISCUSSION 68

ACKNOWLEDGEMENTS 72 LITERATURE CITED 73 SUMMARY AND CONCLUSIONS 76 LITERATURE CITED 79 ACKNOWLEDGEMENTS 87 1

GENERAL INTRODUCTION Filarial nematodes have complex life cycles which usually involve a vertebrate definitive host and an obligate arthropod intermediate host. Mosquitoes serve as the intermediate host for many filarial worms and the filariae develop in several locations within the host. Bruaia spp. and Wuchereria spp. develop in flight muscle; Dirofilaria immitis develops in the Malpighian tubules; and Waltonella (=Foleyella) flexicauda develops in fat

body (Benach and Crans 1973). In general, microfilariae are ingested by the mosquito during bloodfeeding. The microfilariae migrate to the site of development and undergo two developmental stages. Ultimately, the third stage (or infective) larvae migrate to the mouthparts and enter the vertebrate host at the time of bloodfeeding.

While most filarial species will develop in a number of mosquito species, development in a particular mosquito is genetically regulated. Macdonald (1962a,b, 1964) developed a strain of Aedes aeavpti that was highly susceptible to Bruaia malavi and other filarial nematodes that develop in flight muscle, including B. pahanai. Macdonald and Ramachandran (1965) found that genes regulating the development of Bruaia had no influence on the development of Dirofilaria. They suggested that 2

different genetic factors were likely to regulate development in different tissues. Kartman (1953) selected for susceptibility to D. immitis over 8 generations in Ae. aeqypti and McGreevy et al. (1974) demonstrated that development was regulated by a sex linked factor.

Terwedow and Craig (1977a,b) were able to produce strains of aeqypti that were susceptible and refractory to development of Waltonella flexicauda. They found that genetic factors regulating development were different from those that regulate development of either Bruaia or D. immitis. Furthermore, development in males was found to be regulated by different factors than those operating in females (Terwedow and Craig 1977c). The natural definitive hosts of Bruaia pahanai are cats, dogs and other mammals (Edeson 1959). The natural intermediate hosts of B. pahanai are Mansonia annulatus and Mansonia lonaipalpis. However, numerous other species have been identified as laboratory hosts, including Armiaeres obturans. Armiaeres subalbatus. Anopheles barbirostris and Aedes toaoi (Nelson 1959, Edeson et al. 1960, Kan and Ho 1973). Rodriguez and Craig (1973) found that B. pahanai would develop in only 13 of 43 strains of Ae. aeqypti. The Liverpool strain developed by Macdonald (1962a,b) is susceptible to this filarial nematode and is 3 often used as the intermediate host in laboratory studies involving B. pahanai. Adult B. pahanqi and B. malavi are usually maintained in mongolian jirds (Ash and Riley 1970a,b, Ash 1972, McCall et al. 1973). Microfilariae of Bruaia penetrate the midgut and enter the hemocoel rapidly after the mosquito ingests the blood

meal (Christensen and Sutherland 1984, Perrone and

Spielman 1986). The microfilariae migrate through the subcuticular fat body and ultimately penetrate the flight muscle (Esslinger 1962). The midgut epithelium is damaged during penetration and damage to the subcuticular fat body has been demonstrated. The first larval molt occurs after about 4-5 days and the second molt occurs at around 8 days with the third stage larva being immediately infective (Schacher 1962a,b).

Migration and development of B. pahanqi laihrae causes

a great deal of tissue damage and leads to increased mortality in host mosquitoes. Beckett (1971a,b) observed

histological changes in the flight muscle of aeavpti harboring developing filarial larvae. Townson (1971) reported two periods of increased mortality in infected mosquitoes. The first period occurred within 24 hours of

infection and the other occurred after day 8. These times correspond to periods of greatest tissue disruption caused by migrating larvae. Ibrahim and Trpis (1987) found that 4

B. pahanai caused increased mortality in susceptible species of the Aedes scutellaris complex, but had no effect on refractory species. Mortality was greatest in mosquitoes harboring the greatest number of parasites. Dirofilaria immitis occurs naturally in dogs and

other canids, including coyote fCanis latrans) (Weinmann and Garcia 1980), gray fox fUrocyon cinereoaraenteus) (Simmons et al. 1980) and experimentally in ferrets (Mustela putorius furo) (Campbell et al. 1979). After a

period of development, the adult worms are typically found in the right ventricle and pulmonary arteries of the dog (Orihel 1961). Dirofilaria immitis has occasionally been found in humans, typically as a coin lesion in the lung (Lewis and Tinsley 1969, Neafie and Piggott 1971), but also subcutaneously in the eyelid (Font 1980), and in the pectoral muscle (Welty 1963). Tada et al. (1979) report the recovery of an adult female D. immitis from the abdominal cavity of a man. In any event, human infections usually involve undeveloped worms and are relatively rare. Domestic dogs are the most significant host. Dirofilaria immitis is capable of developing in a wide range of mosquito hosts. Ludlam et al. (1970) listed over

60 species, covering 6 genera, in which D. immitis larvae were found to complete development. Arnott and Edman 5

(1978) implicated Aedes canadensis and Aedes excrucians as vectors in Massachusetts. Several studies have implicated Aedes vexans as a natural vector of fî. immitis (Lewandowski et al. 1980, Walters and Lavoipierre 1982). However, Aedes trivittatus has been established as the principal midwestern vector of this filarial parasite

(Christensen and Andrews 1976, Christensen 1977a,b. Finger

1982). Furthermore, dogs were identified as a significant blood host for trivittatus in Indiana (Nasci 1984). Dirofilaria immitis larvae, developing in Malpighian tubules, cause increased mortality in Aedes aeavpti (Kershaw et al. 1953), Aedes sollicitans (Beam 1966) and

Ae. trivittatus (Christensen 1978). Mortality is higher in mosquitoes harboring greater numbers of developing larvae. Christensen (1981) reported a reduction in egg production in trivittatus infected with D. immitis.

but Courtney et al. (1985) reported that aeovtati ingest less blood if the blood contains D. immitis microfilariae, indicating that reduced fecundity may be caused by smaller bloodmeals and not by developing

parasites. Palmer et al. (1986) reported ultrastructural changes, including complete destruction, in the Malpighian tubules of Ae, aeavpti infected with D. immitis. Damage to the tubules undoubtedly upsets osmoregulatory and excretory processes and is a likely cause of the increased 6

mortality observed by other authors.

Despite the great interest in filarial development in mosquitoes, little information is available regarding influences that the parasites have on behavior of host mosquitoes. Almost no information is available regarding the influence of filarial parasites on flight behavior of mosquitoes. Flight is an extremely important aspect of the life history of mosquitoes. Adult mosquitoes locate both carbohydrate and blood meals through flight. Mating is accomplished through flight and male mosquitoes are sensitive to the wingbeat frequency of females. The epidemiology of mosquito-borne disease is markedly influenced by the flight activity of the vector mosquito.

Field studies have indicated that many species of mosquitoes are capable of travelling long distances in relatively short periods of time. For example, in separate studies, marked Culex tarsalis travelled 15.4 and 25 km from a release point (Bailey et al. 1965, Dow et al. 1965). A major problem with field studies is that flight "range" often provides little more than an anecdotal account of distances travelled by a few mosquitoes. Frequently, less than 1% of released

mosquitoes are recovered. Furthermore, mosquitoes become widely dispersed over an increasingly large area as they 7

move away from the release point. The number of traps needed to adequately sample the dispersing population increases geometrically as distance from the release point increases. Logistical difficulties with field studies, along with a need for basic information about mosquito flight, led to the development of a mosquito flight mill (Rowley et al. 1968). Mosquitoes tethered to the flight mill are stimulated to fly by tarsal reflex. The flight mill system was further enhanced by interfacing up to 16 individual mills with a microcomputer (Clarke et al. 1984). This type of flight mill has been used to study the flight ability of several species of mosquitoes, including Culex tarsalis (Rowley 1970a) and Aedes

triseriatus (Clarke and Rowley 1987). Both studies investigated the effect of age and gonotrophic status on the flight performance of mosquitoes. The system has also been used to study glycogen utilization during flight (Rowley 1970b), the effect of temperature, humidity and age on flight performance (Rowley and Graham 1968a,b), and the effect of laboratory colonization on flight ability (Clarke et al. 1983). Ultrastructural changes in flight muscle associated with exhaustive flight were described by

Johnson and Rowley (1972). The circadian pattern of flight activity of various 8

mosquitoes has also been studied. Jones (1964) developed an acoustic system capable of using the wingbeat frequency of mosquitoes to monitor flight activity. In this system, individual mosquitoes were placed in chambers, made from modified reagent bottles (Jones et al. 1967), set atop sensitive microphones. In the original system, data were recorded on strip-charts, making recovery and transcription of the data a time-consuming and laborious process. To simplify quantification of data, Jones et al.

(1967) developed an activity score, representing the number of minutes within each period that a mosquito made at least one flight of any duration. Peterson (1980) improved the system by recording data on a digital cassette which could be loaded onto a mainframe computer for processing. Peterson's system monitored the number of flights, flying time and activity score. The system was further enhanced by Rowley et al. (1987) by interfacing the chambers with a microcomputer. The microcomputer interfaced system records the flying time, number of flights and activity score for a mosquito in each of 32 individual chambers in each 30-minute period. The system does not provide information about individual flights. Peterson (1980) reports that the three variables are highly correlated. However, all variables must be 9

measured unless mosquitoes in different treatments make flights of the same duration.

As noted by Rowley et al. (1987), the acoustic system provides an unobtrusive method for studying spontaneous flight activity. The system is ideal for studying circadian flight behavior of mosquitoes. Acoustic systems have been used to study several physiological factors that affect flight behavior, such as insemination and gonotrophic status (Jones and Gubbins 1978, 1979, Jones

1981, Berry et al. 1989, Clarke and Rowley 1989). The system is also extremely useful in studying subtle modifications of flight behavior that might result from infection of mosquitoes with parasites or pathogens, such as arboviruses (Berry et al. 1987, Clarke 1988). Few previous studies have investigated the influence that developing filarial nematodes have on flight behavior of host mosquitoes and no studies have investigated circadian flight activity of mosquitoes infected with filarial worms.

Bruqia pahanqi microfilariae develop in flight muscle of mosquitoes (Beckett and Macdonald 1970, 1971, Beckett 1971a). Beckett (1971b) observed histological changes in flight muscle of mosquitoes harboring developing filarial larvae. Damage to the flight muscle by developing larvae can have a profound effect on the ability of the host 10

mosquito to fly. Townson (1970) found that more infected Ae. aeavpti were 'nonfliers' than uninfected counterparts. Furthermore, nonfliers tended to have higher infection intensities. He proposed that some mosquitoes might be incapable of transmitting B. pahangi because of an inability to fly. Paige and Craig (1975) and Hockmeyer et al. (1975) confirmed that infection with B. pahanai did influence the flight ability of aegypti. Husain and Kershaw (1971) reported a similar reduction in the flight ability of Aedes togoi infected with Brugia malavi. Aedes aegvpti is not a natural vector of Brugia.

However, Ramachandran et al. (1960) reported experimental infection of aegvpti with Brugia malavi. A strain of Ae. aegypti that was highly susceptible to infection with

Brugia was developed by Macdonald (1962a,b, 1964). Genes regulating the development of Brugia in aegypti have no influence on filarial larvae developing in locations other than flight muscle (Macdonald and Ramachandran 1965). The ease of maintenance of aegypti in the laboratory led to the studies cited above involving flight of this species when infected with filarial nematodes. Aedes aegypti is an unlikely natural vector of Dirofilaria immitis. However, this nematode will develop in the Malpighian tubules of several strains of this 11

mosquito (Buxton and Mullen 1981). Furthermore, Ae. aegyrati can transmit D. immitis by a natural route (Hendrix et al. 1986). The Liverpool strain has been identified as a susceptible strain of aeovpti. An association between Aedes trivittatus and

Dirofilaria immitis in the central United States has been firmly established (Christensen and Andrews 1976,

Christensen 1977a,b. Finger 1982). Dogs are a common host of trivittatus (Nasci 1984). Though trivittatus is a difficult species to work with, a protocol developed by Christensen and Rowley (1978) enables laboratory study of this vector species. The purpose of these studies was to investigate the influence of developing filarial worms on spontaneous flight activity of host mosquitoes. The first study investigated the influence of developing Brugia pahangi on flight activity of Aedes aegvpti. This laboratory model enabled investigation of the effect that filarial larvae developing in the flight muscle had on flight activity of the host mosquito. The flight ability of B. pahangi infected aegvpti had been studied previously (Townson 1970, Hockmeyer et al. 1975). Both the parasite and the host mosquito are easily maintained in the laboratory. The second section dealt with flight activity of Ae. aegvpti infected with Dirofilaria immitis. This system is 12

also a laboratory model, but it involves a nematode which develops in the Malpighian tubules of the host mosquito. Disruption of normal flight activity in mosquitoes infected with D. immitis would not be a direct result of flight muscle disruption. This study enabled comparison of flight activity of mosquitoes when filarial larvae were developing at different locations in the host mosquito. The third study involved Ae. trivittatus and D. immitis. This system represents a natural association between a host mosquito and a filarial parasite, enabling comparisons between natural and model systems.

Explanation of Dissertation Format This dissertation consists of three sections. Section I is concerned with the spontaneous flight activity of Aedes aecfvpti infected with Bruaia pahanai. This section appeared in the July, 1986 issue of the Journal of Medical Entomology 23(4):441-445. Section II examined the

influence of Dirofilaria immitis on the spontaneous flight activity of Aedes aeqvpti. This section appeared in the December, 1987 issue of the Journal of Medical Entomology

24(6): 699-701. Section III evaluates spontaneous flight activity of Aedes trivittatus infected with Dirofilaria immitis. Section III has been accepted for publication by 13 the Journal of Parasitology.

The senior author is responsible for the design and performance of experiments in all three sections of the dissertation. Mosquitoes were infected by Dr. Bruce M. Christensen of the Department of Veterinary Science,

University of Wisconsin, Madison, Wisconsin. Results from all experiments were assembled, interpreted and written into manuscript form by the senior author. Conclusions and recommendations in all sections of the dissertation are those of the author. 14

SECTION I. INFLUENCE OF DEVELOPING BRUGIA PAHANGI ON SPONTANEOUS FLIGHT ACTIVITY OF AEDES AEGYPTI (DIPTERA: CULICIDAE) 15

Influence of Developing Bruaia pahancri on

Spontaneous Flight Activity of Aedes aeavpti

(Diptera: Culicidae)

William J. Berry

Wayne A Rowley Bruce M. Christensen

From the Department of Entomology, Iowa State University, Ames, Iowa 50011 (Berry, Rowley); the Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706. 16

ABSTRACT Aedes aeovpti (black-eyed Liverpool strain) mosquitoes were infected with microfilariae of Bruaia pahanai to evaluate the effect of developing larvae on spontaneous flight activity. Studies using acoustic chambers showed that even low numbers of developing larvae (<10) inhibited mosquito flight activity, especially on days 8 through 10 postinfection. Parasite intensity was inversely correlated with flight activity, and mosquitoes infected with more than 20 larvae could not sustain flight after day 8. Mosquitoes infected with more than 30 larvae did not fly. Low numbers of developing larvae led to an increase in spontaneous flight activity during the first few days after infection. Developing parasites had little influence on the circadian activity patterns of Ae. aeqypti. 17

INTRODUCTION

Development of Bruaia oahanai microfilariae in Aedes aegypti (L.) occurs within the flight muscles (Beckett and

Macdonald 1970, 1971, Beckett 1971a), and damage to these muscles may have a profound effect on the ability of a mosquito to fly. Townson (1970) found that significantly more infected aeavpti were "nonfliers" than uninfected counterparts and that nonfliers had a significantly higher infection intensity. He proposed that some mosquitoes are incapable of transmitting B. pahangi because of a loss of flight ability associated with their parasite burdens. Paige and Craig (1975) also reported that some mosquitoes are incapable of flight after infection with B. pahangi and suggested that 15 developing larvae may cause sufficient destruction of the flight muscles to hinder flight. Hockmeyer et al. (1975) reported that infected mosquitoes flew significantly less than uninfected mosquitoes and that more infected mosquitoes died or were unable to fly. Husain and Kershaw (1971) noted that B. malavi had a similar deleterious effect on the flight ability of Aedes togoi (Theobald). They suggested that measures of the ability of a vector to support a parasite need to be adjusted to account for nonflying individuals.

This study investigated the effect of developing B. pahangi on the spontaneous flight activity of AB. aegypti. 18

Activity of individual mosquitoes was recorded by using a system similar to that of Jones (1964). The original system has been modified so that mosquito flight activity is monitored and recorded by a microcomputer (Rowley et al. 1987). The parameters investigated include the effects of parasite intensity on the number of spontaneous flights made by a mosquito, duration of these flights, circadian pattern of activity, and total flight activity of that mosquito. Activity was measured using an activity score (Jones et al. 1967) representing the number of minutes within each 30-min period in which a mosquito made at least one flight of any duration. Activity scores were used to compare the daily activity patterns of infected and uninfected mosquitoes. 19

MATERIALS AND METHODS Aedes aeovpti (black eyed Liverpool strain) mosquitoes were reared and infected with Bruaia pahanai by following a previously described protocol (Christensen and Sutherland 1984). Infected jirds CMeriones unauiculatus). with microfilaremias ranging from 600 to 800 mf/20 ul and 137 mf/20 ul were use to infect 2 groups of mosquitoes, respectively. Different microfilaremia levels were used to provide a wide range in the number of larvae developing in individual mosquitoes.

On day 2 postinfection (PI), 32 mosquitoes were individually placed into acoustic chambers made from modified reagent bottles (Jones et al. 1967). Bottoms of the bottles were removed and replaced with tightly stretched filter paper. Additionally, the bottoms were covered with a tight layer of plastic wrap to maintain a high humidity within the chamber. A microcentrifuge tube, filled with absorbent cotton saturated with 0.3 M sucrose was suspended from the mouth of each chamber to serve as a source of sugar and moisture. Mosquitoes were randomly assigned to chambers and allowed 1.5 days to acclimate. The number of flights, flying time, and an activity score were recorded for each chamber in each 30-min period by a microcomputer (Rowley et al. 1987). At least twice daily, chambers were examined and dead mosquitoes replaced. All 20

32 mosquitoes were replaced on day 8 PI during the first run (mosquitoes infected on a jird with a high microfilaremia), whereas only dead mosquitoes were replaced during the 2nd run (mosquitoes infected on a jird with a low microfilaremia).

The number of B. pahanqi developing in each mosquito was determined by dissection in Aedes saline (Hayes 1953). Dead mosquitoes were dissected immediately upon removal from the chambers. At the end of an experiment, mosquitoes were anesthetized with CO2 and dissected. Developing 1st- and 2nd-stage larvae (L^ and L2) in the thoracic muscle tissue and 3rd-stage larvae (L3) were counted.

Mosquitoes were classified into 4 groups based on the number of filarial worms found during dissection. Analysis of variance was used to evaluate the total daily activity scores, total number of spontaneous flights, and the total flying time by group for each day PI. Totals are the sum over all periods within each day for each mosquito. Studies began on day 4 and continued through day 13 PI. Daily activity patterns and the changes produced by different parasite burdens were compared. 21

RESULTS Bruqia pahanai had a pronounced effect on the spontaneous flight activity of Aedes aeovpti (Fig. 1). The bimodal daily activity cycle (circadian) was not markedly altered, but the amplitudes of the peaks were significantly different (P<0.01). Mosquitoes in all groups demonstrated peak activity in the hours just before the lights were turned off. Activity remained low through the 8-h scotophase. A smaller activity peak commenced immediately after the beginning of the photophase.

Preceding day 8 PI, infected mosquitoes were at least as active as uninfected mosquitoes. During the first 8 days after infection, mosquitoes infected with fewer than 20 worms were significantly more active than uninfected mosquitoes, with daily activity scores ranging up to three times the scores of control mosquitoes. This increased activity can be seen in the increased amplitudes of the principal peaks (Fig. 1). The spontaneous activity of mosquitoes infected with more than 20 larvae was equivalent to that of uninfected mosquitoes during this period.

After day 8, daily activity scores of infected mosquitoes were lower than those of control mosquitoes. Mosquitoes with the greatest parasite intensity (>20 larvae) showed a steady decline in spontaneous activity Figure 1. Circadian flight activity patterns of Aedes aegypti infected with varying numbers of Brugia pahangi larvae on days 4 through 13 postinfection MEAN HOURLY ACTIVITY SCORE

H (O 24 until day 12 PI, when uninfected mosquitoes were 10 times more active. Mosquitoes with low parasite burdens (l-lO larvae) were less active than control mosquitoes on days 8 through 10. After this period of reduced activity, these mosquitoes became more active, with activity levels near those of uninfected mosquitoes. An intermediate parasite burden (11-20 larvae) caused a decline in activity beginning on day 8. After day 11, the daily activity of this intermediate group remained low. In general, higher parasite burdens caused greater reductions in the spontaneous flight activity of the mosquitoes. The highest correlation between number of larvae and total activity score was -0.53 (P<0.001) on day 11. Differences in the number of flights in each group were similar to differences in the daily activity scores

(Table 1). Before day 8 PI, mosquitoes infected with fewer than 20 worms made more spontaneous flights than uninfected mosquitoes. After day 8, the total number of flights made each day by infected mosquitoes was lower than that of uninfected mosquitoes. Mosquitoes infected with fewer than 20 parasites made the fewest flights on day 10, after which they made increasingly more flights. The total daily number of flights made by the most heavily infected mosquitoes continued to decline through the end of the experiment. 25

Table 1. Mean daily number of flights made by Aedes aegypti infected with Brugia pahangj

No. larvae (Mean no. mosguitoes) Control 1-10 11-20 >20 Dav fl8) fl8) ri3) fl3) F& 4 104 296 206 81 0.46 5 83 292 185 88 0.94 6 81 174 136 76 0.27

7 73 140 113 67 0.29

8b 158 99 42 138 - 9 184 95 102 117 1.49 10 196 57 60 52 4.89*

11 166 89 55 26 8.34** 12 108 94 71 10 4.50** 13 128 95 102 24 3.01* value with 3,58 df (on average). ^F value could not be determined. *P<0.05.

**P<0.01. 26

The mean daily flying time of mosquitoes infected with more than 10 larvae remained low after day 10 (Table 2). Mosquitoes infected with fewer than 10 worms flew less on days 8 through 10 PI. After day 10 these mosquitoes flew increasingly more until they were flying as much as control mosquitoes. The daily flying time for mosquitoes infected with more than 10 larvae began to drop on day 8 and remained low through the end of the experiment. The mean duration of each individual flight also varied with parasite intensity. The mean duration of individual flights made by uninfected mosquitoes or by mosquitoes infected with fewer than 10 larvae remained relatively constant, at about 10 sec per flight, throughout the experiment (Table 3). Mosquitoes harboring 11-20 larvae made shorter flights after day 11 PI. The increase in number of flights (Table l) did not produce an increase in total flying time (Table 2). Heavily infected mosquitoes (>20 parasites) also made shorter flights beginning on day 8. The average duration of an individual flight made by mosquitoes infected with more than 10 larvae was reduced by as much as 50% from the average flight length of lightly infected or control mosquitoes.

Mosquitoes infected with more than 30 larvae did not fly at all after day 11. 27

Table 2. Mean total daily flying time of Aedes aeavpti infected with Bruaia pahanai.

No. larvae (mean sample size) Control 1-10 11-20 >20 Day ri8) fl8) fl3) fl3) pa

4 1,367 2,776 2,365 1,218 0.20 5 1,068 3,035 2,305 1,487 0. 36 6 1,174 2,101 2,078 1,484 0.19 7 1,084 1,522 1,417 1,199 0.03

8^ 1,196 910 605 805 - 9 1,745 813 1,067 585 3.87** 10 1,800 523 689 268 6. H 11 1,629 949 407 175 o 12 1,113 968 366 62 7.02*** 13 1.341 1,224 433 108 7.47***

value with 3,58 df (on average), value could not be determined. **P<0.01.

***P<0.001. 28

Table 3. Mean duration (seconds) of individual flights made by Aedes aeavpti infected with Bruqia pahanqi

No. larvae 0 H Dav Control 1 11-20 >20 4 13.1 9.3 11.5 15.0 5 12.9 10.4 12.6 16.9

6 14.5 12.1 15.3 19.5 7 14.8 10.9 12.5 17.9 8 7.6 9.2 14.4 6.1 9 9.5 8.6 10.6 5.0 10 9.2 9.2 11.5 5.2

11 9.8 10.7 7.4 6.7 12 10.3 10.3 5.2 6.2 13 10.5 12.9 4.2 4.5 29

DISCUSSION

The development of small numbers (<10) of Bruaia pahanai had only a mild effect on spontaneous flight activity of aeovpti. Before day 7 PI, lightly infected mosquitoes were actually more active than uninfected mosquitoes. However, activity decreased markedly on days 8 through 10. This decrease corresponds to the later stages of larval development in the flight muscles (Beckett and Macdonald 1971). Most larvae complete development by day 10 or 11 and leave the muscle tissue. After day 10, the total daily activity score of lightly infected mosquitoes was only slightly lower than that of uninfected mosquitoes. However, this reduction, coupled with slight reductions in the number of flights and total flying time, indicates that even low numbers of developing larvae impair mosquito flight ability to some extent.

A higher parasite intensity had a similar, though more pronounced, effect on the spontaneous flight activity of Ae. aeqypti. Moderately infected mosquitoes (11-20 larvae) were less active than either lightly infected or control mosquitoes. Although their activity score remained low, by day 13 these mosquitoes made more flights, of shorter duration, than their lightly infected counterparts. The total daily flying time of this group 30

was significantly reduced (>60%) compared to that of the control or lightly infected mosquitoes. Although moderately infected mosquitoes were still capable of flight, they were incapable of maintaining flight for an extended period. Their flight activity consisted of numerous extremely short flights. Heavily infected mosquitoes (>20 larvae) never recovered their ability to fly. This is most likely associated with parasite-induced flight-muscle damage as described by Beckett (1971b). Daily activity scores, total flying time and total number of flights were all much lower (>80% reduction) than those of uninfected mosquitoes. Mosquitoes harboring more than 30 worms could not fly.

In all cases, a reduction in flight activity began on day 8 PI, closely corresponding to the time of the second larval molt. Late L2's and early L3•s must cause the most muscle damage. Migration of Ig's out of the muscle and into the head capsule also occurs during this period of reduced activity. Possible irritation caused by migrating larvae also might influence spontaneous activity. In more heavily infected mosquitoes, activity, especially total flying time, does not increase after the infective larvae leave the thoracic muscles. However, activity does 31

increase in mosquitoes with lower parasite burdens. The extent of flight-muscle damage in heavily infected mosquitoes must be such that they become incapable of sustained flight. Paige and Craig (1975) speculated that 15 B. pahancri larvae caused enough flight-muscle damage to render an individual mosquito incapable of flight. We found that mosquitoes infected with comparable numbers of larvae were capable of flight but could not maintain flight. These mosquitoes most likely would be classified as nonfliers in tethered flight studies. It also seems unlikely that mosquitoes infected with 10 or more worms would be capable of flying far enough to find a new host and thereby transmit the infection. Certainly, mosquitoes infected with more than 20 larvae are incapable of transmitting the filarial worms because of an inability to sustain flight. Heavily infected mosquitoes undertook very few spontaneous flights after the worms had completed their development. The increased reduction in more heavily burdened mosquitoes is consistent with previous reports on the effects of B. pahanai on flight ability. Townson (1970) found that significantly more infected mosquitoes were nonfliers and that the infection intensity was significantly higher in these nonfliers. Nonfliers were infected with a mean of 18 larvae, while fliers were 32

infected with a mean of 10 larvae. We found that mosquitoes infected with 10 or fewer.larvae spontaneously fly nearly as much as uninfected mosquitoes. Mosquitoes infected with more than 20 larvae do not fly. Hockmeyer et al. (1975) also found that nonfliers had significantly higher parasite intensities.

Before day 7 PI, mosquitoes infected with fewer than 20 larvae were more active than uninfected mosquitoes. These mosquitoes made up to three times as many flights and had higher activity scores. Spontaneous flights of these mosquitoes lasted as long as those of uninfected mosquitoes. Infection with B. pahanai seems to stimulate flight. One possible explanation for this increased activity is that irritation caused by filariae penetrating the muscle tissue stimulates flight behavior. It is becoming increasingly apparent that in estimating vector competence one must take into account the influence of the pathogen on the vector. Although alteration of flight activity patterns was expected in mosquitoes infected with a filarioid nematode that develops in the indirect flight muscles, the possible effects of filarial worms that develop in other sites within mosquito vectors should be determined. 33

ACKNOWLEDGEMENTS

Jirds used in this experiment were provided by John W. McCall, University of Georgia, through a program supported by the U. S.-Japan cooperative Medical Sciences Program- Naiad. This is Journal paper No. J-12046 of the Iowa Agriculture and Home Economics Experiment Station, Ames,

Iowa, Project 2277. This study was supported in part by National Intitutes of Health grant AI 19769 (To BMC). 34

LITERATURE CITED

Beckett, E. B. 1971a. The distribution of developing Bruaia pahanqi and Bruaia malavi larvae within the dorsal longitudinal flight muscles of Aedes aeovpti and Mansonia uniformis. Parasitology 63: 119-124. Beckett, E. B. 1971b. Histological changes in mosquito flight muscle fibres associated with parasitization by filarial larvae. Parasitology 63; 365-372. Beckett, E. B. and W. W. Macdonald. 1970. The distribution of larvae of Bruaia malavi and Bruaia pahanqj in the flight muscle fibres of Aedes aeavpti and Mansonia uniformis. Parasitology 61: 211-218. Beckett, E. B. and W. W. Macdonald. 1971. The survival and development of subperiodic Bruaia malavi and B. pahanai larvae in a selected strain of Aedes aeavpti. Trans. R. Soc. Trop. Med. Hyg. 65: 339-346.

Christensen, B. M. and D. R. Sutherland. 1984. Bruaia pahanai: Exsheathment and midgut penetration in Aedes aeavpti. Trans. Am. Microsc. Soc. 103; 423-433. Hayes, R. 0. 1953. Determination of a physiological saline for Aedes aeavpti (L.). J. Econ. Entomol. 46; 624-627.

Hockmeyer, W. T., B. A. Scheifer, B. C. Redington and B. F. Eldridge. 1975. Bruaia pahanai; Effects upon the flight capability of Aedes aeavpti. Exp. Parasitol. 38; 1-5.

Husain, A. and W. E. Kershaw. 1971. The effect of • filariasis on the ability of a vector mosquito to fly and feed and to transmit the infection. Trans. R. Soc. Trop. Med. Hyg. 65: 617-619. Jones, M. D. R. 1964. The automatic recording of mosquito activity. J. Insect Physiol. 10: 343-351.

Jones, M. D. R., M. Hill and A. M. Hope. 1967. The circadian flight activity of the mosquito Anopheles gambiae: Phase setting by light regime. J. Exp. Biol. 43: 503-511.

Paige, C. J. and G. B. Craig Jr. 1975. Variation in filarial susceptibility among East African populations 35

of Aedes aeavpti. J. Med. Entomol. 12: 485-493.

Rowley, W. A., M. D. R. Jones, D. W. Jacobson and J. L. Clarke III. 1987. A microcomputer monitored mosquito flight activity system. Ann. Entomol. Soc. Am. 80: 534- 538. Townson, H. 1970. The effect of infection with Bruaia pahanqi on the flight of Aedes aeovpti. Ann. Trop. Med. Parasitol. 64: 411-420. 36

SECTION II. INFLUENCE OF DEVELOPING DIROFIIARIA IMMITIS ON THE SPONTANEOUS FLIGHT ACTIVITY OF AEDES AEGYPTI (DIPTERA; CULICIDAE) 37

Influence of Developing Dirofilaria immitis on

the Spontaneous Flight Activity of Aedes aeqypti (Diptera; Culicidae)

William J. Berry Wayne A. Rowley Bruce M. Christensen

From the Department of Entomology, Iowa State University, Ames, Iowa 50011 (Berry, Rowley); the Department of Veterinary Science, University of Wisconsin, Madison, Wisconsin 53706. 38

ABSTRACT Aedes aecfvpti (L.) (Black-eyed Liverpool) mosquitoes were infected with Dirofilaria immitis (Leidy) to determine if larvae developing in Malpighian tubules have an effect on spontaneous flight activity. Studies using acoustic activity chambers showed that developing larvae decreased flight activity of mosquitoes after day 8 postinfection. Parasite intensity (burden) was inversely related to flight activity after day 12 postinfection. Circadian rhythm (activity pattern) of infected mosquitoes was similar to that of uninfected mosquitoes. 39

TEXT

Developing filarial worms significantly affect host mosquitoes and have been associated with increased mortality in Aedes aeovpti (L.), Aedes sollicitans (Walker), and Aedes trivittatus (Coquillett) infected with Dirofilaria immitis (Leidy) (Kershaw et al. 1953, Beam 1966, Christensen 1978). Mortality is higher in more heavily infected mosquitoes. Larvae of Bruqia spp., which develop in the indirect flight muscles, cause a reduction in flight ability of host mosquitoes (Townson 1970, Husain and Kershaw 1971, Hockmeyer et al. 1975) and the spontaneous flight activity of the Liverpool strain of Ae. aeqypti (Berry et al. 1986). Currently, no information is available regarding the influence that filarial worms developing in locations other than flight muscle have on flight ability or spontaneous flight activity of host mosquitoes.

This study investigated the effect of developing D. immitis on the spontaneous flight activity of aeqypti by using an acoustic activity-chamber system (Rowley et al. 1987). Parameters investigated included the effect of parasite intensity on the number of spontaneous flights made by individual mosquitoes, the duration of these flights, the circadian rhythm (activity pattern), and the total flight activity of that mosquito. Comparisons of 40 daily activity were based on an activity score representing the number of minutes within each 30-min period during which a mosquito made at least one flight of any duration (Jones et al. 1967).

Ae. aeqypti (Black-eyed Liverpool strain) were reared following a protocol reported by Christensen and

Sutherland (1984). Adult D. immitis were maintained in beagle dogs. Mosquitoes were infected by feeding on a water-jacketed membrane feeder (Rutledge et al. 1964) containing dog blood with 86 and 101 microfilariae/ 20 ul for groups 1 and 2 respectively. Before filling, the apparatus was fitted with a mouse skin membrane through which mosquitoes could feed. Control mosquitoes were given a blood meal without microfilariae.

On day 3 postinfection (PI), 32 mosquitoes (eight uninfected and 24 infected) were individually placed in acoustic chambers made from modified reagent bottles. Bottoms of the bottles were removed and replaced with tightly stretched filter paper and a tight layer of plastic wrap. A microcentrifuge tube, filled with absorbent cotton saturated with 0.3 M sucrose, was suspended from the mouth of each chamber to serve as a source of both sugar and moisture. Mosquitoes were randomly assigned to chambers and allowed 1.5 days to acclimate. The number of flights, flying time, and 41 activity score were recorded in each 30-min period. Chambers were examined at least twice daily and dead mosquitoes were replaced. Replacement mosquitoes were allowed at least 24 h before being included in the analysis. Flight activity was monitored on days 4-15 PI.

The temperature during the experiment was 27+2°C.

The number of filarial larvae in each mosquito was determined by dissecting the mosquito in Aedes saline (Hayes 1953). Dead mosquitoes were dissected immediately upon removal from the chambers. At the end of an experiment, mosquitoes were anesthetized by refrigeration and dissected. Both developing larvae in the Malpighian tubules and infective larvae in the head capsule were counted.

Mosquitoes were classified into groups based on the number of developing filarial larvae found by dissection.

Statistical analysis consisted of analysis of variance of the total daily activity score, total number of spontaneous flights, and the total flying time by group for each day PI. Totals are the sum over all periods within the day for each mosquito. Daily activity patterns and the changes produced by different parasite burdens were compared. Differences were considered significant at P<0.05.

The spontaneous flight activity of Ae. aegypti 42 mosquitoes infected with D. immitis was lower than that of uninfected controls. The circadian activity pattern was bimodal in both infected and uninfected mosquitoes. Peak flight activity occurred in the hours immediately before lights off (Fig. 1). There was almost no activity through the 8-h scotophase. A second, but smaller peak of activity occurred at the beginning of the photophase. Infection with D. immitis did not change the timing of the principal peaks, but did reduce the amplitudes of the peaks after day 9 PI. Preceding day 9, there was no reduction in the spontaneous flight activity of infected mosquitoes. After day 12 PI, the amplitude of the activity peaks of mosquitoes infected with low numbers (3- 10) of filarial larvae remained constant. However, mosquitoes infected with >10 larvae showed a continued decline in spontaneous flight activity through the end of the experiment. The total daily activity scores (area under the curves) of infected mosquitoes were significantly lower (f-test; df=2,58; P<0.01) than scores for uninfected mosquitoes after day 9 PI. Heavily infected mosquitoes were significantly less active than mosquitoes with lower parasite intensities after day 14 PI.

On days 9 through 13 PI, infected mosquitoes made Figure 1. Spontaneous flight activity of aeovpti infected with D. immitis on days 6 through 15 postinfection 11 -20 LARVAE 30 Jl 20 10

40 3-10 LARVAE

30 20 10

40 UNINFECTED

30 20 10

7 8 9 10 11 12 13 14 15 DAY POSTINFECTION 45 significantly fewer flights than uninfected mosquitoes

(Table 1). After day 13, mosquitoes harboring 3-10 developing larvae made as many flights as uninfected mosquitoes. However, the mean duration of each flight was considerably reduced (Table 2). Mosquitoes infected with >10 larvae made the fewest flights from day 10 through the end of the experiment. Significant differences between mean total daily flying times of infected and uninfected mosquitoes (f- test; df=2,58; P<0.05) were first detected on day 7 PI

(Table 3). Uninfected mosquitoes flew nearly twice as much as infected mosquitoes from day 8 through day 15.

The decline in flying time in uninfected mosquitoes during the experiment was not as great as that in infected mosquitoes. No difference occurred in the total daily flying time of mosquitoes with different parasite intensities before day 10 PI. After day 9, mosquitoes with >10 larvae flew significantly less than mosquitoes harboring 3-10 larvae. Mosquitoes infected with low numbers of worms (3-10) showed a sharp decline in flight activity on day 9 PI. However, their flight activity did not continue to decline as it did in heavily infected mosquitoes.

The principal peaks in the bimodal activity pattern occurred at the same time in infected and uninfected 46

Table 1. Mean daily number of flights made by aeovpti infected with D. immitis^ O Control 3-10 V

II pb Dav PI fn=18\ fn=23\ to H O

6 379 333 327 0.31

7 294 224 232 0.95 8 197 159 147 1.05 9 206 116 120 4.43* 10 166 107 87 4.92** 11 154 99 87 3.78* 12 145 100 87 3.09* 13 150 84 71 6.40** 14 138 136 47 2.16 15 92 104 47 1.35 SgEM for flights =30. bdf=2,58.

*P<0.05. **P<0.01. 47

Table 2. Mean daily flying time for M- aeqypti infected with D. immitis^

Control 3-10 >10 Dav PI fn=18) fn=23) fn=20l pb 6 3,810 2,983 2,854 0.16 7 3,156 2,087 2,246 4.58* 8 2,559" 1,588 1,574 5.76** 9 2,598 1,265 1,262 13.07** 10 2,116 1,232 930 8.96** 11 1,914 1,103 917 6.76** 12 1,851 1,055 846 8.68** 13 1,811 932 602 14.28** 14 1,636 973 354 9.39** 15 1.090 707 312 7.31** &SEM for flying time = 214. ^df = 2,58.

*P<0.05. **P<0.01. 48

Table 3. Mean flight duration (sec) for aecrsrpti

infected with D. immitis^

Control 3-10 >10 Dav PI fn=18) fn=23) fn=20) pb

6 10.1 9.0 8.7 1.89

7 10.7 9.3 9.7 1.09 8 13.0 10.0 10^7 0.44 9 12.6 10.9 10.5 1.07 10 12.7 11.5 10.7 0.74 11 12.4 11.1 10.5 1.28 12 12.8 10.6 9.7 2.43

13 12.1 11.1 8.5 5.92 14 11.8 7.6 7.5 10.41 15 11.8 6.8 6.6 8.25 ®SEM for flight duration = 0.74. ^df = 2,58.

**P<0.01. 49 mosquitoes. The pattern of activity was consistent with previous reports of the circadian activity pattern of Ae. aeovpti (Taylor and Jones 1969, Trpis .et al. 1973, Jones

1981, Berry et al. 1986, Rowland and Lindsay 1986). The reduction in flight activity seen in all infected mosquitoes (beginning on day 9 PI) corresponded to the development of the second stage larvae of D. immitis in the Malpighian tubules. A second reduction in activity in mosquitoes infected with >10 parasites (beginning on day

14 PI) corresponded with the time when third-stage larvae left the tubules. The decline in spontaneous flight activity must be related to damage to the tubules. The greater the damage (i.e., in heavily infected mosquitoes), the more pronounced the effect on spontaneous flight activity.

Unlike aeovpti infected with low parasite intensities of second- and third-stage Bruaia pahanai (Buckley and Edeson) larvae, which recovered and flew nearly as much as uninfected controls (Berry et al. 1986),

Ae. aeavpti infected with 3-10 D. immitis larvae did not regain their flight activity after infective-stage larvae left the Malpighian tubules. Flight muscle disruption by Bruaia larvae is clearly the cause of the reduction in flight activity in infected mosquitoes. Palmer et al. (1986) reported ultrastructural changes in Malpighian 50

tubules of D. immitis-infected mosquitoes, including a decrease in the density of microvilli and a reduction in the number of mitochondria associated with the microvilli. They suggested that excretory and osmoregulatory functions are disrupted by the developing worms. This disruption may be related to the reduced propensity of infected mosquitoes to fly. Several reports indicate that high parasite intensities increase mortality in D. jjnmitis-infected mosquitoes, but this is the first report dealing with the effects of lower parasite intensities. The reduction in spontaneous flight activity of Ae. aeavpti infected with D. immitis indicates that Malpighian tubule disruption caused by even a few filarial larvae alters the normal behavior of this mosquito. The degree of influence depends on parasite intensity. Reduced flight activity should be taken into account when determining vector competence, especially in heavily infected mosquitoes. Dogs with high microfilaremias probably overload prospective vectors. Increased mortality or reduced flight ability could eliminate these heavily infected mosquitoes as vectors of a pathogen or reduce their ability to disseminate the parasite. 51

ACKNOWLEDGEMENTS

Beagle dogs were provided by Robert Grieve (School of Veterinary Medicine, University of Wisconsin, Madison,

Wisconsin). This is Journal Paper No. J-12345 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Project 2277. This study was supported in part by

National Institutes of Health Grant AI 19769 (to B. M. 52

LITERATURE CITED Beam, F. D. 1966. Mortality of Aedes sollicitans Walker due to a high microfilaremia of dog heartworm, Dirofilaria immitis Leidy. Proc. N. J. Mosq. Exterm. Assoc. 53; 117-126. Berry, W. J., W. A. Rowley, B. M. Christensen. 1986. Influence of developing Brugia pahanqi on the spontaneous flight activity of Aedes aeavpti (Diptera: Culicidae). J. Med. Entomol. 23: 441-445. Christensen, B. M. 1978. Dirofilaria immitis; Effect on the longevity of Aedes trivittatus. Exp. Parasitol. 44: 116-123. Christensen, B. M. and D. R. Sutherland. 1984. Brucfia pahanai; Exsheathment and midgut penetration in Aedes aeoypti. Trans. Am. Microsc. Soc. 103; 423-433.

Hayes, R. O. 1953. Determination of a physiological saline for Aedes aecrvpti (L.). J. Econ. Entomol. 46: 624-627. Hockmeyer, W. T., B. A. Scheifer, B. C. Redington, and B. F. Eldridge. 1975. Bruala pahanai; Effects upon the flight capability of Aedes aeavpti. Exp. Parasitol. 38: 1-5.

Husain, A. and W. E. Kershaw. 1971. The effect of filariasis on the ability of a vector mosquito to fly and feed and to transmit the infection. Trans. R. Soc. Trop. Med. Hyg. 65: 617-619.

Jones, M. D. R. 1981. The programming of circadian flight-activity in relation to mating and the gonotrophic cycle in the mosquito Aedes aeavpti Physiol. Entomol. 6; 307-313. Jones, M. D. R., M. Hill and A. M. Hope. 1967. The circadian flight activity of the mosquito Anopheles aambiae; phase setting by light regime. J. Exp. Biol. 43; 503-511.

Kershaw, W. E., M. M. J. Laviopierre and T. A. Chalmers. 1953. Studies on the intake of microfilariae by their insect vectors, their survival and their effect on the survival of their vectors, I. Dirofilaria immitis and Aedes aeavpti. Ann. Trop. Med. Parasitol. 47: 207-221. 53

Palmer, C. A., D. D. Wittrock, and B. M. Christensen. 1986. Ultrastructure of Malpighian tubules of Aedes aeavpti infected with Dirofilaria iitunitis. J. Invert. Pathol. 48: 310-317. Rowland, M. W. and S. W. Lindsay. 1986. The circadian flight activity of Aedes aeavpti parasitized with the filarial nematode Bruaia pahanai. Physiol. Entomol. 11: 325-334.

Rowley, W. A., M. D. R. Jones, D. W. Jacobson and J. L. Clarke III. 1987. A microcomputer-monitored mosquito flight activity system. Ann. Entomol. Soc. Am. 80: 534- 538. Rutledge, I. C., R. A. Ward and D. J. Gould. 1964. Studies on the feeding response of mosquitoes to nutritive solutions in a new membrane feeder. Mosq. News 24: 407-419.

Taylor, B. and M. D. R. Jones. 1969. The circadian rhythm of flight activity in the mosquito Aedes aeavpti (L.): the phase setting effects of light-on and light- off. J. Exp. Biol. 51: 59-70.

Townson, H. 1970. The effect of infection with Bruaia pahanai on the flight of Aedes aeavpti. Ann. Trop. Med. Parasitol. 64; 411-420. Trpis, M., G. A. H. McClelland, J. D. Gillett, C. Teesdale and T. R. Rao. 1973. Diel periodicity in the landing of Aedes aeovpti on man. Bull. W. H. 0. 48: 623-629. 54

SECTION III. SPONTANEOUS FLIGHT ACTIVITY OF AEDES TRIVITTATUS INFECTED WITH EflROFIIARIA IMMITIS 55

Spontaneous flight activity of Aedes

trivittatus infected with Dirofilaria immitis

William J. Berry Wayne A. Rowley

Bruce M. Christensen

From the Department of Entomology (Berry, Rowley), Iowa State University, Ames, lA 50011; the Department of Veterinary Science (Christensen), University of Wisconsin, Madison, Wisconsin 53706. 56

ABSTRACT Spontaneous flight activity of Dirofilaria immitis- infected Aedes trivittatus was evaluated by using an acoustic activity system. The activity of mosquitoes infected with low numbers of filarial larvae (1-4) was similar to that of uninfected mosquitoes. However, mosquitoes infected with more than 4 larvae became more active than uninfected mosquitoes 8 days after infection. Peak flight activity (circadian) occurred at the same time in both infected and uninfected mosquitoes, but infected mosquitoes were much more active during normal periods of quiescence. Flight activity of mosquitoes infected with more than 4 larvae was suppressed on days 10 and 14 postinfection, corresponding to times of greatest disruption of the Malpighian tubules by the developing larvae. 57

INTRODUCTION Aedes trivittatus (Coquillett) is a natural vector of

Dirofilaria immitis (Leidy) in Iowa (Christensen and Andrews 1976, Christensen 1977a,b) and Indiana (Finger 1982). Aedes trivittatus is a floodwater mosquito with a biology similar to that of Aedes vexans Meigan. In Iowa, Ae. trivittatus is locally abundant and a persistent pest of humans. Dogs were identified as a common host of Ae. trivittatus in Indiana (Nasci 1984), indicating that this species contacts D. immitis under natural circumstances.

Developing D. immitis larvae caused increased mortality of Aedes aeavpti (L.) (Kershaw et al. 1953), Aedes sollicitans (Walker) (Beam 1966) and trivittatus (Christensen 1978). Additionally, aeavpti infected with D. immitis were less active than uninfected counterparts (Berry et al. 1987a).

This study was designed to determine if developing D. immitis larvae alter the spontaneous flight activity and flight behavior of trivittatus. a proven natural vector. A computer interfaced acoustic activity system (Rowley et al. 1987) was used to evaluate flight activity. The parameters investigated included monitoring the circadian activity patterns of mosquitoes with different parasite burdens, flying time, the number of flights initiated and an activity score. 58

MATERIALS AND METHODS Aedes trivittatus were reared from eggs laid by field collected females following the protocol described by

Christensen and Rowley (1978). Adult D. immitis were maintained in beagle dogs. Mosquitoes were infected by feeding on a water-jacketed membrane feeder (Rutledge et al. 1964) containing dog blood with 94 and 168 microfilariae/ 20 ul for experiments 1 and 2 respectively.

Before being filled, the membrane feeder was fitted with a freshly prepared mouse skin through which mosquitoes could feed. Control mosquitoes were given a blood meal without microfilariae. On day 3 postinfection (PI), 32 mosquitoes (24 infected and 8 uninfected in experiment 1; 16 infected and 16 uninfected in experiment 2) were placed individually in acoustic chambers made from modified reagent bottles (Jones et al. 1967). Bottoms of the bottles were replaced with tightly stretched filter paper and a tight layer of

plastic wrap. A 1.5-ml microcentrifuge tube, filled with absorbent cotton saturated in 0.3 M sucrose, was suspended from the mouth of each chamber as a source of both carbohydrate and moisture. The photocycle consisted of 16 hr of light, followed by 8 hr of darkness, with a sharp transition between light and dark. Mosquitoes were randomly assigned to chambers and allowed 1 day to 59

acclimate. The number of flights, flying time and an activity score were recorded for each mosquito in each 30- min period. The activity score represented the number of minutes within a period during which a mosquito made at least one flight of any duration (Jones et al. 1967). Activity chambers were examined at least twice daily. Any dead mosquitoes were replaced with mosquitoes of identical age and infection category. Replacement mosquitoes were allowed at least 24 hr to acclimate before being included in the analysis. Flight activity was monitored on days 4-15 PI. The temperature during the experiment was 27±1°C.

The number of D. immitis larvae in each mosquito was determined by dissecting the mosquito in Aedes saline

(Hayes 1953). Dead mosquitoes were dissected immediately upon removal from the chambers. At the end of an experiment, all mosquitoes were anesthetized by refrigeration and dissected. Developing larvae in the Malpighian tubules and infective larvae in the head capsule or hemocoel were counted to determine the parasite intensity for each mosquito. Mosquitoes were grouped according to parasite intensity. Statistical analysis consisted of analysis of variance of the daily activity score, number of flights 60 and flying time for each day PI. Daily activity patterns and changes produced by different parasite burdens were compared. Differences were considered significant at P<0.05. Dead mosquitoes were deleted from the analysis on the day they died, and replacement mosquitoes were not included on the following day. 61

RESULTS Dirofilaria immitis strongly influenced spontaneous flight activity of Ae. trivittatus (Fig. 1). Uninfected mosquitoes had a large peak of flight activity beginning at the onset of the photophase and lasting for 4 hr. A second, smaller peak of activity occurred at the beginning of the scotophase. Uninfected mosquitoes were inactive through most of the scotophase. Mosquitoes harboring developing larvae had the same pattern of activity as uninfected counterparts early in the infection. However, as filarial worms developed (after day 10), activity of infected mosquitoes continued throughout the photophase. Additionally, infected mosquitoes were active throughout the scotophase, but at a much lower level.

Before day 8 PI, infected mosquitoes were as active as control mosquitoes (Fig. 2a). After day 8, mosquitoes infected with more than 4 filarial larvae were significantly more active than uninfected mosquitoes on most days. Though consistently higher, activity scores of lightly infected (1-4 larvae) mosquitoes were not significantly different from those of control mosquitoes throughout the experiment. Daily activity scores of mosquitoes infected with 5 or more filarial larvae were as much as 3 times higher than those of uninfected mosquitoes. Much of the increase in total flight activity Figure 1. Spontaneous flight activity (circadian) of Aedes trivittatus infected with different intensities of Dirofilaria immitis on days 4 through 15 postinfection; Mosquitoes were maintained on a 16L:8D photocycle 30 20 CONTROL 10 .U M âa ]iJV..jd .jJi. .jJLjiijluL

30-1 1-4LARVAE UJ 20 CC O 10 O CO 01 >• 30 W H 5-9LARVAE > 20 5 '0 <

30 10-18 LARVAE 20 10 2WU 5 6 7 8 9 10 11 12 13 14 15 DAY POSTINFECTION Figure 2. Flight activity of Aedes trivittatus infected with different intensities of Dirofilaria immitis on days 4-15 postinfection; a) Activity scores; b) Number of flights; c) Total flying time; d) Duration of individual flights; numbers in parentheses indicate the number of female mosquitoes in each category 400-1 b

* s s 7 8 9 10 11 12 t3 14 15 DaytPottlntoctlon Days PoitlnlActbn m •Control (24) 20- •1-4(17) • 5-9(14) d g 10-18(8)

4 S 6 7 8 9 10 11 12 13 14 15 Days PostiniscUon Days Poallnfsctlon 66 occurred during periods outside the times when the mosquitoes were normally active. However, activity of infected mosquitoes was equivalent to that of uninfected mosquitoes on day 10 PI. On day 14 PI, flight activity of mosquitoes infected with 5 or more larvae was markedly reduced as compared to the level of activity on day 13 or day 15 PI.

Differences in the number of flights made by mosquitoes in each group were similar to differences in activity scores (Fig. 2b). The number of flights made by individual mosquitoes increased through the experiment in all groups. Mosquitoes infected with 5 or more filarial larvae made more flights than uninfected mosquitoes, but not until after day 8 PI. Infected mosquitoes made fewer flights on day 10 and again on day 14 PI. This change in spontaneous flight activity is reflected in the activity scores for these days (Fig. 2a). Infected mosquitoes spent more time flying than uninfected mosquitoes (Fig. 2c). However, the differences were not as great as the differences noted in the number of times a mosquito actually initiated a spontaneous flight. This indicated that infected mosquitoes made more, but shorter flights (Fig. 2d). The time that infected mosquitoes spent flying decreased markedly on day

10 and again on day 14 PI, especially in mosquitoes 67

harboring 5 or more developing larvae. The mean length of individual flights made by uninfected mosquitoes was 9-10 sec on most days. Mosquitoes infected with fewer than 5 D. immitis larvae made flights of similar duration (9-10 sec) throughout the experiment. Individual flights made by more heavily infected mosquitoes were of shorter duration after day 8 in moderately infected (5-9 larvae)

and after day 6 in heavily infected (10-18 larvae) mosquitoes. 68

DISCUSSION Developing filarial worms have a pronounced effect on the spontaneous flight activity and flight ability of mosquito hosts. Larvae of Bruaia pahanai. developiong in the flight muscle of aegypti. cause a loss of flight

ability (Townson 1970, Paige and Craig 1975). Hockmeyer et al. (1975) reported that B. pahanai-infected Ae. aegypti flew significantly less than uninfected mosquitoes and that a greater number of infected mosquitoes died or were unable to fly. Husain and Kershaw (1971) found that B. malavi had a similar effect on the flight ability of Aedes togoi. Developing B. pahangi and D. immitis

also caused a reduction in the circadian flight activity of infected aegypti (Berry et al. 1986, 1987a, Rowland and Lindsay 1986). All previous studies found that

developing filarial larvae reduced flight ability or flight activity of host mosquitoes. In the study, D. immitis stimulated flight activity of trivittatus. Aedes trivittatus is a natural vector of dog heartworm and the effect of the parasite on this species is markedly different from that observed in the experimental vector, Ae. aegytpj.

The circadian pattern of flight activity of uninfected Ae. trivittatus was similar to that seen in previous laboratory studies using an identical light regime (Berry 69

et al. 1987b). The bimodal circadian activity pattern consisted of a peak of flight activity at lights on and a smaller peak at lights off. Field studies have indicated that these are the times of peak activity of Ae. trivittatus (Thompson and Dicke 1965, Wright and Knight

1966). Beginning on day 9 PI, infected mosquitoes remained active throughout the photophase, and some activity was maintained during the scotophase. The principal peaks (as seen in the controls) were evident, but flight activity continued through periods when this species is normally inactive. In contrast, aecrvpti. infected with D. immitis. were less active than uninfected controls, but the timing of activity was identical in infected and uninfected mosquitoes (Berry et al. 1987a).

Total flight activity of trivittatus increased slightly during the experiment. A similar trend was reported in a previous laboratory study (Berry et al.

1987b). The mean duration of each flight remained the same so the trend was evident in all variables. Activity scores and numbers of flights of the infected mosquitoes increased during the experiment, but the length of individual flights decreased. Consequently, an increase in the number of times a mosquito flew did not lead to proportionately greater amounts of time spent flying. 70

Peterson (1980) found that activity scores, number of flights and flying time tend to yield similar hourly patterns of flight activity. In this study, the duration of individual flights made by mosquitoes infected with 5 or more larvae was shorter than that of uninfected mosquitoes. The pattern for each variable was different. In previous studies, filarial worms, B. pahanai or D. immitis. caused Ae. aeovpti to make shorter flights (Berry et al. 1986, 1987a).

Flight activity of infected mosquitoes was sharply depressed on days 10 and 14 PI. Christensen and Hollander (1978) found that second- and third-stage D. immitis first appear on days 8 and 11, respectively, when infected Ae. trivittatus are held at 26.5°C. The majority of first- stage larvae molt to the second stage between days 8-10, and third stage larvae appear on days 11-14 PI (Christensen 1977b). Similar develpmental periods have been reported for D. immitis in Aedes triseriatus (Say) and vexans (Fortin and Slocombe 1981). Palmer et al.

(1986) found extensive damage to Malpighian tubule cells of D. immitis-infected mosquitoes by days 8-10 PI. The activity of infected mosquitoes was reduced at times corresponding to specific periods in the development of filarial larvae. Reduced flight activity on day 10 PI corresponded with the molt from first to second stage, and 71

the decrease in activity on day 14 corresponded with the time when infective larvae migrated to the mouthparts, but many larvae were still in the Malpighian tubules at that time. In comparison, flight activity of aeavpti was suppressed when B. pahanqi molted but not when D. immitis molted (Berry et al. 1987a)

Increased flight activity of infected mosquitoes could be associated with an irritation related to activity•of the larvae. Alternatively, activity might be stimulated by a reduction in the function of the Malpighian tubules. The periods of reduced activity might be caused by release of waste materials generated by the filarial larvae into the mosquito.

Increased flight activity undoubtedly enhances the possibility that the mosquito will contact a new host.

Flight activity outside normal periods of activity also might enhance contact with host animals. Differences in the influence of D. immitis on flight activity of Ae. aeqypti as compared with Ae. trivittatus probably are related to the natural relationship between Ae. trivittatus and D. immitis. indicating that natural hosts might be influenced differently than experimental vector species. 72

ACKNOWLEDGEMENTS Beagle dogs were provided by Robert Grieve (School of

Veterinary Medicine, University of Wisconsin, Madison).

This study was supported in part by National Institutes of Health Grant No. AI 19769. , This is Journal Paper No. J- 13069 of the Iowa Agriculture and Home Economics

Experiment Station, Ames, lA; Project 2277. 73

LITERATURE CITED

Beam, F. D. 1966. Mortality of Aedes sollicitans Walker due to a high microfilaremia of dog heartworm, Dirofilaria immitis Leidy. Proc. N. J. Mosq. Exterm. Assoc. 53: 117-126.

Berry, W. J., W. A. Rowley and B. M. Christensen. 1986. Influence of developing Bruaia pahanai on spontaneous flight activity of Aedes aeavpti (Diptera; Culicidae). J. Med. Entomol. 23: 441-445.

Berry, W. J., W. A. Rowley and B. M. Christensen. 1987a. Influence of developing Dirofilaria immitis on the spontaneous flight activity of Aedes aeovpti (Diptera; Culicidae). J. Med. Entomol. 24; 699-701. Berry, W. J., W. A. Rowley, J. L. Clarke III, N. S. Swack and W. J. Hausler Jr. 1987b. Spontaneous flight activity of Aedes trivittatus (Diptera; Culicidae) infected with trivittatus virus (Bunyaviridae; California serogroup). J. Med. Entomol. 24: 286-289. Christensen, B. M. 1977a. Observations on the epidemiology of Dirofilaria immitis in urban Ames, Iowa. Proc. Helminthol. Soc. Wash. 44: 226-228.

Christensen, B. M. 1977b. Laboratory studies on the development and transmission of Dirofilaria immitis by Aedes trivittatus. Mosq. News 37: 367-372.

Christensen, B. M. 1978. Dirofilaria immitis: Effect on the longevity of Aedes trivittatus. Exp. Parasitol. 44; 116-123.

Christensen, B. M. and W. N. Andrews. 1976. Natural infection of Aedes trivittatus (Coq.) with Dirofilaria immitis in Central Iowa. J. Parasitol. 62; 276-280.

Christensen, B. M. and A. L. Hollander. 1978. Effect of temperature on vector parasite relationships of Aedes trivittatus and Dirofilaria immitis. Proc. Helminthol. Soc. Wash. 45; 115-119. Christensen, B. M. and W. A. Rowley. 1978. Observations on the laboratory biology and maintenance of Aedes trivittatus. Mosq. News 38: 9-14.

Fortin, J. F. and J. 0. D. Slocombe. 1981. Temperature 74

requirements for the development of Dirofilaria immitis in Aedes triseriatus and Ae. vexans. Mosq. News 41; 625-632. Hayes, R. O. 1953. Determination of a physiological saline for Aedes aeovpti (L.). J. Econ. Entomol. 46: 624-627. Hockmeyer, W. T., B. A. Scheifer, B. C. Redington and B. F. Eldridge. 1975. Bruaia pahanai: Effects upon the flight capability of Aedes aeavpti. Exp. Parasitol. 38: 1-5.

Husain, A. and W. E. Kershaw. 1971. The effect of filariasis on the ability of a vector mosquito to fly and feed and to transmit the infection. Trans. R. Soc. Trop. Med. Hyg. 65: 617-619. Jones, M. D. R., M. Hill and A. M. Hope. 1967. The circadian flight activity of the mosquito Anopheles qambiae; Phase setting by the light regime. J. Exp. Biol. 43: 503-511. Kershaw, W. E., M. M. J. Lavoipierre and T. A. Chalmers. 1953. Studies on the intake of microfilariae by their insect vectors, their survival and their effect on the survival of their vectors, I. Dirofilaria immitis and Aedes aeavpti. Ann. Trop. Med. Parasitol. 47: 207-221. Nasci, R. S. 1984. Variations in the bloodfeeding patterns of Aedes vexans and Aedes trivittatus (Diptera: Culicidae). J. Med. Entomol. 21: 95-99. Paige, C. J. and G. B. Craig Jr. 1975. Variation in filarial susceptibility among East African populations of Aedes aecrvpti. J. Med. Entomol. 12: 485-493. Palmer, C. A., D. D. Wittrock and B. M. Christensen. 1986. Ultrastructure of Malpighian tubules of Aedes aeavpti infected with Dirofilaria immitis. J. Invert. Pathol. 48: 310-317. Peterson, E. L. 1980. The temporal pattern of mosquito flight activity. Behaviour 72: 1-25.

Pinger, R. R. 1982. Presumed Dirofilaria immitis infections in mosquitoes (Diptera: Culicidae) in Indiana, USA. J. Med. Entomol. 19: 553-555. 75

Rowland, M. W. and S. W. Lindsay. 1986. The circadian flight activity of Aedes aeavpti parasitized with the filarial nematode Bruaia pahanai. Physiol. Entomol. 11: 325-334.

Rowley, W. A., M. D. R. Jones, D. W. Jacobson and J. L. Clarke III. 1987. A microcomputer-monitored mosquito flight activity system. Ann. Entomol. Soc. Am. 80: 534- 538.

Rutledge, I. C., R. A. Ward and D. J. Gould. 1964. Studies on the feeding response of mosquitoes to nutritive solutions in a new membrane feeder. Mosq. News 24; 407-419.

Thompson, P. H. and R. J. Dicke. 1965. Sampling studies with Aedes vexans and some other Wisconsin Aedes (Diptera: Culicidae). Ann. Entomol. Soc. Am. 58: 927- 930.

Townson, H. 1970. The effect of infection with Bruaia pahanai on the flight of Aedes aeovpti. Ann. Trop. Med. Parasitol. 64: 411-420.

Wright, R. E. and K. L. Knight. 1966. Effect of environmental factors on biting activity of Aedes vexans (Meigan) and Aedes trivittatus (Coquillett). Mosq. News 26: 565-578. 76

SUMMARY AND CONCLUSIONS Filarial larvae were found to have profound effects on flight activity of host mosquitoes, regardless of the location of the developing larvae. The effects were dependent on parasite intensity in all systems, with flight activity being more strongly influenced in mosquitoes harboring a greater number of developing larvae. Flight activity of a natural vector was altered differently than that of a laboratory vector when the mosquitoes were infected with the same parasite.

Not surprisingly, larvae of Bruaia nahanai. developing in the flight muscle of Aedes aeovnti. inhibited flight activity of the host mosquitoes. Low numbers of parasites (<10) caused a reduction in flight activity on days 8-10 postinfection. Parasite intensity was inversely correlated with flight activity. Mosquitoes infected with more than 20 larvae could not sustain flight after day 8 and those infected with more than 30 larvae did not fly. Mosquitoes infected with less than 10 larvae recovered the ability to fly after day 10. Nevertheless, developing B. pahanai had little influence on the circadian activity pattern of aeavpti. The magnitude of flight activity was altered but not the timing. Working independently and concurrently, Rowland and Lindsay (1986) obtained nearly identical results using the same filarial worm/host 77

mosquito model and a similar method of studying mosquito flight behavior.

The second question related to the location of development of the filarial larvae. Filarial larvae developing in flight muscle could be expected to directly influence flight activity by tissue disruption. However, larvae developing in Malpighian tubules could only indirectly affect flight. Flight activity of Aedes aeqypti infected with Dirofilaria immitis was greatly reduced after day 8 postinfection. As with B. pahanai- infected aeovpti. the reduction in flight activity was greater in mosquitoes infected with greater numbers of parasites. Again, the circadian pattern of activity was not altered. Infected mosquitoes were less active than uninfected mosquitoes, but the activity occurred during the same periods. Both of these systems involved a laboratory host for the filarial larvae; aeovpti is not a natural vector of either parasite.

The final study dealt with a filarial worm and host mosquito that are associated under natural circumstances. Aedes trivittatus is a natural vector of D. immitis. The D. immitis larvae developing in the Malpighian tubules of Ae. trivittatus caused an increase in flight activity.

Low numbers of developing larvae had little influence on 78

flight activity of host mosquitoes. However, mosquitoes

infected with more than 4 larvae were more active after day 8 postinfection. The greatest amount of flight activity occurred at the same time in infected and uninfected mosquitoes, but infected mosquitoes were also active during normal periods of quiescence. This increased activity, both the amount and timing, would undoubtedly enhance contact between infective mosquitoes and potential hosts. The differences in the influence of D[. immitis on aeovpti and trivittatus are probably related to the natural association between D. immitis and

Ae. trivittatus Natural hosts are likely to be influenced differently by parasite infection than laboratory hosts. Future studies should be directed at determining the basis for altered flight activity in parasite infected mosquitoes, especially when the filarial larvae are developing in locations other than flight muscle. However, regulation of flight in mosquitoes is not clearly understood at this time. Flight may be regulated by biogenic amines. Furthermore, comparison of flight

activity of D. immitis infected Ae. aeavpti with that of D. immitis infected gie. trivittatus indicates that different mechanisms may operate in different species. 79

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ACKNOWLEDGEMENTS I would like to thank the members of my committee;

Drs. Wayne A. Rowley, Harold J. Stockdale, Robert E. Lewis, John A. Mutchmor, George W. Beran and Kenneth B. Piatt, for the time and effort required to read this dissertation and examine me.

Most especially, I would like to thank Dr. Wayne A. Rowley, for providing me with an excellent opportunity to develop professionally. I know that in the future I will have fond memories of and good things to say about Iowa State.

I also owe a debt of gratitude to Dr. Bruce M. Christensen of the University of Wisconsin. Dr. Christensen collaborated on all three projects presented here. He provided helpful advice and reviews of the papers.

Several graduate students deserve special mention for their help with ideas and day to day projects, especially Mark Novak, Sayadeth Khounlo and John Clarke. I also must thank the numerous work/study and summer employees; most notably Susan Zylstra. Last, and far from least, I would like to thank ray wife, Carole L. Trybus, for her support and encouragement during our years as Ph.D. candidates.