Aspects of the Ecology of the Common Banded Mosquito, Culex Annulirostris, a Major Vector of Murray Valley Encephalitis Virus

Home , Culex annulirostris

ASPECTS OF lHE ECOLOGY OF lHE CCJrvM>N BANDED r-t:>SQUITOJ CULEX ANNULIROSTRIS~ A MAJOR VECTOR OF MURRAY VALLEY ENCEPHALITIS VIRUS

GARRICK McDONALD

Dip.Ag.Sci. (Dookie), B.Sc. (La Trobe}

A thesis presented in fulfilment of the requirements of the degree of Master of Agricultural Science, in the Faculty of Agriculture and Forestry, University of Melbourne.

December, 1981. TABLE OF CONTENTS

TABLE OF CONTENTS i

ABSTRACT iii

ACKNOWLEDGEMENTS AND STATEMENT OF SOURCES vi

LIST OF FIGURES viii

LIST OF TABLES xi

CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW 1

1.1 CuZex annuZirostris Skuse 1 1.1.1 Importance 1 1.1.2 Distribution 3 1.1.3 Description 4 1.1.4 Life cycle and biological studies 8 1.1.5 Habitat preferences 10 1.1.6 Relative abundance 11 1.1.7 Host preferences 12 1.1.8 Vector status and disease epidemiology 12 1.2 Age specific life and fecundity tables and PoPUlation growth potential 15 1.2.1 The theory 15 1.2.2 Use in mosquito studies 18

CHAPTER 2: EXPERIMENTAL 20

General Introduction 20 2.1 The influence of temperature on development, survival and fecundity of CuZex annuZirostris in the laboratory 21 (a) Juvenile development and survival 22 (b) Adult survival and fecundity 23 (c) ·Age specific life and fecundity tables, and population" statistics 27 2.2 The behaviour of Culex annuZirostris larvae in the laboratory 42 2.3 Field studies of juvenile CuZex annuZirostris 47 2.3.1 .Ecology of Culex annulirostris within a fresh water pond - Mildura 48 (a) Colonization, larval survival and development 50 (b) Time-specific life tables 50 (c) Larval distribution 51 2.3.2 Ecology of Culex annulirostris within a fresh water swamp - Shepparton 63 (a) Development and survival 63 (b) Larval distribution 64 2.4 Field studies of adult Culex annulirostris 68 2.4.1 Seasonal abundance of mosquito populations 69 (a) Culex annulirostris 71 (b) Other species 75 2.4.2 Nightly activity and abundance of Culex annulirostris 78

CHAPTER 3 : GENERAL DISCUSS ION 81

3.1 The bionomics of juvenile Cx annulirostris 81 3.1.1 Larval development 81 3.1.2 Larval survival and predation 82 3.1.3 Colonization 84 3.1.4 · Mosquito control 85 3.2 Population ecology of Cx annulirostPis 88 3.2.1 The effect of temperature on population growth 89 3.2.2 Population development of Cx annulirostris in relation to the transmission of M.V.E. virus 91 3.3 Alternative vectors of M.V.E. virus in the Murray Valley 102 3.4 Conclusions 103

REFERENCES 106

APPENDICES 114 ABSTRACT

In the Mildura area of the Murray Valley, the mosquito Culex annuZiroatris is the dominant summer species. It becomes active in mid-spring and reaches a peak during late February or early March, declining rapidly during April. To examine the population dynamics of

Cx annuZirostris in detail, a laboratory colony was established to measure population growth potential at various constant temperatures, and the results were related to adult and juvenile populations in the field, monitored during 1975-78. In the laboratory, population growth potential was determined by compiling age specific life and fecundity tables at

15°, 20°, 25° and 30°C.

Population growth was positive at 20°, 25°, and 30°C, greatest at

25°C, and negative at 15°C. All larvae died at 10° and 40°C, and survival was greatest at 25°C. Graphic interpolation of the population growth statistic r (the intrinsic rate of natural increase), at r = O, m m provided a means of estimating the temperature threshold of population growth. This temperature was 17.50 c. Field observations confirmed this result as the mean daily temperature at which spring populations commenced growth, and autumn populations ceased activity, was approximately

17.50 c.

Field.studies 'of colonization and juvenile development and survival within an experimental pond and· an established swamp wer.e conducted over two years. Within the pond, colonization by Cx annulirostris occurred within 24 hours of it being filled with water, and larval densities were greatest six or seven days after filling. A gradual decline over the remaining six to eight weeks of the study was attributed to increasing predation and, during the second year, also to declining temperatures.

Larval mortality between egg and adult eclosion was 89% (average) , daily

rates of mortality being reasonably consistent throughout development.

Similar studies in the swamp demonstrated that 75% of larvae survived

to eclosion in the absence of predators, and less than 1% survived in the

presence of predators. Developmental rates of larvae in the pond and swamp

varied considerably between about eight and eleven days. These results

did not appear to comply with laboratory observations, possibly due to

inaccuracies in field temperature records. In the laboratory, studies of

larval behaviour indicated that larvae spent at least 87% of their time

suspended from the water surface. It was suggested, in the absence of

detailed field studies, that developmental rates of larvae may be

influenced strongly by the surface temperature of the water, particularly

if the larvae changed this behaviour in the event of surface temperature

extremes.

Cx annu~irostris was consistently found breeding in shallow and

fresh bodies of water containing dense vegetation. In fresh water,

the occurrence of vegetation was the major influence on larval distribution.

The differences in population growth of Cx annulirostria observed

over the study seasons, and also reported from years of M.V.E. epidemics, were explained using models compiled from laboratory and field data. The

first model was based on the finite rate of natural increase (antilog e r ,. or A.) and provided a prediction of population growth for the three m seasons over which monitoring took place. The actual data used included

the size of the initial spring catches and the mean weekly temperatures recorded for each season. In each case, the predicted population increased at a similar rate to that of the observed populations during spring and early summer of each year. However, beyond December, the predicted rate of growth was substantially greater than that of the observed populations.

This model indicated that (i) the size o.f overwintering populations, (ii) the date of onset of activity in spring and (iii) the availability of breeding grounds during summer may be significant influences on the growth rate and size of summer populations.

A second model indicated how various combinations of temperature and water availability may give rise to abnormally large populations of

Cx annulirostris, and hence assist in triggering epidemics of M.V.E.

Although conjectural, these models may provide a starting point for the design of a programme to predict M.V.E. epidemics in the Murray Valley.

A total of 13 species were identified from adult trapping studies, with Culex pipiens australious being the second most common species.

Circumstantial evidence indicates that this species may be an important amplification vector of M.V.E. virus prior to epidemics. ACKNOWLEDGEMENTS AND STATEMENT OF SOURCES

The results presented in this thesis are from original research

conducted at the Plant Research Institute, Burnley, and at the Horticultural

Research Station, Mildura. I am indebted to my supervisors, Dr. I.W. McLaren,

Mr. G.A. Buchanan and Mr. G.P. Shelden for their helpful advice and

guidance throughout this research project, and for their criticisms

of the manuscript. I am also grateful to Dr. B.H. Kay, Entomologist,

Queensland Institute of Medical Research, and Mr. P.M. Ridland and

Mr. A.M. Smith, Entomologists, Plant Research Institute, Burnley for their

constructive comments on the manuscript; and Dr. E.N. Marks, Entomologist,

Q.I.M.R., and Mr. J. Blyth, National Museum, Victoria for their

identification of mosquitoes and predatory insects.

I am grateful for the valuable technical assistance and

co-operation received from Ms. c. Ricke and Mr. I.R. Smith in performing the

laboratory studies. I would also like to thank Mr. R. Jardine, Biometrician,

for assistance with the statistical content of the thesis, Mr. P. Hardy

and Mr. F. Zudich for assistance in the Shepparton swamp studies;

Ms .. D. Dittmer and Ms. J .. Kelly for their help in preparing the graphs and

Ms. s. Broughton and Mrs. C. Ridland for typing the manuscript.

Finally, I wish to thank Jenny McDonald for her assistance, patience and support during all operations of this res.earch project.

The light t~aps were placed on the properties of the Victorian

Forest Commission, Mildura, and Mr. J. Whiting of Gol Gol (N.S.W.). All of the work described was my own except for the section of the thesis dealing with juvenile development and survival in the laboratory, which was done in co-operation with Mr. I. Smith. Aspects of this project have been submitted and accepted for publication, and these are cited in Chapter 2 and Appendices I and II.

Garrick McDonald LIST OF FIGURES

No. Page

1 Cx annuZirostris egg raft containing approximately 320 eggs. 5

2 Cx annuZirostris larvae. Fourth and third instar. 6

3 Cx annuZirostris adult. 7

4 Water bath designed to maintain a constant temperature for the rearing of mosquito larvae in glass beakers. 24

5 A guinea pig held in a retaining stock to allow uninterrupted feeding of Cx annutirostris females. 26

6 Cages used to study the longevity and fecundity of Cx annuZirostris. 26

7 The relationship between water temperature and rate of juvenile development of Cx annuZirostris. 29

8 The effect of seven constant water temperatures on the survival of the juvenile stages of Cx annuZirostris. 32

9 Age-specific longevity (1 b and fecundity (m ) of Cx annuZirostris at (a) 2~ , {b) 25° and (c)x30°C. 35

10 The intrinsic rate of natural increase (r ) of 1• t · o o o m c.x annu~~ros r~s at 20 , 25 and 30 c. 37

11 The stable age distribution of juvenile and adult 1" t. 0 0 0 0 c:x annu~~ros ~s at 15 , 20 , 25 and 30 c. 38

12 (a) Experimental pond at the Mildura Horticultural Research Station, February, 1977. 49

(b) Layout of vegetation and transects within the pond. 49

13 Daily changes,of mean water temperature and population densities of juvenile Cx annutirostris and aquatic predators during February-April in (a) 1977 and {b) 1978 in a pond at Mildura 53

14 Age-specific stage distribution simulating time specific survivorship curves for juvenile Cx annuZirostris in a Mildura pond during February 1977 •. 54 No. Page

15 The distribution of first instar larvae, and second, third and fourth instar larvae and pupae across the pond. 62

16 Distribution and size of Cx annuZirostris larval populations per sample throughout Reedy Swamp, Shepparton. 67

17 The effect of water depth on the abundance of Cx annuZirostris larvae in Reedy Swamp, Shepparton. 67

18 A 240 volt light and suction trap used to monitor mosquito populations. 70

19 (a) A light trap survey of populations changes of Cx annuZirostris and other mosquito species during 1976-77 in Mildura. 73

(b) Weekly temperatures and precipitation during 1976-77 at Mildura, recorded at the Meteorological Bureau. 73

20 (a) A light trap survey of populations Changes of Cx annuZirostris and other mosquito species during 1977-78 in Mildura. 74

{b) Weekly temperatures and precipitation during 1977-78 at Mildura, recorded at the Meteorological Bureau. 74

21 Changes in population densities of Cx annuZirostris as measured by two light traps at Mildura during 1977-78. 76

22 The effect of five constant water temperatures on the duration of juvenile development of Cx annuZirostris in the laboratory, in comparison to the duration of development in the field at Shepparton. 83

23 A typical habitat for juvenile Cx annuZirostris observed at Reedy Swamp, Shepparton. 83

24 Life cycle and treatment schedule of Cx annuZirostris under optimum field conditions. 86

26 Computer spline curves fitted to the mean weekly temperatures of 1950, 1973, 1974 and a 12 year average at Mildura. 96 No. Page

27 The hypothetical effect of water availability (e.g. depth) on the growth of larval and adult populations of Cx annutirostris. 96

28 Rainfall at Mildura from August to.February in 1950-51 and 1973-74 compared with a 25 year mean. 98

29 River heights and the flood levels for most of the major floods at Mildura since 1950. 98

30 A conjectural model illustrating the combined effects of temperature and water availability (e.g. depth) on population growth and abundance of Cx annuZirostris. 100

31 A contour graph, or plan view of Fig. 30, illustrating how the contrasting conditions within the spring of 1950, 1955 and 1973 produce similar threshold densities of Cx annutirostris and epidemics of M.V.E. 100 - A..L--

LIST OF TABLES

No. Page

1 Survival and rate of development of the juvenile stages of Cx annuZirostris at seven constant temperatures. 31

2 Fertility and size of egg rafts laid by Cx annuZirostris females at four temperatures. 34

3 Population statistics and parameters for Cx annuZirostris derived from life and fecundity 0 0 0 table studies conducted at 20 , 25 and 30 C, and from life table studies at 15°C. 34

4 A comparison of mean adult life time, net reproductive rate and intrinsic rate of natural increase between strains of Cx quinquefasciatus and Cx annulirostris. 40

5 Cx annuZirostris larval behaviour study at 22°c. 45

6 Insect fauna, identified to lowest possible taxon, sampled from the Mildura pond during February, March and April, 1977 and 1978. 55

7 Age distributions of Cx annulirostris derived from instar durations and observed frequencies. 58

8 Life table for immature Cx annuZirostris. 58

9 Mortalities of Cx annulirost~s developmental stages. 59

10 Minimum, maximum and mean daily water temperatures recorded during cage trials in Shepparton and Mildura during February 1976 and February-March 1977 respectively. 60

11 Emergence times of adult Cx annulirostris and percentage of larvae surviving to emergence at Reedy Swamp, Shepparton 1976. 66

12 Numbers of female mosquitoes caught in a light trap at the Forest Commission nursery, Mildura 1975-76. 72

13 Summary of Cx annuZirostris population data as indicated by catches in a light trap between 1975 and 1978. 72 No. Page

14 Species composition of the mosquito fauna occurring in the 1975-78 ·summers in the Mildura district. 77

15 Numbers of Cx annuZirostris caught in a Mildura light trap emptied twice nightly during February and March 1977. 80 CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW

·1.1 CuZex annuZirostris Skuse

The common banded mosquito, CuZex annuZirostris Skuse (1889) is a widespread and abundant mosquito found throughout most temperate and semi-arid zones of Australia, with the exception of southern Victoria and

Tasmania. The species has been the major source of arbovirus isolations in Australia, and in particular, is deemed the major vector of the disease

Murray Valley encephalitis (M.V.E.) (Doherty 1977).

Throughout northern Victoria, Cx annulirostris not only passively disrupts domestic life styles by its persistent and annoying biting, but the diseases it transmits have caused great hardship and financial loss to individuals and communities. In the most recent outbreak of Murray Valley encephalitis in southern Australia, twelve Australians died, and many more suffered permanent brain damage. The costs to the community of this disease arise directly from the care of patients, and indirectly from the dramatic reduction in tourism, an industry from which the entire community benefits.

To control this disease effectively, the vector, Cx annuZirostris, must first be controlled, and this requires a detailed knowledge of its biology and habits. This report investigates some of the biological attributes of the species which help contribute to its success as a major vector of mosquito-borne diseases ·in Australia.

1.1.1 Importance

Cx annuZirostris is an important, if not the major vector of five medically significant arboviruses and eight other arboviruses of lesser significance (Doherty 1977) • It is also a significant vector of at least four veterinary pathogens. During the most recent nation-wide epidemic of Murray

Valley encephalitis (1974), 58 cases and 12 deaths were recorded (Doherty 1977).

Although other arboviruses and diseases spread by this mosquito have gained greater attention in recent years, M.V.E. is still the most significant disease. Many people afflicted with M.V.E. have either failed to recover completely, or have died. Although it is difficult to document all the personal, medical and social implications of thi~, disease, the approximate costs to individuals and the local economy of Murray Valley towns may be determined:

(a) Medical

Average medical costs .for an M.V.E. patient (initial diagnosis,

80-100 days hospitalization, ambulance) $6,290

(This does not include costs required to

rehabilitate or institutionalize patients)

Estimate of medical costs of Victoria's 29 M.V.E. patients

from the 1974 epidemic $182,320*

(b) Tourism

Tourism is the second largest industry within Sunraysia, the

largest being the dried vine fruit industry. Following the

outbreak of M.V.E. in 1974, the net receipts of the businesses

involved with tourism (five·surveyed) fell by approximately

24% over six months. During the financial year 1977-78, it

was estimated the the Sunraysia tourist industry had a

* Survey data provided by Medical Superintendents of the Fairfield

Hospital for Communicable Diseases, and the Mildura Base Hospital. turnover of approximately $70M. If tourism was

affected by M.V.E. during this period in a similar way

to that of 1974, the industry would suffer a loss

in turnover of $8,400,000**

Although the main carriers of this disease appear to be migratory water birds, Anderson (1954) suggests that the only means of controlling the virus is to control the vector. More recently, Cx annulirostris was found to be the likely major vector (Doherty et aZ. 1963) • Despite extensive studies of Cx annuZirostris in Queensland (Kay 1978a) , our understanding of its ecology in the Murray Valley derives mainly from field observations made during studies of myxomatosis during the early 1950's

(Myers 1955). More detailed studies were therefore desirable.

1..1.2 Distribution

Cx annuZistrostris is widely distributed throughout Australia and the

South Pacific. Standfast and Barrow (1968) have observed it inhabiting rainforest, swamp forest, mangrove forest and farmland in northern Australia.

Kay (1978a) found it common or predominant in 23 localities distributed throughout Queensland. Myers (1954) reported that Cx annuZirostris was common across the Riverina, N.S.W., whilst Fenner and Ratcliffe (1965) stated that it occurred in every district on the seaward side of the Great

Dividing Range in N.s.w., and was locally common in s.w. South Australia 1

** Survey data provided by the Victorian Tourist Bureau (Mild.) ,

the Grand Hotel, the Central Flag Motor Inn, th~ Deakin Caravan Park,

and the Cross Roads Caravan Park. although not in S.E. South Australia. In Victoria the species is most

common north of the central highlands (Dobrotworsky 1965). O'Gower (1958)

recorded the species in the Northern Territory and north west Western

Australia, and also observed it in localized regions of south west

Western Australia.

Cx annuZirostris is also distributed widely throughout Indonesia,

the Philippines, the South Pacific islands (Laird 1956, Belkin 1962, Geottel et aZ. 1980) and throughout the low-lands of New Guinea (Peters and Christian

196 3) .

1.1.3 Description

Eggs of Cx annuZirostris are laid on the water surface in an eggraft containing 50-350 eggs (Fig. 1). There are four larval instars that can be distinguished by head capsule measurements (McDonald et aZ. 1977b,

Appendix II) • The fourth instar larva (Fig. 2a) moults to an active but non-feeding pupa.

Some of the distinctive characters of the adult female Cx annuZiros

(Fig. 3) described by Dobrotworsky (1965) are: proboscis dark with white band near middle; palps dark with white tips; wings dark scaled; legs dark, femora mottled, some pale scales on tibiae and narrow basal white bands on tarsi. Sternites white scaled with apical lateral patches of black scales.

However, uninterrupted apical dark banding on abdominal sternites, rather than lateral patches, may occasionally appear as natural variants {Kay 1978b).

The features of the larvae given by Dobrotworsky which most readily characterize the species include pale antennae with dark tips, broad head and a long slender siphon with six pairs of ventral hair tufts. A further unique - .,) -

Fig. 1: Cx annuZirostris eggraft (X 6) containing approximately

320 eggs. (a)

(b)

Fig. 2: Cx annuZirostris larvae. (a) Mature fourth instar (X 18) and

(b) third instar (X 32) . Fig. 3: Cx annu~irost~s adult. - d -

character of first, second, third and early fourth instars is the darkening

of tergites on abdominal segments three and five {unpublished data) (Fig. 2b).

1.1.4 Life cycle and biological studies

Biological studies of the developmental stages of Cx annulirostris

have been fragmentary, and are mostly reported in a wide range of general

publications. However, the following temperature studies were recorded

in annual reports of the Queensland Institute of Medical Research (QIMR)

Cx annulirostris required 29-41 days to complete a generation

(adult-adult) at water temperatures of between 8.5° and 14.5°C {QIMR 1975,

Kay 1979b). Laboratory studies at 30°C showed that Cx annuZirostris laid

eggs three days after receiving a blood meal, and adults emerged nine days

later. At 25°C, oviposition occurred four days after feeding, and a further 13

days were required for development to the adult stage (QIMR 1976) • Under

field conditions, larvae died when the water temperature exceeded 45.5 0 c,

a factor which enabled the species to dominate less tolerant species, such

as Culex fatigans Wiedemann (thermal death point 42°C) (QIMR 1967) •

In the South Pacific, Laird (1950) reported that Cx annuZirostris

could tolerate water temperatures of 390 C.

Age-grading studies of female Cx annulirostris in northern Queensland during the early 1960's showed that the mosquitoes had a probability of 0.85 of surviving one day, indicating a life expectancy of 6.2 days (QIMR 1964).

These studies were based on the number of ovariole dilatations which indicated

the number of three to six day gonotrophic cycles completed by each female

(Clements 1963). More recent studies by Kay (1979a)on Cx annuZirostris from

Kowanyama (north Queensland) and Charleville (south Queensland) indicated - 9 - ! ~ ! + + mean survival rates of 72.1- 10.1% (S.D.) and 73.8- 11.7% per day respectively ... l Over the 1974-1976 study seasons, Kay found that only 12.4% and 18.2%, 1

respectively, of subsampled females contained two or more ovariole dilatations.

This suggested that those females had taken at least three blood meals and were therefore potentially old enough to transmit M.V.E. virus.

Reproductively active females were present in both localities throughout

the year, indicating the possibility of continuous virus transmission in

these areas. In contrast, Shelden et at. (1977) and Marshall (1979) found

very few overwintering Cx annuZirostris in the Murray Valley, and none of

these were reproductively active. There appears to be no indication of

autogeny in Cx annuZirostris (Kay l978a), i.e. the ability of females to lay

their first eggraft without a prior blood meal (Clements 1963) .

Cx annuZirostris adults become active just after dusk (Myers 1954,

Fenner and Ratcliffe 1965) , but flight activity continues during the night with

a second peak of activity occurring at dawn (QIMR 1969). A similar pattern was observed by Standfast (1969) in New Guinea.

Although Cx annutirostris is widely distributed and one of the chief non-domestic pest species in Australia (Marks 1973) , its inability to exist under many environmental extremes on the Australian continent suggests that it is a "soft'' species. In the open plains of the Riverina, it rarely ranges beyond the vegetation which frin~;es its breeding ground,· as it is most sensitive to low humidity (Fenner and Ratcliffe

1965). Marks (1967) suggested that it is not well adapted to the more arid areas of Australia, such as Alice Springs. Hamlyn-Harris (1930) and McDonald et aZ. (1977a) demonstrated that larvae will not tolerate water putrefaction, and Myers (1954) inferred that fresh areas of water will not be colonized unless they become vegetated. In contrast, however, Liehne et aZ. (1976a) and Laird (1956) suggested that the species is ubiquitous in its selection of breeding sites. - 10 -

1.1.5 Habitat preferences

Throughout its range of distribution, there is substantial variation in the habitats selected by Cx annutirostris. In his South Pacific survey, Laird (1956) noted its ability to colonize a wide variety of habitats including ponded streams, transient and permanent ground water pools, large simple containers, brackish and polluted waters. The species did not appear to show a preference for water containing vegetation. In another South Pacific survey, Belkin (1962) reported that the species would readily colonize most forms of ground water including strongly brackish and stagnant water.

In Queensland, Cx annulirostris is less flexible in its habitat requirements. Although Standfast and Barrow (1968) noted that it

"tended to breed in pools polluted with domestic wastes", most reports suggest that it favours transient or permanent shallow grassy pools of rain, irrigation or flood water (Hamlyn-Harris 1930; QIMR 1964, 1967, 1974;

Standfast and Barrow 1969; Marks 1973).

Studies of myxomatosis in southern Australia demonstrated that

Cx annulirostris populations increase rapidly after heavy rainfall or flooding during summer or autumn (Myers 1954, Myers 1955, Fenner and

Ratcliffe 1965). Other reports suggest that irrigated pastures, grassy pools of rain and flood water, water in hoof prints and the weedy margins of waterholes, rivers, irrigation drains and stockwater dams are the most favoured habitats in N.s.w., northern Victoria and s.w. South Australia (Reeves et al. 1954, Dobrotworsky 1965, Edmonds and Goes 1970, Laughlin pers. commun.). - 11 -

1.1.6 Relative abundance

Cx annuZirostris is one of the most common Australian mosquitoes and

is particularly abundant in areas which are closely associated with water courses, which have high annual rainfall or which have been

settled or farmed by man. In northern Queensland, Standfast and

Barrow (1968) found that it was the most common of the species trapped, particularly in the wet season. More recent studies by Kay (l979b) have yielded similar results. Of the 47 species collected at Kowanyama (N. Qld),

Cx annuZirostris made up about 44 +- 17% (S.D.) of the dry and transitional

season collections and 27 ! 11% of the wet season collections. At Charleville

r( ..,. • • + + ( s. Qld), vX annu~~rostr~s cornpr~sed 90 - 8% and 68 - 13% of dry and wet seasons collections, respectively, which included 71,791 mosquitoes in 20 species. Marks (1976b) also reported that it was the main pest species of

the Brisbane area from February to April.

The plains of the Eastern Riverina were dominated by Cx annuZirostris

(88.4%) and Anopheles annulipes Walker (11.6%) during January and

February 1951 (Myers 1954). Reeves et al. (1954) made similar observations, noting that Cx annulirostris made up about 40% of the total catch of

16 species. More recent studies along the Murray Valley during two flood years (1974 and 1975) and a dry year (1976) showed that between

50% and 98% of the mosquitoes trapped were Cx annulirostris (Shelden et al.

1977, Marshall and Woodroofe 1974).

Cx annulirostris dominated the Kimberley's region of north Western

Australia, comprising of between 65-97% of the Ord River Valley catch (Wright et aZ. 1981, Liehne et at. 1981) and 85% of the Derby area catch (Wright 1981) during sampling in 1976-77. Similarly, the species made up 85% of the mosquito population in Darwin (QIMR 1974). - 12 -

In the South Pacific, Belkin (1962) reported that Cx annulirostris was a dominant form actively extending its range throughout the islands.

Laird (1950) suggested that it was the only native species to successfully

rival the two cosmopolitan species Aedes aegypti Linnaeus and CuZex

fatigans Wiedemann. Throughout the low-lands area of New Guinea,

Cx annuZirostris made up 11.5% of the mosquito fauna which consisted of 63 species, but it was not found in the highlands (Peters and Christian 1963).

Cx annulirostris remains active throughout the year in Queensland and north Western Australia (Standfast and Barrow 1968, Kay 1979b, Liehne et aZ. 1976a) whereas in southern Australia the species is common only during late spring, summer and autumn (Dobrotworsky 1965, Myers 1954). The species appears to overwinter as an adult (Myers 1955, Shelden et aZ. 1977,

Marshall 1979).

1.1.7 Host preferences

Studies in Queensland have shown that Cx annuZirostris prefers to feed on calf, pig or dog to man and kangaroo. Domestic fowls were least preferable (Kay et al. 1979a, QIMR 1976) • In southern Australia, the species has been shown to readily attack sheep (Muller and Murray 1977) , jew lizards (Mackerras 1962), cattle and rabbits (Lee et aZ. 1954, Myers 1954,

Reeves et aZ. 1954) and guinea pigs and mice (McDonald et aZ. 1977a) . From bait preference experiments in the Ord River Valley, Western Australia,

Cx annulirostris favoured rabbits over guinea pigs and chickens (Wright i981) .

Kay et al. (1979a) stated that it was surprising that fowls were relatively unattractive to Cx annulirostris in most of these studies, given the suggested role of birds in the epidemiology of M.V.E. virus.

1.1.8 Vector status and disease epidemiology - 13 -

Cx annulirostris has been the most prolific source of arboviruses in the Murray Valley (Marshall and Woodroofe 1974) , Queensland (Doherty

1972) and in Western Australia (Liehne et al. 1981). Kay et al. (1975a) demonstrated that at least 18 and possibly 20 arboviruses from Queensland and

New Guinea multiplied within the tissues of wild Cx annuZirostris in the laboratory after oral infection. These include the medically significant viruses of M.V.E., Ross River, Sindbis, Kunjin.and Sepik. In the field, 94 arbovirus strains and 15 arboviruses have been found and isolated from

Cx annulirostris (Doherty 1977, Doherty et al. 1979). Liehne et al. (1976b) found that 0.8% of Cx annulirostris trapped in the Ord River Valley in 1974 were carrying viruses of the M.V.E. or Kunjin group.

The species also transmits disease organisms of veterinary significance. Cx annulirostris is one of the two main mosquito vectors of myxomatosis in rabbits throughout southern Australia (Myers 1954, Fenner and Ratcliffe 1965) • CUlex annulirostris and Anopheles annulipes were responsible for the initial rapid spread of myxomatosis in rabbits throughout the Murray Valley and Darling basins following the first release of the pathogen in 1950-51. The outbreak of the disease was restricted to the areas of activity of these mosquitoes, and has only been extended with the use of ectoparasites, such as the European rabbit flea (Tighe et al. 1977). Similarly,

Cx annuZirostris is a significant vector of Dirofilaria immitis (heartworm) to dogs (Bemrick and Moorhouse 1968) , a disease which is also restricted to those regions of southern Australia where this mosquito inhabits. Laboratory and field studies have confirmed the probable role of Cx annulirostris in the transmission of bovine ephemeral fever to cattle (Kay et al. 1975a, standfast et al. 1976) and Eperythrozoon ovis to sheep (Daddow 1980).

However the most serious disease transmitted by Cx annulirostris is Murray Valley encephalitis. Epidemics of M.V.E. have occurred in 1917, - 14 -

1918, 1922, 1925 (Australian "X" disease), 1951 and 1974 (Murray Valley

encephalitis). Localized outbreaks have also occurred in 1956 (Mildura),

1971 (Riverina), 1978 and 1980 (Kimberleys, W.A.) (C.D.I. 1978, 1981). The

species was initially incriminated as a potential vector on circumstantial

grounds during the early 1950's (Anderson and Eagle 1953, Reeves et al.

1954), although attempts to isolate virus failed. M.V.E. virus was first

transmitted experimentally using several species including Cx annulirostris~

by McLean (1953, 1957). However the virus was not recovered from wild

Cx annulirostris until Doherty et al. (1963) screened 11,595 females

collected from northern Queensland. The virus was finally isolated from

CX annulirostris in the Murray Valley (Marshall and Woodroofe 1974) during

the 1974 epidemic of M.V.E. Kay et al. (1979b) confirmed the efficiency

of the species in transmitting M.V.E. virus. In comparison with Aedes aegypti~ a significant cosmopolitan vector of arboviruses, Cx annulirostris was highly susceptible to oral infection of M.V.E. virus, and could transmit

virus from 7 days post-infection, with maximum infection after 10 days.

The location of virus reservoirs between epidemics is not clearly

understood, although some virus activity has been demonstrated in northern

Queensland (Doherty 1972) and north west Australia (Liehne et al. 1981)

between epidemics. Miles and Howes (1953) 1 Anderson and Eagle (1953) and

Forbes (1978) postulated that epidemics in the Murray Valley arose through

virus importation, via birds, from the endemic foci of M.V.E. virus in northern Australia. In Queensland, Cx annulirostris maintains breeding cycles throughout the year, and is capable of transmitting virus during all seasons

(Standfast and Barrow 1968, Kay 1980) • · However, attempts to isolate virus during the dry seasons have failed (Doherty et al. 1968) • Marshall (1979) suggests that the inter-epidemic survival of M.V.E. virus may take place in the temperate region of the Murray Valley itself, possibly through transovarian - 15 -

transmission in Aedes spp. In support of this, Gard et aZ. (1976) demonstrated that wild pigs carried antibodies to M.V.E. virus between epidemics.

1.2 Age specific life and fecundity tables and population growth potential

1.2.1 The Theory

The maximum or exponential rate of growth of a population in a defined environment is quantified by the intrinsic rate of natural increase, r (Birch 1948, Andrewartha and Birch 1954). Age-specific life and m fecundity tables compiled by recording all the births and deaths during the entire life of a population are used to calculate r and other related m indices of population growth.

Similar statistics were first used by human demographers (Latka 1925) but were later modified for use in mammalian (Leslie and Ranson 1940) and insect (Birch 1948) populations. However, medical entomologists have recognised more recently, the value of life tables in determining population growth potential of a number of mosquito species and also in distinguishing strains within a species complex (W.H.O. 1967).

To compile age-specific life and fecundity tables, the following records and calculations are required (Southwood 1966) :

1 the proportion of the original cohort surviving at the X beginning of the age class x (where 1 = 1) • For the purpose . 0 of calculating a fecundity table, 1 is the proportion of the X original female cohort surviving to the beginning of age

class x. - 16 -

m the number of live female progeny produced per female during X the age interval x.

In practical terms, 1 and m are determined at the end of each age class; X X the length of the age class depending on the expected life of the population and the accuracy required. Thus 1 and m are commonly recorded at hourly X X or daily intervals, throughout the life of the cohort. After all individuals in the cohort have ceased reproducing, the table can be used to calculate the following parameters relating to the population under study:

(a) Net reproductive rate (R ) 0 This is an estimate of the number of times a population will multiply per generation and is best expressed as the ratio of total female births in two successive generations such that

R where NT is the number of individuals at the end of 0 a generation or at time T and N is the number at the 0 beginning of the generation (or at time 0) .

R may also be determined from the age specific fecundity and survival 0 rates using the formula:

00 R = f 1 m dx, and in practice is calculated using the formula: 0 0 X X

R = El m • 0 X X

(b) Intrinsic rate of natural increase (r ) m

When the population's age structure is st~le, and it is growing under unlimited conditions then

dN dt = where e is the base of natural logarithm and T is time. - 17 -

NT 00 As R = -= f 1m dx 0 N 0 X X 0

00 r x flmdx=e m 0 X X -r x m f ooe 1 m dx = 1. 0 X X

An approximation of this equation increases the ease of solution and in practice is solved by trial and error substitutions of a range of possible r values using r as an initial estimate. m c

(c) Capacity for increase (r ) c This statistic was originally derived by Andrewartha and Birch (1954) as an approximation to the more precise r . However Laughlin (1965) has m re-defined it as the "capacity for increase" , as it is more easily calculated and is still useful in indicating the potential for multiplication.

It is calculated as follows:

log R e o r where T is the mean age of reproductive females in a c T population with a stable age distribution, or cohort

generation time when generations do not overlap.

(d) The finite rate of natural increase {A)

The number of times that the population increases per unit of time is given by N + 1 r T m A = e = antilog r NT e m

(e) Cohort generation time (T ) c T is given by c log R e 0 T c = r m - 18 -

1.2.2 Use in mosquito studies

In an attempt to determine how Aedes aLbopiatus Skuse is able to

competitively displace Aedes poZynesiensis Marks, Gubler (1970) undertook

comparative studies of the reproductive potential of the two species.

His findings were in accordance with De Bach's (1966) suggestion that

competitive displacement will occur when one species has an R value 0 greater than its ecological homologue. However, Hacker (1972) points

out that for multivoltine mosquito populations with overlapping

generations, R does not adequately express the biological potential 0 of a population, as it only describes the number of times the population

will increase per generation and not per unit of time. He demonstrated

that although Ae. aZbopiotus and Ae. poZynesiensis have markedly different

R values, their r values may be similar as Ae. poZynesiensis has a o m shorter generation time.

Another research group, led by Dr. C.S. Hacker, has used life tables

to distinguish strains of mosquito species. Crovello and Hacker (1972)

showed how population growth potential differed markedly between urban

and feral strains of Aedes aegypti. They concluded from this that the

risk of an individual Ae. aegypti surviving to reproduction is higher in

the urban environment than the feral, as the former had consiqerably higher r and R values. They suggested that the forest (feral) habitat m o provided a more ·homogenous and favourable environment, and thus the reproductive pressure on the strain was not as great.

Similarly, Walter and Hacker (1974) have successfully used life and

fecundity tables to demonstrate differences between three geographic strains of CuZex pipiens quinquefasciatus Say. All strains had similar - 19 -

survival curves, but differed in the mean life expectancy, R and 0 r values. It was again suggested that the strains with higher reproductive m potentials arose from more hazardous and less consistent environments than

those with lower R and r values. o m - 20 -

CHAPTER 2 : EXPERIMENTAL

General Introduction

Until recently, attempts to study the biology and vector status of

Culex annuZirostris have been hindered by the failure to establish a laboratory colony (Doherty 1974). Hence, the success in establishing a colony in Victoria (McDonald et aZ. 1977a, Appendix I) provided a means of examining those biological attributes of the species which have enabled it to dominate much of the Australian mosquito fauna.

The population size and growth rate of any animal is influenced by the modifying effects of the environment in which it lives. In the present study, the population growth potential of Cx annuZirostris was measured by compiling age specific life and fecundity tables in the laboratory at different temperatures (Section 2.1). The swimming, eating and resting behaviours of larvae were studied in the laboratory, and the field implications considered (Section 2.2). Environmental factors, particularly temperature, predation, and the presence of vegetation were studied in relation to natural populations of immature Cx annuZirostris in a specifically constructed pond, and in a large permanent swamp (Section 2.3). Light traps were used to monitor adult population densities of Cx ~nnuZirostris over three years, (Section 2.4) as this data provided the basic field information required to complete a study on the bionomics of the species. These studies are discussed in relation to the control of Cx annulirostris in south eastern

Australia, and the prediction of large populations of Cx annuZirostris and subsequent outbreaks of M.V.E. (Chapter 3).

In both laboratory and field environments, it was necessary to the immature and adult populations separately as they exist. in different media. - 21 -

Section 2.1: The influence of temperature on development, survival and fecundity of Culex annulirostria in the laboratory.

Material from this section has been published under the title "The effect of temperature on the population growth potential of Culex annulirostria Skuse (Diptera: Cul.i,.cidae)" in the Australian Journal of Ecology (1980) §_, 379-384. - 22 -

Introduction

The role of temperature in controlling the rate of development and reproduction in populations of poikilothermic animals is fundamental.

In mosquitoes, temperature influences growth rate and survival of larvae, and the survival, longevity, fecundity, host seeking behaviour, reproductive behaviour, diapausing behaviour and the physical size of adults (Clements

1963) . These factors influence the population growth potential of a species (Birch 1948) . To determine the effect of temperature on the population growth potential of Culex annuli~ostris~ it is necessary to construct life and fecundity tables (see Chapter 1.2.1).

Materials and Methods

Mosquitoes used in the laboratory studies were obtained from the colony at the Plant Research Institute, Burnley, and reared according to the methods described by McDonald et al. (1977a) (Appendix I).

(a) Juvenile development and survival

The duration of embryonic development was determined by placing twenty egg rafts into each of four beakers of tap water for incubation at 15 0 , 20 0 , 25 0 and 30 0 c. The numbers of egg rafts hatching were recorded at two hourly intervals during the day for the first three days,. and then twice daily for the next seven days. Egg rafts not hatching after this period were considered infertile or dead. As the eggs hatched synchronously from each raft and thereafter the raft rapidly disintegrated, it was not - 23 -

possible to calculate mortality or fertility of individual eggs.

Larval survival and development rates were studied in 1000 ml beakers

containing 500 ml of tap water. The beakers were maintained at c1 constant

temperature by immersion in a water bath (Fig. 4). Ten replicates of 20

0 0 0 0 first instar larvae were studied at 10 1 15 , 20 and 25 C; and ten replicates

of 30 larvae were studied at 30° 1 35° and 40°C.. A·t daily intervals 20 mg

Dog Chow-Yeast (DCY - Appendix I) was added to the 30 0 , 35 0 and 40 0 C

treatments until pupation wa~ complete or until all larvae had died. Daily

. 0 0 0 0 additions of 20 mg DCY were made to the 10 , 15 , 20 and 25 C treatments

for the first 12 days, but as development was slow, subsequent additions were made on alternate days to prevent putrefaction of the medium due to a build

up of unused food. ~

I/ The media were aerated by bubbling air through 27-gauge dental

needles at 150 ml/min. Fluorescent lighting provided illumination

(1000 lx at water surface) for 13 hours daily, providing conditions suitable for growth of micro-organisms which supplemented the food supply.

All larvae were counted daily and their stage of development recorded. Where necessary, a sample of larvae was removed and the stages identified by measuring the width of their head capsules (McDonald et al. 1977b, Appendix II).

(b) Adult survival and fecundity

The survival and fecundity of adults was studied at 20°, 25° and

30°C, using one cohort of approximately 1000 adults at each temperature. - 24 -

~ /

Fig. 4: Water bath designed to maintain a constant temperature for the rearing of mosquito larvae in glass beakers. - 25 -

An adult study was not conducted at 15°C because the 97% larval mortality

at 15 0 C (see Section 2.1 (a)) made it impracticable to obtain a cohort of

1000 adults.

Similar studies on Aedes aegypti (Crove~lo and Hacker 1972,

Lansdowne and Hacker 1974) and CuZex pipiens quinquefasaiatus (Walter and

Hacker 1974) used small replica cohorts of adults (25 male and 25 female)

which were taken at random .from a large population of adults within one day of

emergence. However this method was not possible with Cx annuZirostris,

which reproduces readily in the laboratory only at high population densities.

+ 0 Temperature control in the rearing rooms was regulated (-1 C) by

a 2400 W fan assisted heating element and a thermostat. Relative !~ humidity was maintained at 60 ~ 5% by a portable steam generator / (C.S.I.R.O. design), a humidistat and a fan which provided continuous

circulation of air and steam. Twin tubed fluorescent banks were

placed along both sides of the room, above the cages. The lighting regime \ was 13 hours full illumination (1000 lx in the cages) , two hours "dusk"

and one hour "dawn" at low illumination (both were 50 lx in the cages)

and eight hours darkness. Guinea pigs, restrained in stocks (Fig. 5), provided

a blood source during the "dusk,. period, six days a week. An oviposition dish (10 em diam., 1 em deep) containing tap water was kept in the cage at all times.

For each of the three temperatures (20 0 , 25 0 and 30 0 C), larvae were grown in rearing trays submerged in water baths to maintain constant temperatures. During the four days after pupation had begun, pupae were removed individually, counted and placed in emergence trays (Appendix I).

These trays, complete with aeration probes, were then placed in 60 x 60 em nylon mesh cages (Fig. 6). Between 800 and 1200 pupae, depending on their - 26 -

Fig. 5: A guinea pig held in a retaining stock to allow uninterrupted feeding of Cx annulirostris females.

Fig. 6: Cages used to study the longevity and fecundity of Cx annuli~ostris. - 27 -

availability, were placed in each cage, and thereafter dead pupae were counted and removed daily, until all had emerged. The number of adults successfully emerging represented the size of the initidl cohort.

Adult mortality was measured by removing dead adults from the cage each day and counting and sexing them. The rate of egg production

(fecundity) was determined by removing and counting egg rafts from the oviposition dishes each morning, and by counting the number of resulting larvae. Rafts which did not hatch were recorded as infertile. These procedures continued until all adults had died.

Age specific life and fecundity tables were compiled for the three temperatures, 20°, 25° and 30°C, using the records of larval and adult mortality and adult fecundity. In addition, a table was constructed for the species at 15°c, but this was done by combining observed juvenile life table data at 15°c with 20°C adult life and fecundity data. The table and statistics derived by this method are only approximations, but do provide an estimate of maximum values at this temperature.

The proportion of the cohort,surviving each day, from egg hatch

(1 ) , was calculated by combining survivorship data from the juvenile X and adult studies using the following formula:

s. . s ax 1 = J X n where s. = proportion of original cohort surviving to adult emergence, J s = number of adults surviving at age x ax and n = size of initial cohort. - 28 -

As the eggs were laid in rafts, and fertility was based on rafts

and not individual eggs, m (number of females born per female on day x) X was calculated thus:

n ex rn = X 2 . S ax

where n number of fertile eggs laid at age x. ex =

This formula assumes a 50:50 sex ratio, which was experimentally confirmed.

The daily 1 and m values for each temperature were then analysed X X

by the "AGSECT" compu~er program, prepared by R. Jardine, Biometrician,

Department of Agriculture. This program was designed to calculate R 0 (net reproductive rate) , r (intrinsic rate of natural increase) , m r (capacity for increase) , (finite rate of increase) , cohort generation c A time and the stable age distribution. A complete description of the

derivations and meanings of the statistics is presented in Chapter 1.2.1.

Results and Discussion

(a) Juvenile

There was a significant linear relationship between rate of larval development and temperatures from 15° to 30°C (P = 0.01) (Fig. 7).

The developmental rate observed at 35 0 C was less than the value derived

from the regression model. This may have been due to the cumulative injurious effects of a continuous exposure to high temperatures

(Huffaker 1944). Stinner et al. (1974) demonstrated that a sigmoidal curve would more accurately account for the reduction in development rate which is usually found at both temperature extremes. 0· 14 ,...... -I>.._ 0· 12 ..... c: Q) .'> E 0· 10 Ojo Q. / .2 0·08 Q) " > ~+ Q) ~"' . "'C 0·06 ~ '+- 0 ~ 0·04 Q) ..... ('(I a: 0·02

0·00 0 10 20 30 40 Temperature CCl

Fig. 7: The regression of water temperature and rate of juvenile development of Cx annuZirost~s (P = 0.01, coefficient of determination 100R2 = 98.3%) 0 - data not included in regression model. - 30 -

Larval survival was low at 15°C, but increased rapidly at 20°c, being greatest at 25°c. Survival was still high at 30°C, but declined

to zero at 40°C. At each temperature except 25° and 30°C, mortality was greatest during the first instar (Table 1, Fig. 8) • It was not technically possible to determine egg mortality as non-viable egg rafts may have been the result of either egg mortality or infertility.

At all temperatures, about 14% of the immature development time was required for egg development. The larvae spent more time in Lhe fourth instar than in any other stage of development. This was also observed by Brust

{1967) with three other species (Aedes vexans (Meigen), Aedes nigrimaauZis

(Ludlow), and CuZiseta inornata (Williston)). Clements (1963) suggests that the lengthening of the fourth instar may reflect an increase in differentiation during that stage.

The theoretical threshold of development, extrapolated from Fig. 7, was 9.5°C. However this is lower than the actual thermal death point which was between 10° and 15°C {Fig. 8).

For each temperature, a thermal constant (k) (Clement9 1963), was calculated using the equation:-

k = t (T - C) where t = development time (days) T = temperature and c = threshold of development (oC)

T°C 15° 20° 25° 30° 35° k(degree - days) 176.0 153.5 178.0 157.6 186.6

The mean thermal constant was 166.6 degree - days, calculated from the four temperatures which were linearly related to rate of development. - 31 -

Table 1: Survival and rate of development of the immature stages of Cx annuZirostris at seven constant temperatures.

Temp. Stage Time (days) % time No. alive at % mortality Proportion (OC) take to spent the end of in stage surviving complete in stage until end stage from stage of stage oviposition (lx) (Mean ± SE)

10° Egg 1st 0 100 0

15° Egg 5.00 14.0 *200 1.00 1st 12.57±0.08 20.1 75 62.5 0.39 2nd 18.01±0.21 14.8 45 15.0 0. 26 3rd 24.13±0.36 16.5 23 11.0 0.14 4th 33.60±0.88 25.7 9 7.0 0.05 Pupae 37.00±0.39 9.2 6 1.5 0.03

20° Egg 2.25 13.4 200 1.00 1st 5.27±0.05 18.0 147 26.5 0.72 !!!:: 2nd 7.17±0.05 11.3 141 3.0 0. 71 / 3rd 9.85±0.09 15.9 140 0.5 0.70 4th 13.83±0.12 23.8 128 6.0 0.64 Pupae 16.82±0.11 17.8 118 5.0 0.59

25° Egg 2.00 14.8 200 1.00 .,.,. 1st 4.33±0.03 17.3 179 10.5 0.90 ~ 2nd 5.88±0.04 11.5 174 2.5 0.83 3rd 7.47±0.08 11.8 150 12.0 0.78 4th 11.80±0.14 32.1 135 7.5 0.67 Pupae 13.48±0.11 12.5 131 2.0 0.65

30° Egg 1.37 14.8 300 1.00 1st 2.37 10.8 290 3.0 0.97 2nd 3.37 10.8 261 9.6 0.75 3rd 4.37 10.8 191 23.3 0.64 4th 7.28±0.08 31.5 178 4.3 0.59 Pupae 9.25±0.07 21.3 168 3.0 0.56

35° Egg 1.25 14.6 300 1.00 1st 2.25 11.7 106 64.6 0.35 2nd 3.25 11.7 99 2.3 0.33 3rd 4.25 11.7 93 2 •. 0 0.31 4th 6.84±0.13 30.2 85 2.6 0.30 Pupae 8.57±0.07 20.2 77 2.6 0.26

40° Egg 0 100 0 1st

* Egg mortality was not calculated. - 32 -

1.0 ,. / 1st ins tar .;' 2nd 0.9 3rd c:n 4th c: pupae ·:; ·:;... 0.8 \ :;, \ en \ 0.6 c: .Q ...... 0 0.4 Q. 0 a.. Q. 0.2

0 40 10 15 20 25 30 35 Temperature (oC)

Fig. 8: The effect of seven constant water temperatures on the survival of the juvenile stages of CUlex annulirostris. - 33 -

This constant provides a means of comparing developmental rates of different

species; it also provides a means of calculating the duration of development

under any combination of temperature and time, and therefore has application

in planning control schedules where the timing of larvicide treatments are

critical.

The following mean thermal constants were calculated from similar

studies on other species:

114.0 (Aedes aegypti Bar-Zeev 1958)

146.4 (Aedes triseriatus Jalil 1972)

271.0 (Aedes aomrrrunis (d') Haufe and Burgess 1956)

318.0 (Aedes aommunia (~). Haufe and Burgess 1956) . i:

.-1'

(b) Adult

0 0 0 Fertility was high at 20 and 25 C, and lower at 30 C. Fecundity

{eggs laid per raft) did not vary significantly between temperatures (Table 2) .

At 20° and 25°C, male mortality was markedly greater than female mortality, particularly during the first 14 days following adult emergence

(Fig. 9). At these temperatures, there was approximately ten days difference between male and female longevity. There was no appreciable difference between mean longevity of males and females at 30 0 C, and the males had a

similar longevity to those at 20° and 25°C. In addition, the females maintained at 20° and 25°C had a mean longevity of approximately 15 days

0 0 more than at 30°C (Table 3) • At 20 and 25 c, the longest surviving adults

died about 70 days after emergence, and at 30°C died 38 days after emergence.

Walter and Hacker (1974) and Lansdowne and Hacker (1974) also showed

that mean expected longeyi ty of males was significantly lower - 33 -

This constant provides a means of comparing developmental rates of different species; it also provides a means of calculating the duration of development under any combination of temperature and time, and therefore has application in planning control schedules where the timing of larvicide treatments are critical.

The following mean thermal constants were calculated from similar studies on other species:

114.0 (Aedes aegypti Bar-Zeev 1958)

146.4 (Aedes triseriatus Jalil 1972)

271.0 (Aedes aommunis (~) Haufe and Burgess 1956)

318.0 (Aedes aommunis (~). Haufe and Burgess 1956).

(b) Adult

0 0 0 Fertility was high at 20 and 25 c, and lower at 30 C. Fecundity

(eggs laid per raft) did not vary significantly between temperatures (Table 2) •

At 20° and 25°C, male mortality was markedly greater than female mortality, particularly during the first 14 days following adult emergence

(Fig. 9). At these temperatures, there was approximately ten days difference between male and female longevity. There was no appreciable difference

0 between mean longevity of males and females at 30 C, and the males had a similar longevity to those at 20° and 25°C. In addition, the females maintained at 20° and 25°C had a mean longevity of approximately 15 days

0 0 more than at 30°C (Table 3) • At 20 and 25 c, the longest surviving adults died about 70 days after emergence, and at 30°C died 38 days after emergence.

Walter and Hacker (1974) and Lansdowne and Hacker (1974) also showed that mean expected longeyity of males was significantly lower - 34 -

Table 2: Fertility and size of egg rafts laid by Cx annuZirostris females at three temperatures.

Temperature Fertility Eggs per raft (OC) No.+ % No.* Mean (±SE)

20° 15/17 89.3 10 152.2 (±12.6)

25° 235/266 88.4 26 151.4 (±17 .4)

30° 32/47 68.1 6 148.8 (±15. 2)

+ Number fertile/Number tested * Number counted

Table 3: Population statistics and parameters for Cx annuZirostris derived from life and fecundity table studies conducted at 0 0 0 . 0 20 , 25 and 30 c, and from l~fe table studies at 15 c.

Parameter

Larval development time (days) 32.00 16.80 13.50 9.30 Mean adult longevity (

* Data unavailable. - 34 -

Table 2: Fertility and size of egg rafts laid by Cx annuti~ostria females at three temperatures.

Temperature Fertility Eggs per raft (OC) No.+ % No.* Mean (±SE)

20° 15/17 89.3 10 152.2 (±12 .. 6)

25° 235/266 88.4 26 151.4 (±17 .. 4)

30° 32/47 68.1 6 148.8 (±15. 2)

+ Number fertile/Number tested * Number counted

Table 3: Population statistics and parameters for Cx annutirostris derived from life and fecundity table studies conducted at 20°, 25° and 30°C, and from life table studies at 15°C.

Parameter

Larval development time (days) 32.00 16.80 13.50 9.30 Mean adult longevity (d') (days) * 14.00 15.70 13.60 Mean adult longevity ( ~ ) ( " ) * 23.40 25.30 14.70 Mean ~ longevity (from hatch) (days) * 40.20 38.80 24.0 Pre-oviposition period (days) * 19 7 10 Net reproductive rate Ro 0.16 3.63 26.03 2.10 Intrinsic rate of natural increase (rm) -0.0221 0.0215 0.0812 0.0250 Finite rate of natural increase C\) 0.9781 1.0215 1.0815 1.0250 Capacity for increase (rc) -0.0226 0.0207 0.0685 0.0246 Finite capacity for increase (Ac) 0.9776 1.0208 1.0685 1.0246 % increase of rm over rc -2.34 3.55 18.44 1.69 Cohort generation time (Tc) 80.23 62.18 47.57 30.14 t Parameters derived by combining 15°C 3uvenile life tables with life adult and fecundity tables at 20 C.

* Data unavailable. - 35 -

-)( E (a) 1.0 E lO ill juverule he - 0,8 . adull I• 16

0.6 12 > c: ill C) 0.4 0 c: 8 ...... 2 c. 0 0.2 ill c. 4 c; 0... E 0... 0 OJ 0 u. 0 10 20 30 40 so 60 70 8 0 90 Days

)( (b) E 1.0 20 - ..... Juvenll• ht E (1) o..• _ ...adult I• 16 ..... ~ 'i 'i... 0 .6 12 :::l (I) c: 0.4 .2 • ...... &. 0.2 4 ...0 a.. 0 0 0 1 0 20 30 40 50 60 70 80 90 Days

(c) - 1,0 )( E ill ·"juvenile lx ..... 0) 0.8 2.0 .•• adult llc 1... (1) .5 c. .~ >... 0.6 > :::l c: (I) OJ C'J 0,4 0 c: 1, 0 I 0 C. ·;;... 0 0.2 Q. ...0 0... 0 0 10 20 30 40 so 60 70 80 90

Days

Age specific longevity (1 ) and fecundity (m } of Fig. 9: X X Cx annuZirostris at (a) 20°, (b) 25° and (c) 30°c. - 36 -

than that of females in Cx quinquefasaiatus and Ae. aegypti

respectively.

0 0 0 . At 20 , 25 and 30 , egg production per surviving female (m ) X increased as the cohort aged, and usually reached a maximum after most of the

females had died (Fig. 9). Generally, the pre-oviposition periods were long

(7-20 days) in contrast to the four or five days observed in thP

laboratory colony (McDonald et aZ. 1977a). In additionr large numbers of

females died within the pre-oviposition period.

Complete life and fecundity tables for three temperatures (20°,

0 0 25 and 30 C) are presented graphically in Fig. 9 and tabulated in

Appendices III, IV and V. The statistics derived from the tables are listed in Table 3, together with the estimated statistics for 15°c.

The intrinsic rate of natural increase was positive for the

0 0 0 temperatures 20 , 25 and 30 C. A negative population growth coefficient was recorded at 15 0 C (Table 3) . The net reproductive rate (R ) and intrinsic 0 rate of natural increase (r ) were greatest at 25 0 C and markedly lower at m . 20° and 30°C. Using the approximate r value for 15°C (r = -0.022), it m m is possible to estimate the threshold of population growth (where r = 0, T 17.5°C) (Fig. 10). Althouth the statistic r is easier to m = c calculate, the use of the more accurate estimate of population growth potential, r , was justified at 25°c as r was 18.4% higher than r • m m c

Fig. 11 illustrates that at temperatures above 20°C, a relatively large proportion of the population exists in the adult stage at a stable age distribution. Larval mortality did not vary markedly between instars. In - 37 -

.10

.08 •

rm .06

.04 • . 02 • / ~/ / / 0 / / 25 30 35 15 ,.;' 20 -.02 / 0 Temperature rc)

-.04

The intrinsic rate of natural increase (r ) of Cx Fig. 10: rn annuZirostris 0 0 0 0 at 20 , 25 and 30 c. An r value was also derived at 15 c, m 0 based on juvenile life tables at 1~ , and adult life and fecundity tables at 20°c. - 38 -

50 300

60 250 50 40

20 1-z 10 w 0 a: 80 83.18 w 200 Q. 50 40

20 10 - 60

50 150

egg ht 2nd 3rd Ath pupoe adult

Stage

Fig. 11: The theoretical stable age distribution of Cx annuZirost.ris at 150 , 20 0 , 25 0 and 30 0 C.

I - 39 -

0 contrast, at 15 C, the greatest proportion of the population existed in the

larval stages, particularly in the earlier stages. This is presumably due

to the high mortality (97%) and long development period (32 days).

This study clearly indicated that under the laboratory conditions

described, 25°C was the most favoured temperature for population growth

of Cx annuZirostris. At this temperature, larval survival was greatest

(65%), as were adult longevity (mean (o) = 25.3 days) and reproductive + potential (R = 26.03, r 0.0812). Although larval survival was only o m = marginally lower at 20°C and 30°C (59% and 56% respectively), reproductive potential was considerably lower (r 0.0195 and 0.0250 respectively). m =

Walter and Hacker (1974) have compared the reproductive potentials of three strains of CUZex p. quinquefasaiatus (= Cx fatigana) (Table 4) ,

'/., and despite similar mean longevities, all differed markedly in the R and 0 r . In comparing Cx annulirostris with any of these strains, R does not m o reflect the difference in reproductive potential between the two species to .A<• M the same extent as r , particularly considering the shorter life time m of Cx annuZirostris (c. f. Hacker 1972). This suggests that, as the contribution to r is greatest when the females are youngest (Birch 1948) , m the reproductive peaks for the Cx quinquefasoiatus populations occurred early in the population's life, and not late as was illustrated with

Cx annuZirostris.

The large differences in reproductive potentials between Cx quinquefasciatus and Cx annuZirostris may be explained by the differences in ecological niches. Walter and Hacker (1974) point out that a mosquito population (species) which lives in a. variable environment, or where predation and parasitism is hea~, may be expected to display a ~igher reproductive - 40 -

Table 4: A comparison of mean adult longevity, net reproductive rate and intrinsic rate of natural increase between strains of Cx quinquefasciatus (Walter and Hacker 1974) and Cx annuZirostris.

Species and Mean adult Net Intrinsic longevity Reproductive rate of strains ( ~ ) (days) Rate natural (R ) increase 0 (r ) m

Cx p. quinquefasciatus (27° ± 1°C) Bangkok 35 16 0.17 Houston 40 so 0.29 Vera Beach 45 75 0.29 Cx annuZirostris (25°± 1°C) 25 26 0.08 - 41 -

potential than a species which inhabits a less harsh and more consistent environment. This may be true of Cx quinquefasciatus (habitat: old, stagnant, often putrid water) and Cx annuli~ostris (habitat: fresh, non-stagnant grassy water) .

In addition, compared with the easily reared Cx quinquefasaiatus~

Cx annuli~ostris is extremely sensitive to environmental changes in the laboratory, and will only reproduce under optimum conditions. Consequently, the calculation of population growth potential of Cx annulirostris under laboratory conditions is more likely to be an under-estimate than that of Cx quinquefasciatus~ which is easily cultured. - 42 -

Section 2. 2: The behaviour of Cx annul,irostris larvae in the laboratory - 43 -

Introduction

The swimming and diving movements of mosquito larvae are probably

in response to temperature, light, vibration and availability of food

(Clements 1963) . These behaviour patterns also vary between species (Bates

1949) .

In the field, the thermal stratum which the larvae usually .occupy

will have the most influence on development rate. For this reason, an

experiment was undertaken to determine the length of time that Cx

annuZirostris larvae spend at the surface and floor levels of their

aquatic habitat, under constant conditions.

Materials and Methods

The behaviour of larvae was studied in the laboratory to determine

the proportion of time which they spend:

(i) resting and breathing on the surface

(ii) swimming to or from the surface and

(iii) eating or resting on the aquarium floor.

Petri dishes (100 rnm diam. x 20 mm depth) were filled with a

larval medium containing grass infusion (Appendix I) . Two larvae were observed in each of four sessions for the first, second and third instars

and for ten sessions for the fourth instar. Each session continued for

30 min. A stop watch was used to measure the time that larvae spent resting - 44 -

on the floor, feeding on the floor, swimming, or on the surface. The·

following behaviour classifications were used:

- resting on the floor: larva lies motionless ori the floor.

- feeding on the floor: larva moves across the floor., mouth

brushes active, abdomen and siphon usually above the floor

level, but head on the floor sieving sediment with mouth

brushes.

- swimming: larva actively moves away from or towards the

surface ..

- surface: larva rests or feeds with siphon placed at the

water surface.

Fluorescent lighting was used and the water and ambient temperatures

0 were 22 C. For the period of the study, the movements of the observer were maintained at a minimum to avoid disturbing the larvae.

Results and Discussion

Table 5 shows that activities away from the surface occur more often as the larvae aged. The first instar larvae never ventured away from the top two millimetres of water, and spent most time in one position.

Although fourth instar spent much more time swimming, feeding and resting away from the surface, 87.7% of their time was still spent at the surface.

Cx annulirostris larvae were therefore shown to spend most time on the water ~urface, under the conditions described. These results suggested that large thermal gradients within field habitats could - 45 -

Table 5: Cx annuZi~ostris larval behaviour study 22°C.

Percentage of total time spent

INS TAR Bottom Swimming Surface

Resting Feeding

1st 0 0 0.3 99.7

2nd 0.3 0 6.3 93.4

3rd 2.6 4.0 3.2 90.2

4th 4.3 4.3 3.7 87.7 - 46 -

markedly influence larval development rates. Water surface temperatures may be substantially warmer or cooler than the remaining depth of water, particularly in pools exposed to direct sunlight or wind action.

Haufe (1957) has shown that Aedes communis (Deg.) larvae aggregate in zones where the surface temperature approaches their preferred optimum.

Similarly, Standfast et aZ. {1964) suggested that Cx annuZirostris avoided surface temperatures of 40°C, and therefore death, by spending most of the time in the cool lower layers.

It would be useful to continue this investigation in an attempt to determine whether larval behaviour varies at different temperatures, and to determine the preferred optimum temperature in relation to the biological optimum of the species. - 47 -

Section 2.3: Field studies of juvenile Culex annulirostris.

l ii

Material from this section has been published under the title nThe mosquito and predatory insect fauna inhabiting fresh-water ponds, with particular reference to Culex annulirostris Sktise (Diptera: Culicidae)" in the Australian Journal of Eoology (1981) ~ 21-27. - 48 -

Introduction

Culex annuZirostris has been the major target of mosquito control

programmes conducted in the Murray Valley since 1974. Control is based on

the extensive use of Abate R . To ensure the most effective and economical

use of Abate R , a detailed knowledge of ·the larval ecology of

Cx annuZirostris in the field is required.

Laird (1956) described the habitat preferences of Cx annuZirostris

and associated insects in the islands of the South Pacific. However,

similar comprehensive studies of Cx annuZirostris in Australia are lacking.

The following study describes the colonization and population densities of

Cx annuZirostris and associated insects in man-made ponds at Mildura in

the Murray Valley, and in an established swamp in Shepparton in the Goulburn

Valley.

2.3.1 Ecology of Cx annuZiroatris within a fresh water pond - Mildura

Materials and Methods

The Study Area

A 10 M x 3 M x 16 em deep pond with sloping edges was constructed at the Mildura Horticultural Research Station (Fig. 12a). ·The floor was lined with black plastic to prevent seepage, and covered with a thin layer of earth. Turfs, obtained from a dried up swamp and comprising mainly water couch (PaspaZum distichum L.) and bull rushes (Typha angustifoZia L.), were placed down the centre of the pond (Fig. 12b). The pond was filled - 49 -

Fig. 12a: Experimental pond at the Mildura Horticultural Research Station, February 1977.

Transect No.

. 1 • 2 • 3 . 4 . 5 . 6 . 7 . 8 . 9 ,10. 11 .12 . 13 .14 . 15. 16 . 17 . 18. 19. 20.

vegetation

Fig. 12b: Layout of vegetation and transects within the pond. - 50 -

and the level maintained with town water through a hose-mounted ball

valve. Twenty 0.5 M wide transects were marked laterally along both

sides of the pond (Fig. 12).

(a) Colonization, larval survival and development

Populations of mosquito larvae and their predators in the pond were

studied during February-April in both 1977 and 1978. A sample of

water (200 ml) was ladled from the centre and from the edge of each transect

at daily intervals after the initial filling of the pond with water. The

numbers and developmental stages of the different mosquito species were

recorded for each sample, together with the number of taxa of the predators.

Each sample was returned to the original transect after recording. A

clockwork recorder with two probes provided continuous records of air and

water temperature.

(b) Time-specific life tables

As eggs were laid in the pond continually, resulting in overlapping

generations, the age structure and rate of mortality of the juvenile

population at one point in time may be estimated using time-specific

life tables (Service 1976) •

It was necessary to calculate the mean duration of larval development over that period of the pond study for which the life tables were compiled.

This experiment was undertaken during the 1977 pond study, ten days after

the pond was filled. Two predator-exclusion cages (30 x 30 x 30 em) were constructed with 50 denier nylon mesh on all six sides and a sleeve on top. They were placed in the side of the pond opposite to where the daily - 51 -

sampling activities (section (a)) were being conducted. Six hundred newly hatched first instar Cx annulirostria larvae were placed in each cage. For each of the following 12 days, ten samples (200 ml) were ladled from each cage, the number and stage recorded per sample, and the contents returned.

The results from both cages were pooled to calculate the mean durations of each instar. These were derived by using the frequency dependent mean, i.e. the time (t ) half the larvae took to reach that instar. 50

The results from the routine daily sampling along the pond (section

(a)) for the 12 days over which the above experiment was run were collated and used as a basis for constructing time-specific life tables. To construct life tables, stage specific age distributions were first obtained by dividing the total numbers of each instar sampled by the instar duration

(Service 1976). Service proposed that if the population was not undergoing a rapid increase or decrease in size, the "smoothed" age-specific age distribution curve joining the stage histograms should approximate time specific survivorship curves for the species under those conditions (Fig. 14).

From the curve, daily mortalities were read off, and life tables constructed.

As the instar durations were short, it was reasonable to assume that mortality throughout each was constant (Dempster 1956). On this assumption, instar mortalities were also estimated.

The sampling procedures described in section (a) provided a means of comparing larval densities in relation to vegetation and water depth. - 52 -

Results and Discussion

(a) Colonization, larval survival and development

In 1977, first instar Cx annutirostria were first recorded on day 2, which suggests that eggs were laid in the evening of day 0.

After this, the numbers of larvae rose steadily until day 7 when an average of 24 larvae per dip was recorded (Fig. 13a). Fourth instar· larvae were first recorded on day 5, the larvae having progressed one instar per day from day 2. The first pupae were recorded on day 7

(Appendix VI). Larval population densities fluctuated markedly for the following twenty days, although a gradual decline was apparent. For the remaining ten days, larval numbers diminished steadily.

In the 1978 study, the trends were very similar to those described above, particularly during the first week. However, larval populations reached a peak of 30 larvae per dip at day 6 and thereafter declined more rapidly and steadily than in 1977 (Fig. 13b). As in 1977, larvae progressed through one instar per day until the fourth instar which lasted for two days. (Appendix VI).

Table 6 lists the species composition of the mosquito and predatory insect fauna colonizing the pool during both study periods. The relative proportion of these species, also given in Table 6, indicated that

Cx annutirostris was the dominant mosquito species in the pond during both. years. Beetle larvae of the family Dytiscidae were also very common, only being outnumbered in 1977 by damsel fly nymphs (Odonata: Lestidae). All - 53 -

---t (a) i ::]~------·.. _·_···..... _ ..

10 1977 28 . Mean Daily Water Temp. 10 . Cx annulirostris larvae c. ·Predators 60 :.0 24 I\ ,._ I en I I c. CIJ c. I \ ~ 20 50 0 CIJ ctl > ~ .... Q) ~ 16 II 40 c. : E VI ca 'i: en t; 12 30 ... 0 Q) c. ·=3 c: c: 20 ~ ca .....0 ('Q )( "0 (.) 4 10 ...Q) 0..

Days

•' .. ~ 301······ ...... """····· '•·"'···· E~ 20~------~·~··~~----~--~--~ CIJ {b) ~ 10 1978 .~ . . . Mean Daily Water Temp. 28 \ ·-· Cx annulirostris larvae 70 c. . ·- -. Predators " :.0 24 1 60 ~' en ... 1 I I c. c.CIJ :.0 20 50 0 CIJ ('Q I\ ; i t:! > ... Q) ~ 16 40 c. VI I ·;:::: E ..... ('Q VI I ~\ en 0 12 30 .... Q) ·=3 . \ .'\ i : . Q, c: c: ,, ~ .'· • I \ ,·, .· : i', ('Q 20 ~ 0 )( ... (.) j. . ('Q ..J ··... 'T. \!.)//\ \ J\ "0 10 Q) ,..· '.,· ....· ~ · :...... ,.. : t .. -.... ol' ·, , , , \ r\ 0...... \.' ·-...--- ..... :/ ~' \.' .... - ...... ,. ----··.-'· --- ... :.. 0~~--~---~~~~~T-~~---r-~~~--~~-r~--__.0--- 0 12 18 24 30 38 42 48 Days

Fig. 13: Daily changes of mean water temperature and population densities of juvenile Cx annuZirostris and aquatic predators during February-April in (a) 1977 and (b) 1978 in a pond at Mildura. - 54 -

,..... c .2 ~ c ::) 300 0 a -:l ..0 ~ ...... -= ., >- - u 0 c •::) G) fT 0) • < ~ ~ 100 ~-=

I II m :m: p 0 0 10 12 14

Fig. 14: Age-specific stage distribution simulating time-specific survivorship curves for juvenile Cx annuLirostris in a Mildura pond during February 1977. - 55 -

Table 6. Insect fauna, identified to lowest possible taxon, sampled

from the Mildura pond during February, March and April

in 1977 and 1978.

1977 1978

Mosquitoes Total % of Total % of No. Total No. Total

Culex annulirostris 6782 97.6 2980 89.8 Culex australicus 64 0.9 208 6.3 Anopheles annulipes 101 1.5 165 5.0

Predators

Coleoptera (larvae) Dytiscidae Undetermined Genus 99 11.7 18 4.0 - Discus sp. 24 2.8 - Hydroporous sp. 68 8.1 193 43.4 Hydrophilidae - Helochares sp. 124 14.7 34 7.6 - Berus sp. 1 0.2 Coleoptera (Adults - unidentified) 59 7.0 60 13.5 Odonata (Zygopteran nymphs) Lestidae 237 28.1 66 14.8 Hemiptera (Adults and nymphs) Notonectidae Anisops inconstans 164 19.5 44 9.9 Corixidae Agraptocorixa sp. 66 7.8 30 6.7 - 56 -

insects listed as predatory, with the exception of adult Coleoptera, had

been observed or were known to prey on mosquito larvae (Hinman 1934,

Laird 1956). Due to the difficulty of in situ identifications of the

adult beetles, these were not identified and therefore they may not

have all been predatory.

In both 1977 and 1978, mosquito and predator populations in the

pond increased most rapidly during the first seven or eight days

(Figs. 13a, 13b). Although mosquito densities declined after this time,

predator densities continued to increase. In the 1977 study, there was

a significant inverse relationship between mosquito larval densities and

predator densities (P = 0.01, coefficient of determination 100R2 = 30.2%).

However, in 1978, there was no significant relationship between mosquito

and predator densities, and both mosquito and predator densities decreased

after day 28. This decrease in both populations may be attributed to

seasonal conditions. The mean daily temperature was approximately 4°C

cooler during the final two weeks of the 1978 study than the corresponding

period in the 1977 study (Fig. 13b).

Although there was no attempt to assess the number of mosquitoes

consumed by each predatory species in the pond, predation by notonectids,

coroxids, and dytiscids was observed frequently.

Hinman (1934) and James (1965) have shown that dytiscids are the most

voracious of all the coleopteran predators, probably because they are far more effective swimmers than the other common family; Hydrophilidae (Laird

1956) • Laird also reports that the dytiscids were the most common of the

aquatic coleopterans in the South Pacific regions. Hinman (1934) considers - 57 -

that the notonectids are the most voracious of the aquatic forms of

Hemiptera. Sailer and Lienk (1954) suggest that Corixidae are only

accidentally predacious, although observations on corixidan species

in this study indicate that attack on mosquito larvae was more

purposeful. Damsel fly nymphs were also efficient predators in

studies by El Rahah and Abu Shama (1977) which showed that a matured nymph would potentially eat 858 small larvae per day.

(b) Time-specific life tables

The instar duration times, given in Table 7, were calculated from day 10 to day 22 of the pond study, when there was an average pond temperature of 240 C (Table 10). The instar durations derived in this study differ slightly from those calculated in the laboratory at a constant water temperature of 25 0 c. However, completion of development took approximately 11 days in both field and laboratory experiments.

The age distribution, derived from the instar sampling results collat over the 12 day experiment (Table 7) , are presented graphically to simulate a time-specific survivorship curve (Fig. 14) . A life table was constructed from this curve (Table 8) •

Over the 12 day sampling period, the.overall mortality between egg and adult eclosion was estimated at 89% (Table 8). Service (1973) found that approximately 96% AnopheZes gambiae died in the pre-adult stages in natural ponds.

The survivorship curve is of Type III of Slobodkin (1962) , implying that the mortality rate was constant throughout the study period. The estimated mortality rates (1000 qx in Table 8) are in fact reasonably constant (approx. mean 18% per day) ,. except for the second instar larvae

(Table 9) which appear to have slightly higher mortalities. Service - 58 -

Table 7: Age distributions of Cx annuZirostris derived from instar durations and observed frequencies.

Age distribution Stage duration Total number Stage (No. larvae (days) collected entering stage per day)

1st 1.78 728 419 2nd 2.26 617 273 3rd 2.40 279 116 4th 2.93 280 96 Pupae 1.91 128 67 Total 11.28

Table 8: Life table for inunature Cx annuZirostris

X n 1 d 1000~ e X X X X

0 450 1000 178 178 4.47 1 370 822 145 177 4.33 2 305 677 140 207 4.15 3 242 537 137 255 4.10 4 180 400 78 195 4.33 5 145 322 67 208 4.26 6 115 255 33 129 4.25 7 100 222 34 154 3.80 8 85 188 33 176 3.40 9 70 155 18 116 3.02 10 62 137 22 161 2.35 11 50 115 22 193 1.70 12 42 93 1.00 (11.3 50 111)

(x = age in days; n - no. of larvae surviving to age x; X 1 = no. per 1000 larvae surviving to age x; d = mortality X X between ages x and x + 1; 1000~ = mortality rate per 1000 alive at beginning of age x (days) ; e = mean life time X expected for those attaining age x (days). Mean eclosion time, x = 11.3 days). - 59 -

Table 9. Mortalities of Cx annulirostris devE>1opmental stages.

Deaths in Relative Stage Age at No. entering beginning of stage stage proportion stage (days) dying in stage

130 0.289 1st 0 450 140 0.437 2nd 1.78 320 71 0.394 3rd 4.04 180 41 0.376 4th 6.44 109 18 0.264 Pupae 9.36 68

Adult 11.28 50 - 60 -

Table 10. Minimum, maximum and mean daily water temperatures

recorded during cage trials in Shepparton and Mi1dura

during February 1976 and February -March 1977 respectively.

1976 - Shepparton 1977 - Mi1dura

Date Min. Max. Mean Date Min. Max. Mean

10/2 17.8 23.5 20.7 24/2 21.0 33.0 27.0

11/2 17.8 22.0 19.0 25/2 18.5 32.0 25.0

12/2 18.9 22.1 20.5 26/2 19.0 29.5 24.0

13/2 19.7 22.0 20.9 27/2 16.0 32.0 24.0

14/2 19.5 23.0 21.3 28/2 10.0 31.0 20.5

15/2 18.2 22.4 20.3 1/3 14.0 31.0 22.5

16/2 20.7 23.5 22.1 2/3 17 .o 31.0 24.0

17/2 21.9 24.2 23.1 3/3 17.0 32.0 24.5

18/2 19.5 25.0 22.3 4/3 19.0 28.5 24.0

19/2 14.9 22.6 18.8 5/3 20.0 36.0 28.0

20/2 18,8 21.6 20.2 6/3 16.0 30.0 23.0

21/2 18.1 22.7 20.4

Mean 20.8 24.4 - 61 -

(1973) found mortality highest in fourth instar An.gambiae and Southwood

et aZ. ( 1972) found the age of highest mortality in Aedes degypti was

dependent on the season: fourth instar and first instar having highest

mortalities in cold and hot seasons, respectively.

The life tables derived from these studies are useful in providing

an indication of age and instar specific mortality under the conditions

described. This information is particularly valuable for designing chemical and biological control programs, and provides a basis for

comparing different habitats and regions.

For the duration of the 1977 study, there was a marked preference shown by all stages of larvae for the central area of the pond which contained vegetation. The edge o~ the pond (2-6 em deep) contained 15.1% of the larvae in comparison to 84.9% found in the centre (10-16 em deep).

Fig. 15 demonstrates that larvae were also most commonly found in the grassy area of the pond and not in transects 17, 18, 19 and 20 where there was no vegetation.

Fig. 15 shows the distribution of first instar, and second, third and fourth instar and the pupal stage across the length of the pond. It was assumed that most first instar were less than one day old and therefore would not have moved far from the egg hatch sites. On this basis, their distribution is indicative of the egg distribution within the pond.

Transect 6 contained about 50% more larvae than the average for the rest of the pond and transects 10 and 11 also had large numbers. Transect 6 - 62 -

:l . 4 6 H

_...-·----· .. - --·-- ·--,~ ...... ~ ------

First Ins tar

500

400

300 ~

'lOO -0 0 z 100

Transect No.

Total larvae (less 1st in star) and pupae soo

400

Q,) 300 !0 > '- s 100 -0 ci 100 z

Transect No.

Fig. 15: The distribution of first instar larvae, and second, third and fourth instar larvae and pupae across the length of. the pond. - 63 -

was bordered by bullrushes (transect 5) and a heavy clump of couch (transect

7) • Transects 10 and 11 contained thick couch. Insufficient evidence exists to draw conclusions regarding influence of grass type or quantity on oviposition. However, one factor common to all 3 transects was their close proximity to the tallest grasses within the pond.

2. 3. 2 Ecology of Cx annuZirostris within a fresh water swamp - Shepparton

Materials and Methods

(a) Development and survival

This study was undertaken in February 1976 over a period of 12 days at Reedy Swamp, Shepparton, an area known to be suitable for breeding of

Cx annuZirostris.

Recording instruments were installed at the site to record air and water temperature.

Two types of cage were used. They consisted of frames measuring

30 x 30 x 60 em onto which 50 denier nylon mesh was glued. One type was completely covered in order to exclude predators, and the second, used to measure the effect of predators on larval survival, had the floor uncovered.

The predators present in the swamp were Hydra spp., PZanaria spp., rotiferans, leeches, Gambusia affinis and se_veral families· of the following insect orders: Hemiptera, Odonata, Coleoptera, Diptera and

Ephemeropter a. - 64 -

Four cages of each type were placed at random i~ swamp water about

20 em deep, and allowed to settle for about 24 hours. About 2000 first

instar larvae of Cx annulirostris hatching synchronously from field collected egg rafts were then placed in each cage. Nylon mesh emergence cages with perspex doors and sleeved access holes were fixed to the top of the cages to collect emerging adults. Adults were collected twice daily with an aspirator, counted and sexed.

(b) Larval distribution

A larval grid survey was conducted over Reedy Swamp during February

1976 to ascertain the larval distribution pattern. Reedy Swamp is approximately rectangular with an area of about 380 ha.

Using a flat-bottomed row boat, nine traverses at 200 M intervals were made across the swamp in an east-west direction. Samples were taken at approximately 30 M intervals on each traverse. Each sample consisted of ten 200 ml dips. The number and species of mosquito larvae were recorded for each sample. The water depth was determined at each sampling site and recorded in one of the following categories (em): 0-15, 16-22,

23-30, 31-45, 46-60, 61-90, 91-120 and 120 and over. The presence or absence of vegetation was also noted. A total of 113 samples were taken.

Results and Discussion

(a) Larval development and survival

The mean water temperature for the 12 day period was 20.8°C (Table

10}. Adult survival in the absence of predators ranged from 67.4% to - 65 -

89.1% for the four cages, with a mean of 74.5% (Table 11). Peak emergence

of adults occurred eight days after hatching of eggs. Mean time to

emergence of adults was 8.25 days, females taking slightly longer than

males.

In all four cages which did not exclude predation, only one adult

Cx annuZirostris emerged. Presumably, this extremely high mortality was

a direct consequence of the diverse predatory fauna inhabiting the swamp.

(b) Larval distribution

Larvae were not regularly distributed throughout the swamp (Fig. 16),

63.4% of the larvae sampled being found within 10M of the swamp's edge.

The other larvae collected were near outcrops of vegetation in the swamp.

Over 90% of larvae collected were Cx annuZirostris.

The percentage of samples containing Cx annuZirostris larvae

decreased significantly when taken at water depths of 15, 22, 30 and 45 em

respectively (Fig. 17). The percentage of samples containing larvae were

very low at water depths of between 45 and 120 em and did not differ

significantly.

Most Cx annuZirostris larvae were found in water less than 15 em deep. The larvae were not recorded in open water, and were most common in

areas with a grass cover but not near rushes. - 66 -

Table 11. Emergence times of adult Cx annulirostris and percentage

of larvae surviving to eme.rgence at Reedy Swamp, Shepparton,

in February, 1976.

Numbers of adults collected at Mean survival Mean to emergence emergence 6 7 8 9 10 11 Total (%) time (days) days after hatching

Male 1 135 496 86 7 1 726 8.00

Female 1 47 349 332 32 4 765 8.50

Total 2 182 845 418 39 5 1491 74.5 8.25 - 67 -

Sampling Site 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

4 2 3 2 9 16

4 16 1 QJ N ~ 5 2 Reedy Swamp QJ > ~ 6 2 1- 7

8 80 52 2 9 29 2

Fig. 16: Distribution and size of Cx annulirostria larval populations per sample throughout Reedy Swamp, Shepparton.

~ 30 .....0

QJ c 0 20 0) c c .E c 0 u 10

15 30 45 60 75 90 Water Depth (em)

Fig. 17: The effect of water depth on the distribution of Cx annulirostria larvae in Reedy Swamp, Shepparton (S.E. =·± 6.5%). - 68 -

Section 2. 4: Field studied of adult Culex annulirostris.

Material from this section has been published under the title .. Population studies of Culex annulirostris Skuse and other mosquitoes (Diptera: Culicidae) at Mildura in the Murray Valley of southern Australia" in the Journal of the Australian EntomoZogicaL Society (1980) ~ 37-40. - 69 -

Introduction

Information on the seasonal abundance of adult Cx annutirostris

in the Murray Valley was necessary to complement labora·tory studies of the

effect of temperature on the population growth potential of the species.

However, long term trapping of Cx annutirostria in the Murray Valley, the

major centre of M.V.E. outbreaks, was also an obvious prerequisite

to any attempts to predict further disease outbreaks. A three year study

of adult populations at Mildura is discussed in this section.

2.4.1 S.easonal abundance of mosquito populations

Materials and Methods

Adult mosquito populations were monitored using a 240 volt light

and suction trap .(Fig. 18). The unit was switched on from dusk to dawn each night by a time clock. A neon pilot globe provided a

favourably attractive light source (0.1 lx illumination) to adult mosquitoes, and a 19 em diam. fan mounted below the globe, sucked all insects flying around the globe into a container of methyl alcohol (70%).

Mosquito populations at Mildura were monitored during 1975-78 using a trap (LTl) permanently stationed at the Mildura Forest

Commission nursery, situated about one kilometre from the Murray

River and the Mildura sewage treatment ponds. During late 1975 and early 1976, the trap ran for irregular intervals, but ran continuously after March 1976. - 70 -

Fig. 18: A 240 volt light and suction trap used to monitor mosquito populations. - 71 -

A second light trap (LT2) was placed on a private property

adjacent to the Murray River in Gal Gal (4 km from Mildura). This

trap began operation in July, 1977, and ran continuously from that

time.

The contents of both traps were emptied weekly, or in summer,

twice weekly. All mosquitoes caught were counted and the species identified.

Temperature records were provided by the Meteorological Bureau.

Results and Discussion

(a) Culex annuZirostris

In all three seasons, Cx annuZirostris females were taken in the

trap from mid-October (19/10/75, 24/10/76 and 17/10/77) (Table 12,

Figs. 19 and 20). The weekly re-appearance of a species within the trap was interpreted as indicating that breeding had commenced. During 1976,

single Cx annuZirostris females were also taken in August and in September.

In addition, LT2 also caught five females during mid-August 1977. These small and irregular catches suggest that Cx annuZirostris overwinters as an adult, although actual breeding was most unlikely, and no larvae could be located in normally acceptable habitats.

The short-term fluctuations in population densities noted in late summer/autumn 1977 and 1978 (Figs. 19a and 20a) may be attributable to the four major irrigations within the district and prevailing climatic conditions including temperature, precipitation (Figs. 19b and 20b) and relative humidity. - 72 -

Table 12. Numbers of female mosquitoes caught in a light trap

(LTl) at the Forest Commission nursery, Mildura, 1975-76.

Mean numbers of females caugh·t per night Period Cx annuZirostria Other Culex Anopheles Aedes

14-19/10/75 0.3 33.2 4.8 11.2

20-26/10/75 1.1 20.4 27.3 10.4

27/10-2/11/75 0.8 47.2 53.2 33.2

3-9/11/75 0.9 36.0 35.3 16.4

10-13/11/75 4.3 80.3 32.3 65.3

22-29/1/76 36.5 1.4 6.1

5-10/2/76 40.8 2.0 2.2 17.3

17-24/2/76 89.6 2.1 2.5 38.4

2-3/3/76 51.10 10.5 2.5 5.0

Table 13. Summary of Cx annuZirost~s population data as indicat~d by

catches in a light trap between 1975 and 1978.

Season Population peak - Mean daily catch % of total catch females per day {catch period)

33.0 (Oct-Nov) 1975-76 89.6 66 .o (25/1-3/3) 71.8 (Jan-Mar)

1976-77 33.5 10.7 (7/1-28/3) 40.4 (Oct-May)

1977-78 38.2 12.1 (3/1-28/3) 33.5 (Oct-May) - 73 -

(a) E Jo .2> c: .... <1) Cx annulirostris 0. Other spp. . (I) 8 ?0 ·:;+-' r:::r (I) 0 E

0 10 i 11 '· . ,... r\i \ /\ F/,·~· I '\/) :~\ \ " ./...... •. ·•. i \ :\... • •• •• 0 l \ //'\. ! .-· · i\ \I \ 1 z \i \ r! • ·-.';·; i • J i I • V i 1 ._..... •• : •• :'! '1 \! ., \ / \ ./ . ~ ! ··,\•. .,! \ ·-· ; \ t y ...... f \, • .. : •. \ :/ .../ '· . ;,, \; ...\...... •.·~ •... 0 ...... SEP OCT NOV DEC JAN FEB MAR APR MAY JUN 1976 1977

30 (b)

25 E -!-> E - 2 c: .2 1a ·a..... 15 0 ·c:; Q)... 0.

SEP OCT NOV DEC JAN FEB MAR APR MAY·# JUN 1976 1977

Fig. 19: (a) A light trap surve'y of population changes of Cx annulirostris and other mosquito species during 1976-77 in Mildura.

(b) Mean weekly temperatures and precipitation during 1976-77 at Mildura, recorded at the Meteorological Bureau. The l7.5°C temperature reference line, derived from life and fecundity tables, indicates a theoretical cessation in population growth of C:c annuZi110Stri.s.· - 74 -

40 ·-· Cx annulirostris (a) ...... Other spp.

...... \ 30 !, t 1 i \ ; I ~ ..

rJ') 20 I \ 1\ '1 . II Q,) !\ • · = i • I\ f\ ·· : : i i I \ i i !i • : : I l j !\ 0 f \ J \ f \ 1 \ J \ .~ : ~ ' ' 0i • \:: i \ . \ :J f \ t \ ! •' / '· i I • • I : C1' I . i i YV \i \ f \ q .. rJ') ~·''\ 0 \ E {\ , .I \ / \/ \;1 ·:-\ 1~1.;\.J /\1 \ /\. .... 10 \ ! '. 0 ! 1 \f \1 ··~ !/ ·-·\ , ·\\.r .\ \ ,!i v \j\ zci \ \ I . / ...... _. ___;· '~" \...... ~ ...... _. 0~----~~~----~----~----~--~----~~~·-~~=-~----~ SEP OCT NOV DEC JAN FEB MAR APR MAY JUN 1977 1978

30 .

25 E E

/V\-~-iYV\_~· c (b) Q) 20 ;· . ·;;0 ~ ., :J ca ~ ·-· ~ co ·a ~ '(3 ~ 15 60 Q) ~ E 0.. Q) ,/.\ .... . > \ 40 :i .?: 1 Q) .X ·-· Q) Q) ~ ~ 5 20 Cii..., c:: co 0 Q) .... ~ 0 SEP OCT NOV DEC JAN FEB MAR APR MAY JUN 1977 1978

Fig. 20: (a} A light trap survey of population changes of Cx annuZirostris and other mosquito species during 1977-78 in Mildura.

(b) Mean weekly temperatures and precipitation during 1977-78 at Mildura, recorded at the Meteorological Bureau. The 17.5°C temperature reference line, derived from life and fecundity tables, indicates a theoretical cessation in population growth of Cx annuZirostris. - 75 -

During the 1975-76 season, which was a second consecutive flood year,

Cx annuZirostris populations were considerably larger than those of the

following two years (Tables 12 and 13). The mean daily catch of Cx

annuZirostris was approximately ten times greater during that season, and

represented a far greater proportion of the ·total catch than did those caught

in the following years (Table 13). In addition, the data given .in Table

13 are probably underestimates as the results are not available for much

of February and March when the species is normally at its peak.

Results from LT2 are clearly different to those of LTl (Fig. 21).

Although both traps demonstrated a population peak in late December, only a

small increase in the Cx annulirostris population was apparent in the vicinity of LT2 during February. The difference between traps is probably

related to the proximity to the breeding areas, the first light trap being close

to known breeding areas.

(b) Other species

The light trap caught 13 species of mosquitoes - 5 Culex, 6 Aedes,

1 Coquilletidia and 1 Anopheles (Tab~e 14). Other than Cx annuliroatris,

Cx australiaus was the most dominant species, with catches often as large or larger than Cx annulirostris. Generally, Aedes populations did not

fluctuate markedly throughout the October to May period, although very large populations were recorded during spring in both 1975 and 1976. Anopheles and

CoquilZetidia populations were generally largest during spring.

Reeves et al. (1954) collected 18 species from the Mildura area, which included 5 species not caught in the present light trapping studies. Other species caught included Mansonia unifo~is, Culex cyZindriaus,

Ae.vigilax, Ae.banaroftianus, and Tripteroides atripes. However, Reeves - 76 -

40 •

•-• L .T. 1 Cx annulirostris ..... 30 .J: L.T. 2 Cx annulirostris .~ c

'- Q) 20 c. • • i .J:... I 'i en • ::s C'O 10 (J rJ: . • . 0 • z ... ~~~...•.• ( •... . / . . . •••• t ... -~: ~ 0 .... s 0 N 0 J F M A M J

1977 1978

Fig. 21: Changes in population densities of Cx annutirostris as measured by two light traps at Mildura during 1977-78. - 77 -

Table 14. Species composition of the mosquito fauna ocuurring in

the 1975-78 summers (November - April) in the Mildura district,

as measured by a light trap.

1975-76 1976-77 1977-78

No. No. No.

Culex annuZirostris 1408 33 939 45 1370 32

Cx austraZicus 680 16 882 42 2244 53

Cx fatigans 52 2.5 279 6.6

Cx moZestus 99 2 3 0.1 14 0.3

Cx gZobocoxitus 3 0.1 5 0.1

Aedes theobaldi 79 3.8 44 1.0

A~ camptorhynchus 26 1.2 8 0.2 Ae.. sagax 1115 26 4 0.1 Ae. trerrrulus 2 >0.1

Ae.aZternans 41 2.0 96 2.3

Ae.vittiger 1 >0.1

Anopheles annuZipes 982 23 68 3.2 174 4.1

CoquiZZettidia ZineaZis 5 0.1 - 78 -

used several other sampling techniques which did not rely on the species

being attracted to a light source.

Cx australiaus feeds on birds and small mammals, but is not a

pest of man (Dobrotworsky 1965). However, populations of this mosquito

may play a significant role in maintaining the cycle of M.V.E. virus

through local and migrating bird populations. In contrast, Cx annulirostris

which has a broad host range includi.ng man, is considered the major vector

of this disease (Doherty 1974). The prevalence of Cx annulirostris in

Mildura, during wet and dry seasons 1 supports this view on epidemiological

grounds.

2.4.2 Nightly activity and abundance of Cx annulirostris

Mat~rials and Methods

A light trap was operated from a private property on the eastern

side of Mildura during February-April, 1977. Collections from this trap

were made twice nightly; at 12.30 a.m. and after sunrise. A

thermohygrograph, placed next to the trap, provided a continuous record

of temperature and relative humidity.

To determine the effect of temperature and relative humidity on

flight activity (and not abundance), regression analyses were performed on the a.m., p.m. and total catch data. The analyses compared the effect of relative humidity (a.m. and p.m.) and temperature (a.m. and p.m.) on

flight activity. Acti~ity was measured as the deviation away from the running mean of the daily catch data. - 79 -

Results and Discussion

Large variation in nightly activity and abundance of Cx

annutirostris was demonstrated throughout the two month study period (Table

15). However, there was no significant relationship demonstrated between

the environmental factors considered and the catch data. Possibly, a

combination of factors such as wind speed and humidity are related more

closely to activity than are individual environmental factors.

Over the two month period of trap operation, more female Cx

annutirostris were caught per hour before midnight (1.91 females per hour)

than after midnight (1.03 females per hour). This is in contrast to results

from bait trapping studies conducted at Shepparton in 1976, where equal

numbers of mosquitoes were caught before and after midnight (Shelden 1977).

However, Standfast and Fieldhouse (1963) reported that Cx annutirost.ris

had a peak activity just after sunset, and a smaller peak prior to

sunrise. Results from the present study support these observations.

The mean nightly catch for this trap was 15.3 female

Cx annutirostris per night, which was substantially greater than the Forest

Commission nursery trap (LTl) over the corresponding period (11.7 females per night) • - 80 -

Table 15. Numbers of Cx annuZirostris caught in a Mildura light

trap emptied twice nightly during February and March, 1977

8-12.30 12 .. 30-7.00 8-12.30 12.30-7.00 date p.m. a.m.* total date p.m. a.m.* total

2/2 8 8 16 10/3 28 24 52 3/2 6 5 11 11/3 6 22 28 4/2 2 1 3 12/3 10 4 14 5j2 4 7 11 13/3 11 2 13 6/2 4 2 6 14/3 38 32 70 7/2 8 7 15 15/3 0 0 0 8/2 16 15 33 16/3 1 0 1 9/2 9 5 14 17/3 2 0 2 10/2 14 13 27 18/3 2 0 2 11/2 9 10 19 19/3 3 0 3 12/2 1 3 4 20/3 10 4 14 13/2. 13 17 30 21/3 4 0 4 14/2 13 8 21 22/3 44 23 67 15/2 3 9 12 23/3 2 2 4 16/2 13 16 29 24/3 13 3 16 17/2 7 12 19 25/3 18/2 1 1 2 26/3 19/2 3 2 5 27/3 0 1 1 20/2 6 17 23 28/3 0 0 0 21/2 5 1 6 29/3 11 2 13 22/2 4 3 7 30/3 3 2 5 23/2 6 5 11 24/2 6 9 13 25/2 8 5 13 Total 455 356 811 26/2 4 8 12 27/2 1 4 5 28/2 7 1 8 1/3 21 '9 30 2/3 6 4 10 3/3 5 4 9 4.3 5 3 8 5/3 15 10 25 6/3 7/3 7 5 12 8/3 10 6 16 9/3

* a.m. catch = morning following the evening catch on the date shown • - 81 -

CHAPTER 3: GENERAL DISCUSSION

3.1 The bionomics of juvenile Cx annuZirostris

3.1.1 Larval development

Laboratory studies clearly indicate that temperature is most

critical in determining rates of larval development. However, at Shepparton

the mean duration of development (egg hatch to adult) was 8.25 days at

20.80 c. This was considerably lower than the predicted value based on

laboratory studies (Fig. 22). There may be several explanations for this:

(1) Laboratory observations during this study have shown that Cx

annuZirostris larvae spend 90% or more of their time at the water surface.

If the larvae were to behave in a similar manner in the field, their

developmental rate would be strongly influenced by the surface te~perature, which was not recorded in the Shepparton experiment. However, Standfast

et aZ. (1964) noted that Cx annuZirostris avoided temperature extremes at

the surface by remaining submerged for prolonged periods. This suggests

that, although it remains on the water surface under moderate conditions,

the species may select and remain within a preferred temperature zone when

extremes of temperature exist in the upper or lower thermal strata. The

20.8°C recorded in the Shepparton experiment was the mean water temperature of the swamp floor, where the recording probe lay. If the preferr~d optimum temperature was greater than 20. 8 0 C, and occurred above the floor

level, larval development rates would be proportionately greater. This aspect of the study warrants further investigation. - 82 -

(2) Headlee (1942) demonstrated that fluctuations of moderate

temperatures accelerated larval developmental rates of Aedes aegypti3

whereas the reverse was true for high and low temperatures. A similar

situation may exist with Cx annuZirostris, which would thereby contribute to

the observed discrepancy.

(3) In contrast to the simulated rearing conditions in the laboratory,

the swamp undoubtedly provided an ideal environment for larval growth, particularly with regard to food. This factor may have also increased

developmental rates beyond those predicted for 20.8 0 c.

3.1.2 Larval survival and predation

In the laboratory, larval survival was approximately 60% at temperatures ranging between 20°C and 30°C. Field trials at Shepparton showed that about 75% of larvae survived in the absence of predators, and less than 1% in the presence of predators. Eleven percent survival was recorded at Mildura in the presence of predators. Although it is inappropriate to draw any final conclusions from these sets of data, it is interesting to note that survival was at least 50% greater in the absence of predators.

A comparison may be made between the stable age distribution of pre-adult stages in the laboratory and in the field (c.f. Figs. 11 and 14).

0 0 0 At 20 , 25 and 30 c (lab.) there was less than 5% difference between the proportion of the juvenile population found in each stage. As mortality was far higher in the field (about 18% per day), the stable age distribution derived from the pond demonstrated a much greater proportion of young larvae than older. Approximately 35% difference in numbers was observed between first instar and pupae. - 83 -

,...,. Cl) 40 • LABORATORY > v 5HEPPAR TON

..... ~ 0 c: CU lO c: E 0 a. ~ ..2 10 • :J cu c "'C 0 15 20 25 30 35 Temperature (OC )

Fig. 22: The effect of five constant water temperatures on the duration of juvenile development of Cx annutirostris in the laboratory, in comparison to the duration of development in the field (Shepparton) .

Fig. 23: A typical habitat for juvenile Cx annuLirostris observed a~ Reedy Swamp, Shepp~ton. - 84 -

The greater mortality observed in the field may have been a result of a combination of factors, including the action of pathogens and predators.

During the pond studies, peak densities of between 20 and 30 larvae per

2 sample (equivalent to 15,000-22,000/M ) were only attained within the first seven days of pond formation. Between day 6 and day 30 in both years, mosquito densities decreased five fold. In the first year a sign~ficant negative relationship was demonstrated between the density of mosquito

larvae and predator populations in the pond, suggesting that predators were

largely responsible for the high mortalities. The exclusion of predators

(but not necessarily pathogens) from larvae in the Shepparton studies also demonstrated the importance of predators in reducing mosquito numbers~

The age distribution data, from which mortality rates were calculated, were compiled after the pond had been filled for ten days. The impact of predator populations on larval densities would become more pronounced as the pond aged, because predator populations continued to increase (Fig. 13).

As Reedy Swamp (Shepparton) was a permanent swamp, and predator densities were high, it is not surprising that less than one per cent larval survival was recorded in the presence of predators.

3.1.3 Colonization

Field studies at Mildura and Shepparton demonstrated that Cx annulirostris readily colonizes fresh shallow bodies of grassy water immediately after they form (e.g. Fig. 23). However, Myers (1954) noted that the species does not colonize pools of floodwater until the pools become well vegetated, confirming the suggestion that vegetation is essential before colonization can occur. - 85 -

Reeves et aZ. (1954) and Dobrotworsky (1965) have reported that

Cx annuZirostris inhabits semi-permanent and permanent bodies of water.

Although these observations are consistent with the findings in this study, it

has not been previously recogpised that much of the success of the species

must be attributed to its ability to rapidly colonize fresh and temporary

pools of water containing vegetation. The species can attain extremely high

densities in a very short period after pool formation, and successfully

complete a generation before many temporary pools dry up. This is particularly

relevant to irrigation districts where pools of water are often supplemented

for periods of up to two weeks during irrigation. The additional benefits of being able to readily colonize temporary pools of this nature is that predators, which are very often univoltine, are slow in colonizing and

thus mosquito survival is high. However, permanent swamps, such as the

Reedy Swamp at Shepparton, still contribute significantly to mosquito populations because of their large size, although larval survival is low.

3.1.4 Mosquito control

Larval development and survival trials conducted in this study have highlighted several areas which are significant in terms of control strategy.

Previously, workers have assumed that Cx annuZirostris required at least two weeks to complete development. Hence, larvicide treatments were re applied every two or three weeks. However, this study has shown that the species can successfully complete development in approximately eight day~ under optimum conditions (late January-early March). Continuous control of larvae during these periods with AbateR would therefore require re-application at weekly intervals (Fig. 24). - 86 -

1 LIFE CYC LE ,\ND TRE1~f~lENT SlHEDl.rLE f1JLEX ANNULIR<>STRIS APPLY ABATE PROPOSED RETREATMENT TIME

ABATE NEED 110 EFFECTIVE REAPPLY ABATE

t''d Jrd

EGGS LAID EGGS LAID I NSTARS & 2ND & 3RD EST BLOOD MEAL B.OOD MEAL EGGS MATE & LAID BLOOO ~L t EGGS PEAK AllJU POSSIBILITY HATCH FOR VIRUS E~RGEf\CE TRANSMISSION

Fig. 24: Life cycle and treatment schedule of Qx annulirost~s under optimum field conditions. (Data relating to Abate persistence and virus 'transmission are from Cumming l977d and Kay et at. 1976 respectively) .- - 87 -

Aquatic insect predators are of fundamental importance in planning

mosquito control programs. Although there is limited potential for their

commercial use in control (Notestine 1971} , their existence in all mosquito

habitats should be carefully protected against the effects of insecticides.

Currently, the recommended mosquito larvicide is AbateR , a short

lived organo-phosphate contact and stomach poison which is only lethal to

filter-feeding dipterous insects, such as mosquitoes and chironomids,at

normal field use rates. The larvicide is non-toxic to non-target

organisms when used at the recommended dosages in water containing organic

sediment (Kay et aZ. 1973, Cumming 1977a). However, overdosing with AbateR may

destroy many invertebrate communi ties, resulting in a temporary resurgence

of mosquito breeding. Aerial spreading of AbateR appears to be reasonably

reliable provided the pilot-operator does not repeat previous flight paths

\,• (Cumming l977b). However, the use of hand-operated and motorized granule

spreaders., which are now extensively used in the Murray Valley during non-

epidemic disease seasons, are not as reliable. All spreaders that have

been studied by the Department of Agriculture, particularly the motorized

spreader, applied the larvicide unevenly, and either under- or over-dosed, or both (Cumming 1977~, Shelden pers. comm.). The dosage rate was also determined by the walking pace of the operator. In terms of environmental

conservation and mosquito control, dosage rate is extremely important, particularly in permanent and semi-permanent water bodies such as swamps.

This is because most of the predatory invertebrates have long generations, and are often univoltine. Recolonization of swamps by beneficial organisms is therefore slow, enabling several mosquito generations to be completed in the absence of large numbers of predators.

An alternative approach to mosquito control, now practiced widely throughout Europe and Asia, is source reduction. Although previous I - 88 -

Victorian control campaigns have relied heavily on the use of chemicals, it

is important that habitat modification or removal be recognised as an

important aspect of control programs. As the dominant mosquito, Cx

annulirostris3 readily colonizes fresh and grassy pools of water, priority

should be given to locating and treating these types of pools; and preferably

further action taken to prevent their formation. Reeves et aZ. (1954) also

recognised the importance of establishing source reduction as the principal

means of control in northern Victoria irrigation districts, such as Mildura.

3.2 Population ecology of Cx annuZirostris

Regarding future studies in Queensland, Kay (1978) stated

"Definitive studies relating the activity and abundance of Cx annuZirostris

with climate and meteorology could also provide the basis for firstly, the

recognition and secondly, prediction of abnormally large Cx annuZiroatria

populations such as occurred in the Murray Valley and Riverina in 1974".

The present study used a light-suction trap as the basic sampling

tool for monitoring populations of Cx annuZiroatpis in the Mildura area of the Murray Valley. This is one of several well-recognized techniques available for measuring the prevalence, seasonal abundance and composition of mosquito populations (Service 1976). While it is conceded that the light trap has several failings in monitoring populations (Husbands

1976) , it is the most consistent and convenient means of conducting a long term study of mosquito populations. For the purposes of this study, it is assumed that the light trap data characterizes the seasonal trends in population growth of Cx annuZirostria. The complex and short term effects of daily temperature, humidity (Section 2.4.2), moon phase, windspeed, - 89 -

photoperiod, etc. are not easily distinguished and therefore are not

considered further within this study.

Population growth of Cx annuZirostris is now discussed in relation

to aspects of the seasonal climate and meteorology. This provides a

possibility for the prediction of "abnormally large Cx annuZirostris

populations".

3.2.1 The effect of temperature on population growth

Laboratory life and fecundity studies demonstrated clearly that population growth potential of Cx annu"liros-tPis is strongly influenced

0 0 by temperature. Population growth was low at 20 and 30 c, and maximum at 25°C (Fig. 10). Adult monitoring studies readily support these findings

(Figs. 19 and 20). In both years, ,mean weekly temperatures increased rapidly from 20°C in late November to temperatures as high as 26°C in December.

Similarly, rapid increases were observed in populations of Cx annulirostris in December. This trend was reversed during March 1978. However in 1977, despite a rapid decrease in temperature, populations of Cx annuZirost~s continued to increase. This is probably attributable to the high rainfall which occurred during late February (Fig. 19b) • The large pools of re~idual rainwater were readily colonized in February, producing large populations of Cx annuZirostris during early March.

In the laboratory, the theoretical temperature at which the number of Cx annulirostris was expected to remain constant (where r = 0) was m found to be 17.5°C (Fig. 10). In practice, this was precisely the temperature at which field populations of the species began to steadily increase.

Similarly, when the mean weekly autumn temperatures fell below 17;.5°C - 90 -

Cx annulirostris disappeared (Figs. 19 and 20). In 1976, populations

began to consistently increase from mid-November, and in 1977 the population

increase began one month earlier in mid-October. On both occasions,

the temperature had reached or exceeded 17.5°C. At the end of both seasons,

the species disappeared during early April, as temperatures fell below

0 17.5 c.

Although 17.5°C is theoretically the temperature at which a

population becomes constant and does not disappear, the monitoring technique

used probably did not detect very small populations, such as overwintering

populations. Hence, the consistent reappearance of the species in the trap

during spring is interpreted to mean that the species has begun breeding at

a rate which is greater than its mortality rate, and thus r > 0. In addition m to those females taken in the light trap during winter (Section 2.4.1),

females which were actively searching for a blood meal have also been recorded

along the Murray Valley during winter (unpublished data) • It was unlikely

that these females were contributing to positive population growth, even if they were reproductively active.

In the 1978 Mildura pond study 1 it was also apparent that oviposition, as indicated by the distribution of first instar larvae, almost ceased when the mean water temperature fell below 17.5°C (Fig. 13, Appendix

VI). In 1977, oviposition continued to the end of the study. In this case, temperature did not fall below 17.5°C.

In summary, these findings show that temperature can be used as a means of accurately predicting when Cx annulirostris populations will begin

to increase (l7.5°C) 1 and when they will achieve peak densities (25°C). An extension of this principle can be applied to overwintering populations. - 91 -

In regions where the mean weekly winter temperatures approach or exceed

17.50 C, such as Queensland, the species would not be expected to. enter into

a state of hibernation. Cx annuZirost~s does continue to breed in northern

Australia throughout most of the year (Standfast and Barrow 1968).

3 .. 2.2 Population development of Cx annuZirostris in relation to the transmission of M.V.E. virus

Attempts to explain outbreaks of M.V.E. in southern Australia

(Anderson and Eagle 1953; Miles and Howes 1953; Forbes 1978) have placed

emphasis on the incidence of particular rainfall patterns in the major water

catchment areas of eastern Australia. These were thought to induce the

southerly migration of virus-infected birds, assuming the virus comes from a northern enzootic focus. However, while very large mosquito populations

are an obvious pre-requisite for outbreaks, regardless of the enz·ootic

focus/foci of M.V.E. virus, little consideration has been given to the

conditions necessary for mosquito breedingt particularly at the actual site of the outbreak.

Temperature and the availability of water are probably the most critical factors influencing the rate of population growth of Cx annuZi~ostris.

These will now be examined,by using various models, in relation to the light trapping seasons of 1975-76, 1976-77 and 1977-78, and the M.V.E. outbreak years of 1951 and 1974.

{a) Temperature

• 0 0 0 0 At each of the temperatures stud~ed {15 , 20 , 25 and 30 C) , the population growth statistic A (the number of times the population multiplies itself per day) was plotted to interpolate the equivalent A values for weekly - 92 -

mean field temperatures during the three study seasons. From these values, the theoretical rate of population growth was calculated for each of the three seasons, starting from the time when Cx annuZirostris was observed in spring. This model illustrates the maximum potential for population growth using actual field temperatures and assuming no other limiting factors.

In each season, the actual rate of population growth of Cx annuZirostris observed from September to December closely approximated that· of the predicted population (Fig. 25). However, after December the theoretical populations continued to grow rapidly until the end of March, and then decreased at a similar rate. In contrast, the observed populations gradually increased until March, and February, respectively, and then decreased rapidly. Hence, over spring and early summer, temperature appeared to be the major limiting factor of ·significance, as the actual population growth achieved the maximum possible rate under the available field temperatures.

However, the sudden decline in population densities during March-April rather than April-May, as predicted, demonstrates that other important factors, other than temperature, also affect population growth. In this case, declining photoperiod and increasing predator populations are likely to have had an overriding influence.

Comparison of the three theoretical populations also illustrates the importance of the (i) size and (ii) date of initial activity of overwintering or early spring populations on the potential growth of summer populations. This model supports the suggestion of Marks (1976) that the size of summer populations, and consequently the likelihood of arbovirus epidemics in that season, is influenced by the temperature {"mildness") of the preceding winter. The spring populations of 1975 and 1977 began to increase consistently from mid-October (Table 12, Fig. 20), whereas, in 1976, - 93 -

population growth commenced in mid-November, and the initial population was

also smaller. Because of the late start the 1976 population had fewer warm

days during which it could grow. Hence, the predicted populations of

1975-76 and 1977-78 were three or four times greater than that of 1976-77.

In fact, the actual difference in magnitude between the observed populations

of 1976-77 and 1977-78 was not as great as predicted (Table 13) , presumably

due to other limiting factors such as the availability of breeding grounds.

However, as predicted, there was a considerable difference between the

populations of 1975-76 and 1976-77, presumably because water (breeding grounds)

was not lacking in 1975-76, which was a flood year.

Thus, based on this temperature-dependent model, the three most

important factors which contributed. to the development of large populations observed in the first light-trapping season appeared to be (i) the early

start of the 1975-76 population (ii) the size of the initial spring population and (iii) the abundance of breeding grounds caused by flooding.

Comparative light trap data are not available for the seasons in which M.V.E. outbreaks occurred, and so it is necessary to consider meteorological records alone. The mean weekly temperatures in spring 1950 were above the long term average (Fig. 26}, with the critical temperature of 17.5°C being reached 2-3 weeks earlier than average. This would have allowed the development of an extra generation. Temperatures were slightly above average in 1973 and well below average in the spring of 1974 (a wet but non-epidemic spring) (Fig. 26) • Spring temperatures may also influence virus transmission and epidemics in other ways. Temperature probably has a similar affect on the mosquito species which occur earlier in the season, some of which may contribute to the early amplification of virus. The timing and occurrence of warm temperatures in spring affects the size and movement - 94 -

of predator populations, which may have a significant but indirect bearing on mosquito population size. Similarly, the extrinsic incubation period of virus (i.e. the incubation period within the mosquito) is also temperature dependent. Hess et at. (1963) demonstrated that the accumulation of temperature in day-degrees above 75QF in spring favoured the increase in transmission of St. Louis encephalitis virus. During winter in southern Australia, mean weekly temperatures fall well below 17.5°c, so even above-average winter temperatures are unlikely to contribute to increasing population densities. However, the longevity of overwintering adults may well be increased. Winter temperatures in the Murray Valley in

1950 and 1973 were average (Fig. 26).

(b) The availability of water

Although there is no empirical measure of the effect of water on population growth, Cx annu~irostris has strong preferences for shallow, grassy pools of water. The origin of this water may be rain, flood or irrigation.

A comparison of light trap data collected during the flood year 1975-76

(Fig. 25c, Tables 12 and 13) with that of the non-flood years of 1976-77 and 1977-78 (Figs. 25a and 25b, Table 13) demonstrates how the large areas of breeding grounds which arise from floods can increase population densities.

The populations in the flood year were two to three times greater whilst temperatures were not greatly different (Table 13). Population growth during flood years is closer to that predicted by the temperature-based model than in non-flood years (Fig .. 25).

However, as with temperature (Fig. 10), the relationship between population growth and the amount and/or persistence of water is most likely to be represented by a normal curve (Fig. 27). A deluge of rain or a large flood from creeks and rivers produce enormous areas of surface water, (c) (a) (b) .

J 5 3.5 3.5 • -· Predttttd Model

•• .• Ob~erved /,/·-·-·,. JO 3.0 -· Preo.cred MOoel 3.0 -· Pr•d•cted Model \ I •.•. Otnerved / •• -· ObliiYtd ...... ·...... 2 5 2.5 I 2.5 ,/·-·-., / I .". / L L 01 ~ .!!' 1/ \ z I i i 20 2.0 • 2.0 i CL ; .CL I /·, ,/ .1· ,/ ',·, .. .. . 0 / 0- -= C!> ·~1 ~-~· ·' '\ 31 I ~ ~1-5 \!) I ~ :.1.s I .:.. 0 U1 ·, 0 'I l•ts ;·-·' ~ ~ ~ :. I \ \ I 0 0 .;-·· 0 ' 0 l I / .. 1.0 l I ci z .-)' z 1.0 1.0 ..···,_;· ...... / I __ ,.,-'j/ ~ t I·. ..- ...... 0.5 ./·. 0.5 0.5 ... _-·~,/- ..... ~ I :')/ ______,... .: I MAll API .!_./· OCT NOV DEC ;AN fEJ MAll API OCT NO-.' DEC JAN fEI 1977· 7i OCT NOV DEC JAN fEI MAll ...... 1976•77 1975·76

1050 I I 25 / I 1073 / MEAN / / ,'1874 I I I I 20 / I 4) '- / ;:) / I -0 8. E ...! 15

JAN FEB MAR APR MAY JUN JUL AUG SEP OCT NOV DEC Month Fig. 26: Computer spline curves fitted to the mean weekly temperatures of 1950, 1973, 1974 and a 12 year average at Mildura (data for Jan.-July, 1950 unavailable).

AVAILABLE WATER ( eg. flood over grassland - em)

Fig. 27: The hypothetical effect of water availability (e.g. depth) on the growth of larval and adult populations of Cx annutirostris. - 97 -

but often these are totally unsuitable for breeding because of the water depth, surface movement, or lack of vegetation.

Persistent rain, flooding or irrigation has a moderating influence on the rate of mosquito breeding. Established water bodies tend to produce less suitable vegetation, and hence attract fewer ovipositing females.

Similarly, predator populations readily increase in persisting ponds, and account for large pre-adult mortalities. In contrast, mortality is low in short-lived pools of grassy water (Fig. 13). The source of water for breeding sites may also influence their productivity. Kay (1978) noted that flooding may flush existing larval populations downstream, as well as introduce predatory fish into grassland pools. Following floods on the

Murray, Myers (1954) observed that breeding of Cro annutirostris dia not begin until ponds stabilized and vegetation developed.

Hence, short term but extensive pools of shallow surface water, originating from rain, irrigation, or minor flood, heavily favour maximum rates of mosquito production.

In view of this, consider the flood and rainfall conditions which preceded the 1951 and 1974 outbreaks. Despite the excessive rainfall received in all major water catchment areas in spring 1950, completing the

pre-epidemic pluvial rainfall pattern (Forbes 1978) 1 the Mildura-Swan Hill area of the Murray Valley received below average rainfall (Fig. 28). In contrast, the rainfall of spring 1973 was excessive throughout the valley.

The flood heights shown in Figure 29 reflect the general rainfall situation in the Murray Valley since 1950. The slight flood of 1950, caused by rainfall in the distant headwaters, together with minimal local rainfall, produced only moderate numbers of breeding grounds. In certain areas, irrigation also - 98 -

40 1950- 51

0 NOV DEC JAN FEB E E

1973-74 0 MEAN RAINFALL 120 ACTUAL ·RAINFALL 100

0 AUG SEP OCT NOV DEC JAN FEB Fig. 28: Rainfall at Mildura from August to February in 1950-51 and 1973-74 compared with a 25 year mean.

RIVER HEIGHTS 40 MILDURA WEIR, RECORDS 38 1956 ,•"'*,...... 36 ,, ...... 34 ...... ·::.--...... -:. -·-·-· -·-. -· .... 1950-51 r.s--&~--- 1955-56 32 .. -- , • e...._.n ..... _., ,,• 1- ,• I w 30 .. , •"" ·~ ...... ' ...... 1958-57 w '• ,. o/ i-"-a ...... ' ...... 28 '•• ·-•' ./• '•, 'a;:~~ ...... 1973 -74 •, 1974 ,, •""a •-• li~Q ,, 28 1974 -75 ; / .. 1- .- ..... -. ., ' , ·-·· :I: 24 ~ ~.., \\ ,, Q 22 ...~.' / \ Q ~\ w ,.• ~~c ,,,••"'•\ • '• :z: 20 FLOOD t ,.-· ~~., ....1?-~.q ...... u••··• ••.. ' FLOOD+ ec:: 18 / • ,....._,a LU • _,. e .• > 16 .... •"'/ CJ..., n...... • ~ 14 .::7-~ ...... ·· 12 10 81

JUL AUG SEP OCT NOV DEC JAN FEB MAR MONTH

Fig. 29: River heights and the flood levels for most of the major floods at Mi1dura since 1950. - ~9 -

cont.ributed to mosquito breeding (Reeves et aZ. 1954). The moderate flood of 1973 resulted in extensive surface water and abundant breeding grounds.

The flood receded in December and reformed in mid-January, creating many more predator-free habitats. Both spring periods prec-eded large outbreaks of M.V.E., and in December 1950, the first major outbreak of myxomatosis in rabbits also swept throughout southern Australia.

The springs of 1956 and 1974 experienced floods of unprecedented heights, covering wide areas of already water-logged soil with much greater depths of water than in preceding years. Flooding of this scale was clearly not conducive to the rate of Cx annuZiroatria breeding experienced during epidemics. A mosquito trapping study in the Murray Valley during February

1975 indicated that populations were only 3% of those monitored in February

1974 (Marshall, pers. carom.).

{c) Combined effects: Temperature and water

In terms of temperature, rainfall and flooding, the epidemic years of 1950-51 and 1973-74 were clearly different. However, the spring of both years preceded seasons of unusually high populations of Cx annuZirostris and epidemics of M.V.E. To explain this, these meteorological factors can be combined to illustrate, hypothetically, their joint effect on population growth using a three dimensional model (Fig. 30). The summit represents maximum population growth or size, and the upper zone represents threshold densities below which virus transmission cycles are unlikely to be maintained.

The shape of the model and placement of threshold densities are largely conjectural. Reeves (1968) has clearly shown that Western Equine encephalitis virus transmission requires certain threshold densities of

Cx taPsaZis, and these are monitored using light traps. Similarly,

Kay (1978} has shown, at a basic level, how threshold densities of - 100 -

TffiESOO..D IINSITIES

} Virus overflow - lftlman cases. Maintenance of primary vi ru:~ transmission cycle.

AVAILAB.£ WATER (e.g. flood over grassland)

Fig. 30: A conjectural model illustrating the combined effects of temperature and water availability (e.g. depth) on population growth and abundance of Cx annuZirostria. Threshold densities in respect to the two variables are shown by hatching on the summit. ~00 GiOml CCMQ.JRS -

'lliRESfl)[D a!NSITUS [ill] •

AVAILAll.E WAlER (e.g. flcx:Xt over gra5slono) Fig. 31: A contour graph, or plan view of Fig. 30, illustrating how the contrasting conditions within the springs of 1950, 1955 and 1973 produce similar threshold densities of Cx annuliroa~s and epidemics of M.V.E. There was no epidemic in 1956-57 or 1974-75. - 101 -

Cx annulirostris may be determined for the different ages of adults and for

varying periods of extrinsic incubation of virus which is temperature

dependant.

A simpler version of this model is illustrated by a contour graph (Fig. 31), the inner contours depicting an increasing population size, and the innermost contour representing threshold densities. As described previously,

the spring of 1950 in the Murray Valley was unusually hot but surface water was limited. In contrast, the spring of 1973 and 1955 experienced slightly

above average temperatures, but large floods produced extensive areas of

surface water. From Figure 31, it is clear how varying climatic conditions may mutually compensate to produce populations of Cx annuliPostria large

enough to support an outbreak of M. V.E.. The conditions of spring 1956 and

1974 were undoubtedly too wet and/or cool to produce the mosquito populations necessary for an outbreak in the corresponding summers. While the model is simplistic and conjectural, the calculation of real values for each variable may provide the basis of a useful predictive tool for future outbreaks.

Above all else, the model demonstrates that there may be a basic fallacy in assuming that excessive rainfall and/or flooding automatically leads to abnormally large mosquito and bird populations, and ultimately M.V.E. outbreaks. This was the case in the theories of Anderson and Eagle (1953) ~

Miles and Howes ( 1953) and Forbes (1978). The growth of Cx annu'lirostria populations depends on temperature and the availability, source and persistence of surface water during spring. - 102 -

3.3 Alternative vectors of M.V.E. virus in the Murray Valley

It is significant that, following appropriate meteorological

conditions, Cx annuliros tris is consistently found at high densities in those

regions experiencing at least some M.V.E. virus activity (Marshall and

·woodroofe 1976, Kay 1979b). Virus isolations from this mosquito during the

1974 epidemic confirmed its role as a major vector (Marshall and Woodroofe

1975). Yet, in the Murray Valley, the species is not recorded in the same high densities in the months which normally precede the first clinical

cases of M.V.E. Field studies have confirmed that populations of

Cx annulirostris gradually increase when average temperatures exceed 17.5°c in late October or November, but rapid population growth does not begin until late November or December (Fig. 19; Myers et al. 1954). As a result, this species alone is unlikely to account for disease outbreak in early

January. Thus the required level of pre-epidemic virus amplification among host populations may be dependent on other species of mosquitoes. Reports from the Center for Disease Control, USA, suggest that the cool season mosquito, Cx reatuans, plays a major role as an enzootic vector and participates in early ampli'fication of St. Louis encephalitis virus months before an outbreak (Anon. 1975). In the·Murray Valley, Aedes theobaldi and

Cx auatralieus fill this cool season niche.

Ae. th~obaldi responds to the early spring floods. or rai~s with one large generation. In view of the isolation of Ross River virus from this species early in 1979, (Marshall 1979) and successful experiments with transovarial transmission of M.V.E. virus (Mahon, pers. comm.) r its role in the primary cycle transmission must be considered. Reports of very large Aedes populations during the spring of 1950 support this argument (Anon.

1951) • - 103 -

The second species, Cx auat~aZiaus is reproductively active at temperatures well below that of Cx annuZirostris. It breeds consistently throughout the year, attaining peak densities in spring and autumn (Figs.

19 and 20). Although conclusive vector competence studies with M.V.E. virus in the species have yet to be undertaken, isolates of Sindbis and

Kunjin viruses were extracted for the first time from field collected

Cx australicus during the summer of 1979 (Marshall 1979) • Unlike Cx annulirostris, which favour mammals as hosts (Kay et at. 1979a), Cx austratieus is a non-man-biting mosquito which prefers birds and small mammals (Dobrotworsky 1965}. In flood years, it is less dependent on the development of vegetation in flood pools (Fenner and Ratcliffe 1965) and will respond immediately to floods. These factors all favour Cx auatraticus as an important vector in the primary bird-mosquito or pre-epidemic ,· amplification cycles in the Murray Valley. Similarly, it may also contribute to the maintenance of enzootic cycles. Mild and wet winters are more likely to influence population growth of these cool sea'son mosquitoes than Cx annulirostris.

3.4 Conclusions

l. In the Murray Valley, CULex annutirostris achieves very large population densities during the hottest months of each year, when the population densities of other species decline. The success of the species is largely attributable to:

(a) a population growth potential which is maximum at mean daily temperatures of 25°C. The mean weekly temperatures during mid-summer regularly approach but rarely exceed 25°c. - 104 -

(b) the readiness of the species to colonize shallow pools of grassy

water within hours of formation. This characteristic enables the species

to take advantage of many temporary pools before they dry up, and also

reduces larval mortality through avoiding predators. Short-lived pools

of this nature are very common within the irrigation and flood zones of the

Murray Valley.

2. The growth of Cx annulirostris populations commence when mean daily

spring temperatures exceed 17.5°C, and similarly populations rapidly

decline when mean daily autumn temperatures fall below 17.5°C.

3. The size of surruner populations of Cx annulirostris appears to be

influenced by the availability of breeding grounds, the size of the

overwintering or initial spring populations, and the. date of initial

activity of those populations.

4. The juvenile development of Cx annu~irostris~ from egg deposition,

may be completed in eight days when water temperatures approach 30°C.

5. Improved mosquito control in the Murray Valley requires a

re-examination of the current techniques and schedules used in Victoria,

particularly in relation to the biology and habits of Cx annutirostris.

Specifically:

(a) The application of the larvicide AbateR at weekly intervals is

justified during periods of high temperatures.

(b) The careful and controlled use of the larvicide at recommended

, rates will avoid high predator mortality, and resurgence of mosquito

populations. - 105 -

(c) Mosquito control in temporary water, where breeding is most intensive, may be gained most effectively by employing techniques in source reduction, such as drainage, cultivation or empoundage of temporary pools.

(d) Long term abatement programs should be established so priority may be given to improved water management facilities within irrigation districts.

6. Outbreaks of Murray Valley encephalitis appear to be activated by abnormally large populations of the vector and intermediate hosts. As the

growth of Cx annu'lirostris populations depends on temperature and the availability, source and persistence of surface water, a model which

incorporates these criteria would provide a means of·predicting threshold populations of Cx annuZirostris~ and hence M.V.E. epidemics. - 106 -

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APPENDICES

Appendix I

Laboratory Rearing of Culex annuZirostris Skuse (Diptera Culicidae)

by G. McDonald, I.R. Smith and G.P. Shelden

Published in The Journal of the Austratian EntomologiaaZ Soaiety (1977) 16: 353-358. - 115 -

J. Aust. ent. Soc., 1977, 16: 353-358 353 LABORATORY REARING OF CULEX ANNULIROSTRIS SKUSE (DIPTERA: CULICIDAE) G. McDoNALD*, I. R. SMITH and G. P. SHELDEN Plant Research Institute, SH"'Cll1 Street, Burnley, Victoria, 3121.

Abstract A successful technique was developed for laboratory rearing of Culex cmnulirostris. The larval rearing medium, containing water, crushed dog biscuit and yeast, reqtlired continuous aeration to prevent putrefaction. An emergence cage was designed to improve rearing and operational efficiency. Adults were fed on boiled sultanas, and females received their blood meals from guinea pigs. The culture room was kept at 26°C ± 1oc, and the relative humidity was alternated from 60% to 80% for three clay periods to maximize reproduction and survival. Simulated dusk and dawn enhanced feeding and mating. Observations from culturing studies are reported.

Introduction The mosquito, Culex wuwlirostris Skuse, is considered the major vector of Murray Valley encephalitis1' (Doherty et al. 1963; Doherty 1974). Although several other Culex spp. can be reared in the laboratory (Gerberg 1970), no satisfactory technique has been developed for Cx. annulirostris (Standfast et al. 1964; Doherty 1974). As a laboratory culture was required for a study of the biononiics of this tnosquito, the factors influencing survival of laboratory reared Cx. annulirostris were investigated. This paper describes the influence of water quality, food, and larval density on survival of Cx. annulirostris in the laboratory. This information was then used to develop a successful technique for culturing the species.

Materials and Methods Pupae and fourth instar larvae of Cx. annuliroslris were collected at Shepparton, Victoria in February 1975. Pond water rich in organic matter wus used for rearing these specimens to the adult stage and mating of adults was then obtained, with partial success, using the method described for Cx. tarsalis Coquillett (Brennan and Harwood 1953). All larval experiments were conducted at 26" ± 1"C, using 13h fluorescent lighting (1000 lx). b?fiuence o.ffood, aeration, and 1vater quality on larval and pupal survival Survival was evaluated for five types of food, added to beakers containing 500 ml of distilled water and 50 n~wly hatched first instar larvae, as follows:-- ( 1) Grass infusion: Sg of cocksfoot (Dactyli,\' glornerata L.) foliage was washed under tap water and placed in beakers 24h before adding larvae. (2) Blood: 2 drops of heparinized sheep's blood were added daily. (3) "Dog Chow"-Yeast (DCY): Finely crushed "Dog Chow" brand dog biscuits mixed with equal weight of Torula yeast. lO mg added daily. (4) "Liquifry": Balanced liquid branded fish food. 2 drops added daily. (5) Wheat Infusion: 3g of wheat boiled in 50 ml of water and 25 ml of filtrate added. All media were tested without aeration, or with aeration by bubbling compressed air through a 27 gauge dental needle at 150 ml min- 1• In addition, DCY was tested with and without aeration in shallow rearing trays ( 46 x 42 x 5 em) containing 8/ of water, to which 100 mg DCY was added per day. 500 larvae were placed in each of these trays. Four replicates of each treatment were used. After the adults had emerged, pH and the amounts of H2S, NI-1 3 and total dissolved solids (TDS) were measured for each medium. To determine the etfect of aeration on dissolved oxygen (D.O.) levels, 20 first ins tar larvae were placed into each of twenty 11. glass beakers containing 500 ml distilled water, and 10 mg DCY added daily. Ten of the beakers were aerated continuously as above, and D.O. levels measured daily in all beakers for five days with a YSI Model 54 Oxygen Meter. Dissolved oxygen was also measured in situ with this meter in a range of field habitats which appeared suitable for Cx. annulirostris. Influence of larval density on survival Beakers containing 500 ml of distilled water were stocked with either 25, 50, 75 or 100 first instar larvae using four replicates per treatment. Ten mg of DCY was added daily, and rates oflarval development and larval and pupal survival recorded.

*Present Address: Horticultural Research Station, Box 460, Irymple, Victoria, 3498. tAustralian arbo-encephalitis, National Health and Medical Research Council. 1974, Report of seventy eighth session, p. 8. rl!! - 116 - ('! F'··--·

Appendix II

Identification of instars of Culex annulirostria Skuse (Diptera Culicidae)

by G. McDonald, I.R. Smith and G.P. Shelden

Published in The Journal of the Australian EntomologiaaZ Soaiety (1977) 16: 359-60. - 117 -

J. Aust. ent. Soc., 1977, 16: 359~360 359 NOTE IDENTIFICATION OF INSTARS OF CULEX ANNULIROSTRIS SKUSE (DIPTERA: CULICIDAE) G. McDONALD*, I. R. SMITH and G. P. SHELDEN Plant Research ln~·titute, Swan Street, Burnley, Victoda, Australia 3121.

Abstract Head capsule width is shown to provide a reliable and rapid method for identifying the four larval instars of Culex annulirostris.

Accurate distinction between the four larval instars of the mosquito Culex annulirostris Skuse, the major vector of Murray Valley encephalitist (Doherty 1974), was required during field and laboratory studies of the species. Measuring th~ growth of sclerotised parts was a useful means of separating instars of Anopheles sergenti Theobald (Kettle 1948) and Cx.fatigans Wiedemann (Sen and Das Gupta 1958). Peters (1943) found that head capsule widths, but not body lengths, were relatively constant in each of the four instars of An. walkeri Theobald. We have investigated whether measurements of body length or head capsule width could enable rapid and accurate distinction between the four larval instars of Cx. annulirostris. Since the aim was to identify Cx. annulirostris larval instars from different habitats and varied environmental conditions, half the larvae were obtained from laboratory cultures, whilst half were sampled from a range of field environments. The laboratory larvae were sampled from cultures reared at temperatures ranging from l5°C to 35°C and in several nutrient media (the laboratory culture which provided these larvae was derived from larvae collected at Shepparton, Victoria), About 400 larvae, representing a wide range of body lengths and age groups, were immobilized by cooling to 3°C for two minutes. Using an eye~piece micrometer at 25 x, head capsule width was measured at the widest section of the head at the level of the eyes, and body length along the dorsal surface from the anterior extremity of the head capsule to the junction of the eighth and anal segments. The growth ratio (Dyar 1890) for head capsule width was derived by dividing the mean head capsule width in one instar by that in the preceding instar. Body length varied widely and the ranges overlapped substantially between consecutive ins tars (Table). In contrast, head capsule width varied only slightly Within each instar and the ranges did not overlap (Table).

TABLE HEAD CAPSULE WIDTHS AND BODY LENGTHS OF FOUR LARVAL INSTARS AND GROWTH RATIO FOR HEAD WIDTH IN SUCCESSIVE INSTARS DURING LARVAL DEVELOPMENT OF CX. ANNULIROSTRIS

Instar Mean and range· Coefficient of Growth (mm) variation ratio

1 Head capsule 0.300 (0.25~0.32) 2.00 Body length 1.104 (0.80-1.80) 20.02 1.67 2 Head capsule 0.501 (0.48-0.55) 2.40 Body length 2.219 (1.80~3.15) 11.36 1.59 3 Head capsule 0.799 (0.70-0.89) 4.26 Body length 3.078 (2.10~4.60) 14.88 1.61 4 Head capsule 1.285 (1.18~1.45) 5.06 Body length 5.862 (4.00~7.80) 13.75

Thus bead capsule width is characteristic for each stage of development and provides a reliable criterion for identifying the ins tars of Cx. annu!irostris, regardless of rearing conditions. Body length was too variable for this purpose, apparently being markedly influenced by temperature and nutrition. The growth ratio was constant between second and third, and third and fourth ins tars, while a higher ratio was observed between first and second instars (Table). Gaines and Campbell (1935) also found that growth ratios are often higher during early larval development. It is possible that other sclerotised structures (e.g. saddle, siphon) may be suitable for larval instar determination, but they were not measured in this study. For criticism of the manuscript we are grateful to Dr E. N. Marks and Mr B. H. Kay, Queensland Institute of Medical Research, Brisbane, and Dr I. McLaren, Plant Research Institute, Burnley. Funds were provided jointly by the Commonwealth and State Departments of Health.

*Present address: Mildura Horticultural Research Station, Box 460, lrymple, ViCtoria 3498. tAustralian arbo~encephalitis, National Health and Medical Research Council, 1974, Report of seventy~ eighth session, p. 8. - 118 - Appendix III Age specific life and fecundity tables fo~ Cx ·annuZiPostPis at 20° ±1°c.

Developmental Stage Mean _age during age interval x t * (days} lx mx lx. mx

egg 2.2 0.870 0.0 0.0 1st in star 5.2 0.730 o.o 0.0 2nd ins tar 7.2 0.715 0.0 0.0 3rd ins tar 9.7 0 .. 705 o.o 0.0 4th ins tar 13.7 0 .. 665 o.o 0.0 pupae 16.7 0.610 0.0 0.0 adult 17.5 0.590 o.o 0.0 18.5 0.585 0.0 0.0 19.5 . 0.570 0.0 0.0 20.5 0.550 0.0 0.0 21.5 0.540 0.0 o.o 22.5 0.530 0.0 0.0 23.5 0.495 0.0 0.0 24.5 0.445 o.o 0.0 25.5 0.400 0.0 o.o 26.5 0.375 0.0 0.0 27.5 0.355 0.0 0.0 28.5 0.320 o.o o.o 29.5 0.285 0.0 o.o 30.5 0.260 0.0 0.0 31.5 0.250 o.o 0.0 32.5 0.245 o.o 0.0 33.5 0.240 0.0 0.0 34.5 0.240 0.0 o.o 35.5 0 .. 240 o .. o 0 .. 0 36.5 0.240 0.0 0 .. 0 37.5 0.230 1.033 0.238 38.5 0.215 0.0 0.0 39.5 0.195 0.0 0.0 40.5 0.175 0.0 0.0 41.5 0.155 o.o 0.0 42.5 0.155 1.481 0.230 43.5 0.135 0.0 0.0 44.5 0.130 o.o o.o 45.5 0.130 3.738 0.486 46.5 0.130 0.0 0.0 47.5 0.130 0.0 0.0 48.5 0.130 0.0 0.0 49.5 0.130 o.o 0.0 50.5 0.130 0.0 0.0 51.5 0.125 0.0 o.o 52.5 0.120 0.0 0.0 53.5 0.115 0.0 0.0 54.5 0.110 J.243 0.247 55.5 0.110 0.0 0.0 56.5 0.110 0.0 0.0 57.5 0.105 0.0 o.o 58.5 0.100 o.o o.o 59.5 0.095 o.o 0.0 t lx - proportion of the original cohort surviving to the age interval x * mx - number of female offspring produced per surviving female in the age interval x. - 119 -

Stage Mean age

adult 60.5 0.085 8.411 61.5 0.175 0.080 0.0 62.5 0.0 0.075 0.0 o.o 63.5 0.070 o.o 64.5 . o.o 0.070 0.0 0.0 65.5 0.065 o.o o.o 66.5 0.060 67.5 0.0 o.o 0.060 4.132 0.248 68.5 0.060 o.o 69.5 o.o 0.060 o.o 0.0 70.5 0.050 0.0 71 .. 5 o.o 0.040 o.o 0.0 72.5 0.035 14.273 73.5 0.500 0.030 8.722 0.262 74.5 0.025 o.o 75.5 o.o 0.020 o.o 0.0 76.5 0.020 o.o o.o 77.5 0.020 78.5 o.o o.o 0.020 22.429 0.449 79 .. 5 0.015 o.o 0.0 80.5 0.010 81.5 o.o 0.0 0.010 26.167 0.262 82.5 o.oos o.o o.o - 120 - Appendix IV Age specific life and fecundity tables for Cx annulirostrie at 25° + 1°c

Developmental Mean age t * stage during age interval X (days) lx IDx lx. mx

egg 2.0 0.965 0.0 0.0 1st ins tar 4.5 0.915 0.0 o.o 2nd ins tar 6.0 0.865 0.0 o.o 3rd in star 7.5 0.805 0.0 o.o 4th ins tar 9.2 0.760 0.0 0.0 Pupa. 11.7 0.705 0.0 o.o adult 13.5 0.660 0.0 o.o 14.5 0.645 0.0 o.o 15.5 0.635 0.0 0.0 16.5 0.613 0.0 o.o 17.5 0.579 o.o 0.0 18.5 0.551 0.0 0.0 19.5 0.535 0.0 o.o 20.5 0.522 0.0 ,0.0 21.5 0.496 0.468 0.232 22.5 0,469 o.o 0.0 23.5 o.452 0.499 0.225 24.5 0.434 1. 303. 0.566 25.5 0.419 2.441 1 .. 022 26.5 0.407 0.0 o.o 27.5 0·.400 0.289 0.116 28.5 0.~90 o.o o.. o 29.5 0.383 1.810 0.693 30.5 0.'377 4.516 1.700 31.5 0.369 2.176 0.802 32.5 0.365 0.312 0.114 33.5 0.360 2.855 1.028 34.5 0.356 4.53~ 1.611 35.5 o.3so 0.975 0.341 36.5 0.340 0.671 0.228 37.5 0.334 1.037 0.346 38.5 0.327 0.0 o.o 39.5 0.312 o.o o.o 40.5 0.297 1.139 0.338 41.5 0.282 0.796 0.224 42 .. 5 0.271 0.4!29 0.116 43.5 0.257 0.437 0.112 44.5 0.240 '0.936 0.224 45 .. 5 0.228 1.435 0 .3,26 46.5 0.219 1.490 0.326 47.5 0.211 2.66S Q ,§6:3 48.5 0.206 2.78$ 0.512 49.5 0.203 4.524 6~.916 50 .. 5 o.2oo "4.586 ,· 6.915· 51.5 0.192 4.072 C). 780, 52.5 0.180 10.609 l.9f>'4··· .. 0.166 s.•. ~se, iq,,,$75 53.5 ··· ··.,,m'•<>'.92.' 54.5 0.149 1 . .3:5,5' •6 B6S 0 .6''7¢· 55.5 0,133 ."'

0 t lx - proportion of the original female cohort sUJ:viving,.' 1 ' tq ' ,i' ',)'th~J~~~E!'' I'" ':, ,'J;' ,, ,i ! ~(:;' ', ',, ~.o~e~v'aa:x•',, ,'' ,,',',I ~ ;·,~·, i * rnx - number of female offspring produoed per ew;viv;:ta, fi,xii~e/ !'~))~,}~~~,, ln,~~'i:t'v~l ~· i ; ' ~ - 121 -

·Stage Mean a•Je 1x mx lx· mx adult 56.5 0.120 5.507 0.685 57.5 0.104 8.104 0.843 58.5 0.091 8.533 0.772 59.5 0.080 5.288 0.423 6o.5 0.070 1.498 0.104 61.5 0.065 8.666 0.563 62.5 0 .. 060 o.o 0.0 63.5 0.055 4.001 0.222 64.5 0.052 8.319 0.433 65.5 0.048 0.0 o.o 66.5 0.046 2.441 0.114 67.5 0.045 5.031 0.0 68.5 0.043 o .. o o.o 69.5 0.043 2.691 0.116 70.5 0.040 2.691 0.109 71.5 0.036 5.998 0.216 72.5 0.034 6.787 0.231 73.5 0.034 13.564 0.461 74.5 0.026 o.o 0.0 75.5 0.018 70 .. 003 1.260 76.5 0 .. 015 o.o 0.0 77.5 0.011 9.750 0.102 78.5 0.008 o.o 0.0 79.5 0.007 o.o 0.0 80.5 0.007 o.o 0.0 81.5 0.006 o.o 0.0 82.5 0.002 o.o o.o - 122 -

Appendix V

Age specific life and fecundity tables for Cro annuZi~ostris at 30° ± 1°c.

Mean age during Developmental stage age interval X 1xt mx* lx·mx (days}

egg o.s 1.000 0.0 0.0 1.2 1.000 0 .. 0 0.0 1st ins tar 1.7 0.985 o .. o. 0.0 2nd instar 2.7 0.860 o.o 0.0 3rd ins tar 3.7 0.695 0.0 0.0 4th ins tar 5.0 0.635 0.0 0.0 7.2 0.610 0 .. 0 o.o pupa 9.2 0.575 o .. o 0.0 adult 10.5 0.560 o.o o.o 11.5 0.555 o.o o.. o 12.5 0.550 0.0 0.0 13.5 0.525 0.0 0.0 14.5 0.475 o.o 0.0 15.5 0.440 o.o 0.0 16.5 0.410 o.o 0.0 17.5 0.375 o.o o.o 18.'5 0.350 o.o 0.0 19.5 0.330 o.o 0.0 20.5 0.315 0.392 0.123 21.5 0.305 0.0 o.o 22.5 0,290 0.0 0.0 23.5 0.265 0.680 0.180 24.5 0.245 1.359 0.333 25.5 0.240 0.534 0.128 26.5 0.225 0.297 0.067 27.5 0.190 1.227 0.233 28.5 0.170 0.722 0.123 29.5 0.160 q.377 0.060 30.5 0.130 o.. o o.o 31 .. 5 0.105 1.174 0.1~3 32 .. 5 0.• 100 o.o 0.0 33.5 0.095 Q.O o.o 34.5 0.085 1.452 0.12~ 35.5 o.oso 0.0 0.0 36.5 0.075 2 ... 470 .0.11;3.$ 37.5 0.065 0.951 0.062 38.5 0 .0'60 1.985 0~119 39.5 o.oso 1.164 o.osa 40.5 o.o~s o.o 0!'0 41.5 0. 0.30. 3.97S 0.119 42.5 o.~a~ 2.l~8 D.osa 43.5 0.01$ o.o o.o 44.5 o .• o·Q:$ o.. o o.o

to th~ ~ge t lx - proportion of the original female interval x. * mx - number of female age interval x • •. - 123 -

Appendix VIa

A summary of daily population estimates of mosquito larvae and predators in the Mildura pond in February and March 1977.

Day Date Total Av./dip. In star (Cx annuZir'O.cJ·tr1itJ) Total No. Larvao Pred. lst 2nd 3rd 4th Pupae

1 13/2 2 14/2 56 2.8 56 3 3 15/2 52 2.6 11 41 0 4 16/2 158 7.9 99 3 13 5 5 17/2 262 1'3.1 48 100 l)2 14 12 6 18/2 301 15.0 117 54 107 17 14 7 19/2 474 23.9 254 76 56 70 15 8 8 20/2 365 18.2 130 76 32 64 21 5 10 22/2 134 7.7 39 5~ 26 47 11 a 11 23/2 157 7.8 37 53 19 19 25 16 12 24/2 14 7 7.3 6 33 57 31 18 17 13 25/2 281 14.0 154 38 46 32 11 9 14 26/2 265 13.2 96 94 20 57 15 21 15 27/2 178 8.9 37 59 9 36 7 13 16 28/2 332 16.6 87 132 14 30 16 13 17 1/3 315 15.7 103 91 59 26 25 8 18 2/3 253 12.7 100 53 33 15 29 19 19 3/3 96 4.8 13 39 22 24 6 20 20 4/3 122 6.1 72 22 3 10 0 27 21 5/3 165 8.4 60 58 16 25 1 24 22 6/3 167 8.3 98 53 11 4 1 7 23 7/3 181 9.0 83 49 8 13 8 23 24 8/3 216. 10.8 49 10~ 28 12 7 23 25 9/3 197 9.9 78 71 22 12 14 24 26 10/3 341 17.0 267 35 25 3 9 43 27 11/3 411 20.5 355 27 23 1 4 36 29 13/3 34H 17.4 110 184 28 -;1.4 8 28 30 14/3 118 5 .'9 29 65 9 5 6 35 31 15/3 106 5.3 5 57 27 10 4 47 32 16/3 113 5.6 58 33 15 2 1 49 33 17/3 211 10.5 124 45 24 9 7 15 34 18/3 145 7.2 37 26 a 4 8 34 35 19/3 114 5.7 ~1 33 10 6 3 62 36 20/3 75 3.7 42 19 5 4 3 56 37 21/3 91 4.5 28 31 24 4 2 ~so 40 24/3 61 3.0 23 24 6 6 0 37 - 124 -

Appendix VIb

A summary of daily population estimates of mosquito larvae ' and predators in the Mildura pond during February to April, 1978.

Day Date Total In star (Cx annuZirostrie-) Total Av./Dip. No. Larvae Pred. 1st 2nd 3rd 4th Pupae

1 17/2 0 0 2 18/2 202 12.6 200 3 4 20/2 253 15.8 96 128 10 6 5 21/2 302 23.9 161 109. 54 6 9 6 22/2 478 29.9 257 87 79 3 12 7 23/2 184 11.5 65 62 35 4 2 13 8 24/2 298 18.6 134 99 . 35 6 4 24 11 27/2 148 9.3 49 22 60 5 1 8 12 28/2 131 8.2 14 46 51 4 1 10 13 1/3 137 8.6 6 70 36 10 3 7 14 2/3 121 7.6 12 60 27 6 a 4 15 3/3 172 lO.a 78 42 11 20 8 19 la 6/3 11a 7.4 29 43 17 9 2 20 20 8/3 76 4.8 29 35 12 10 5 9 21 9/3 as 5.3 12 26 19 5 8 22 22 10/3 64 4.0 30 13 7 5 4 9 26 14/3 113 7.1 10 44 31 24 3 58 27 15/3 126 7.9 5 13 15 18 a 62 28 16/3 34 2.1 0 11 6 9 7 29 29 17/3 26 1.6 0 3 14 2 . 4 7 32 20/3 0 0 0 0 0 0 0 0 33 21/3 32 2.0 8 0 1 6 13 11 34 22/3 25 1'.. 6 12 1 1 9 4 24 35 23/3 17 1 .. 1 0 4 2 8 8 3 36 24/3 20 1.3 0 10 3 4 8 10 38 26/3 8 0.5 0 5 l l 8 9 41 29/3 15 0.9 0 0 13 ·1 6 11 42 30/3 10 0.6 0 1 7 1 2 15 43 31/3 15 0.9 0 0 12 1 0 5 46 3/4 9 0.6 0 0 6 2 4 11 47 4/4 4 0.3 0 0 2 2 1 3 48 5/4 4 0.3 0 0 2 2 1 10 49 6/4 8 0.5 0 0 0 2 1 6 54 11/4 3 0.2 0 0 0 0 1 2

Minerva Access is the Institutional Repository of The University of Melbourne

Author/s: MCDONALD, GARRICK

Title: Aspects of the ecology of the common banded mosquito, Culex annulirostris, a major vector of Murray Valley encephalitis virus

Date: 1981

Citation: McDonald, G. (1981). Aspects of the ecology of the common banded mosquito, Culex annulirostris, a major vector of Murray Valley encephalitis virus. Masters Research thesis, Faculty of Agriculture and Forestry, The University of Melbourne.

Publication Status: Unpublished

Persistent Link: http://hdl.handle.net/11343/35845

File Description: Aspects of the ecology of the common banded mosquito, Culex annulirostris, a major vector of Murray Valley encephalitis virus

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