The common saltmarsh mosquito, Ochlerotatus vigilax (Skuse), is an important vector of Ross River and Barmah Forest viruses in coastal areas throughout northern Australia. Although vector survivorship, or life span, is one of the most important determinants of arbovirus transmission, there have only been a few investigations of vector survivorship in Australia. Therefore, investigations were conducted to evaluate the suitability of currently available mosquito age grading methodologies for Oc. vigilax, and to investigate new methods that could lead to the development of practical and reliable tools for age grading insects.
Evaluations were made of the suitability of mosquito age grading methods based on changes to morphology; including the Detinova ovarian tracheation, Polovodova ovariole dilatation, ovarian injection and daily growth line methods. Application of the Detinova technique to laboratory reared Oc. vigilax mosquitoes in a blinded trial enabled successful identification of nulliparous and parous females in 82 to 88% of specimens. The majority of nulliparous females could also be identified by observing the presence of midgut meconium. However, application of the Polovodova method only enabled 58% of nulliparous, 1-parous, 2-parous and 3-parous females to be identified and ovarian injections were found to be unfeasible. Additionally, poor correlation was achieved between the number of growth lines per phragma and the calendar age of laboratory reared Oc. vigilax females.
Given that nulliparous/parous classifications were the most accurate of the morphological techniques for Oc. vigilax, investigations were conducted into autogeny (development of the first batch of eggs without blood) in southeast Queensland populations of this species; a factor that can seriously bias estimates of survivorship based on nulliparous/parous classifications. Field and laboratory investigations were conducted to determine the seasonal prevalence of autogeny in Oc. vigilax at Wellington Point and Donnybrook between January 2001 and January 2002. Autogeny rates varied between 71 and 100% at Wellington Point and 63 and 100% at Donnybrook. A laboratory study was conducted to investigate the influence of larval nutrition and adult diet (water versus sucrose) on the expression of autogeny. The autogeny rate for a low diet treatment varied between 73 and 90% without sucrose and was 100% when sucrose was provided. Immatures fed on a high larval diet developed into autogenous adults.
The cuticular hydrocarbon technique of age grading mosquitoes was evaluated for use on laboratory reared Oc. vigilax. Gas Chromatography / Mass Spectroscopy was used to determine the relative abundance of five alkanes in hexane extracts from the legs of individual mosquitoes. However, there was an absence of suitable age related trends from the hydrocarbon levels. In contrast to Oc. vigilax, age related changes to relative hydrocarbon abundances were observed from Anopheles farauti (Laveran) and Aedes aegypti (Linn.), vectors of malaria and dengue, respectively. Female An. farauti were classified into two age categories (1 to 5 and ≥5 days old at 27°C) and Ae. aegypti into three age categories (1 to <5, 5 to <9 and >≥9 days old at 27°C) based on these changes.
Simulation modelling was used to construct sequential sampling guidelines for the application of this technique to estimate the survivorship of Ae. aegypti and An. farauti populations. These guidelines define the relationship between the survival rate, number of mosquitoes sampled, cuticular hydrocarbon based predictions of age and the accuracy of survival rate estimates. They demonstrated for example, that if 19% of a population of Ae. aegypti is estimated to be≥9 days old by cuticular hydrocarbon analysis, an estimate of the daily survival rate from the exponential model should be based on a sample of 200 mosquitoes for the survival rate estimate to be within 5% of the actual rate. However, if only 10% of the population is estimated to be ≥9 d old, 500 mosquitoes would need to be analysed for the survival rate estimate to be of equivalent accuracy.
Investigations were made to evaluate changes to the transcription levels of mosquito genes as potential age markers. In particular, investigations were made of a small heat shock protein (SHSP) gene and a muscle actin gene (Aeact-1). A new SHSP gene was cloned and sequenced from Ae. aegypti cDNA, however, transcription of the gene was unrelated to age in this species. In contrast to the SHSP gene, transcription of the Aeact-1 gene decreased rapidly in young Ae. aegypti and could potentially be used as a marker of age. Additionally, total RNA abundance was shown to decrease with age in young Ae. aegypti. An. farauti and Oc. vigilax.
The results presented in this thesis demonstrate the need for an integrated approach to determining mosquito survival rates, whereby the influences of accuracy and precision of age estimates are considered, as well as factors including mosquito behaviour and sample sizes on which estimates are based. The most accurate methods for determining vertical survival rates of Oc. vigilax populations are assessments of parity and the presence of meconium in the midgut. Behavioural factors related to autogeny represent complicating factors in these analyses, alternative age grading methods were found to be unsuitable. However, for other species, cuticular hydrocarbon analysis provided the most accurate estimates of age out of the methods examined. There is large potential for applying cuticular hydrocarbon analysis, as well as sequential sampling guidelines, in future investigations of the survival rates of important Australian mosquito vectors.