Study site
Malahlapanga, (22°53'S; 31°02'E) is a fresh water geothermal spring situated in the Northwestern region of the Kruger National Park (Figure 3). The spring is surrounded by Colophospermum mopane and Acacia nigrescens trees [21]. Warm water (~37°C) from the eye of the spring flows downstream, creating a wide wetland with a profusion of suitable breeding sites for mosquitoes. The spring supports a perennial, geographically isolated, population of An. arabiensis[21]. Proliferation of mosquitoes is supported by abundant ruminant and antelope herds which use the pan as a water source.
Anopheles arabiensis laboratory colony
The An. arabiensis laboratory colony, MALPAN, originates from material collected at Malahlapanga in 1994. It is maintained in the Botha DeMeillon insectary, Vector Control Reference Unit (VCRU), National Institute for Communicable Diseases (NICD), Johannesburg, South Africa, under standard insectary conditions of 25°C, 85% relative humidity and a photo period of 12: 12 hour light/darkness, with a 45-min dawn and dusk light regime.
Wild mosquito collections
Mosquitoes were collected in June 2010, November 2010 and February 2011 from Malahlapanga. Host seeking females were collected outdoors by human landing catches and CO2 bait net traps between 18h00 and 23h00 over three successive nights in each collection month. Only six adults were collected in June 2010 as a consequence of adverse weather conditions, so larvae were collected instead. Larval sampling was based on the standard dipping method [22]. Only anopheline larvae were retained and preserved in 70% ethanol for further identification to species level in the laboratory. Adult mosquitoes were morphologically identified using a taxonomic key [23]. Those identified as members of the An. gambiae complex were pooled and maintained live in 250 ml paper cups for transport to the VCRU laboratory in Johannesburg. All wild-caught females were offered two blood meals in order to procure progeny for the series of experiments described below.
Species identification
Specimens morphologically identified as members of the An. gambiae complex were identified to species level using the polymerase chain reaction (PCR) assay [24]. To get an overview of anopheline species composition in Malahlapanga other anophelines collected in February 2011 were identified to species level. Specimens identified as members of the An. funestus group were processed using the multiplex PCR assay with slight modifications [25].
Measurement of fitness components
Comparative fitness between wild An. arabiensis from Malahlapanga and the MALPAN laboratory colony was assessed using the following fitness parameters:
(i) Fecundity
Individual An. arabiensis females collected from Malahlapanga were transferred into oviposition vials following blood feeding. Each oviposition vial contained a moistened Whatman filter paper disc (Cat No. 1001125) to induce oviposition. A total of 70 newly emerged males and 50 females from the MALPAN colony were allowed to mate for 7 days in 5-litre plastic adult cages before being offered three blood meals over a five day period. Immediately after the third blood meal, 30 randomly selected blood-fed females were placed in oviposition glass vials to induce oviposition. Oviposited eggs from each female were counted using a hand-held magnifying lens. Mean number of eggs laid were calculated. Fecundity of wild collected females and laboratory females could not be compared directly because wild-caught females were of unknown age. Owing to variation in the number of egg batches produced by each female, fecundity was scored as the number of eggs laid by each female per gonotrophic cycle.
(ii) Egg hatch rates
For each wild-caught egg batch and MALPAN female, eggs from the first gonotrophic cycle were transferred into plastic bowls (27 cm × 16 cm × 6.5 cm) containing 150 ml of distilled water and allowed to hatch. Upon hatching, larvae were counted and transferred to a new bowl of water. This was done daily for 10 consecutive days. The mean number of days taken to hatch and proportion of eggs hatching was determined and compared between the wild-caught and MALPAN samples.
(iii) Larval survivorship
Fifty randomly selected newly hatched larvae from eggs oviposited by each female cohort were placed in larval bowls (34 × 27 cm) containing 150 ml of distilled water. The larvae were fed daily on approximately 30 mg of larval food ((a mixture of brewers yeast (Vital Health Foods, South Africa) and finely ground dog biscuits (West's traditional crunching biscuits treats, Martin and Martin, South Africa)) prepared at a ratio of 1:3. Larvae were maintained under standard insectary conditions. Each day the numbers of surviving larvae were counted and their stage of development was recorded. Pupae that emerged were counted and transferred into pupal emergence vials (35 mm × 57 mm) placed in 10-litre adult emergence cages. Larval survivorship was calculated as the proportion of first-instar larvae which pupated. Mean larval survivorship was compared between the wild-caught and MALPAN samples.
(iv) Adult emergence and sex ratio
Pupae were counted by family and transferred into plastic vials filled with 50 ml distilled water and kept in 10-litre plastic cages for adult emergence. Pupae were monitored daily and the number and sex of emerging adults was recorded for each family. Only adult mosquitoes that successfully emerged and were capable of flying were scored as emerged. The mean proportion of pupae surviving to the adult stage was calculated for each strain and compared between the wild-caught and MALPAN samples.
(v) Adult longevity
Newly emerged F1 progeny from field collected females were pooled from at least 10 families, separated by sex and placed in mosquito cages (50 for each sex). For the laboratory colony (MALPAN), one-day old males and females (50 of each) were set up in cages as described above. All adults were then maintained on 10% sugar solution soaked on cotton wool at standard insectary conditions. Survival was assessed daily until 100% mortality was reached. The experiment was replicated three times.
(vi) Wing length measurements
Wing length gives a good approximation of body size [26]. Wing lengths were measured from all females used for fecundity studies as well as 50 males each from MALPAN and the F1 progeny of wild caught females. One wing was dissected from each individual and placed on a clean glass slide. Wing-lengths (wing tip to thorax joint) were measured under a calibrated dissecting microscope. Mean wing-length was compared between the wild-caught and MALPAN samples by gender.
Mating compatibility
Mating compatibility between the F1 progeny of wild-caught samples and MALPAN was determined by combining fifty newly emerged females and 70 newly emerged males in 5 litre cages using the following combinations (i) MALPAN females × F1 males, (ii) MALPAN males × F1 females, (iii) F1 females × F1 males and (iv) MALPAN males × MALPAN females. Each cross was set up in duplicate. Mosquitoes from each cross were allowed to mate for seven days post-emergence. During this period they were maintained on a 10% sucrose solution under standard insectary conditions. After seven days the females were offered three successive blood meals with one day in between each blood meal. The following parameters were used to assess mating compatibility and fitness: insemination rate, fecundity, egg hatch rates, larval survivorship, pupal survivorship, emergence rates and adult survivorship of progeny from each cross.
(i) Insemination rates
After the third blood meal a sub-sample of 10 females from each cross described above was removed in order to determine the proportion/rate of insemination. Each female's spermatheca was dissected and the presence of spermatozoa was assessed under a dissecting microscope (Wild, Heerbrugg M5-71661, Switzerland) at 200 × magnification. The proportion of inseminated females was calculated for each cross enabling direct comparisons between cross-mating permutations.
Fecundity, egg hatch rates, larval survivorship, pupal survivorship, emergence rates and adult survivorship for each cross were determined as previously described above and a comparison between the crosses was carried out.
(ii) Hybrid polytene chromosome assessments
Seventy newly emerged F1 males from wild caught females and fifty one day old virgin females from MALPAN were pooled into a single 5 litre plastic cage and allowed to mate for seven days. Hybrid progeny accruing from this cross were raised to adults, placed in a cage and allowed to mate for seven days. After the seventh day females were offered three successive blood meals over a period of a week. After the third blood meal a petri dish painted black on the outside and filled with 40 mls of distilled water was put into the cage to induce oviposition. Two days after oviposition 10 hybrid females which had completed their gonotrophic cycle were randomly selected, transferred into a 250 ml paper cup and were offered another blood meal. Their ovaries were dissected at the half gravid stage and were then fixed in Carnoy's fixative (3 parts absolute alcohol: 1 part glacial acetic acid) for at least three days. Polytene chromosomes were then prepared for analysis [27, 28]. The hybrid chromosomal preparations were examined under a phase contrast compound microscope (OLYMPUS BX50) in order to assess chromosomal homogeneity and to monitor abnormalities in the hybrid chromosomes such as asynapsis that could indicate a genetic discontinuity between the parental groups.
Statistical analysis
Data on fecundity, adult sizes (wing length), hatch rates, larval survivorship and adult emergence was summarised as mean wing lengths, mean number of eggs produced, mean proportion of eggs hatching, mean proportion of larvae surviving to pupae and mean proportion of pupae surviving to adult stages respectively. A student-t test in Statistix 7®was used to analyze differences in mean egg production and adult sizes between wild-caught F1 progeny and MALPAN females. One way ANOVA was used to assess the differences in mean egg production between the crosses followed by Bonferroni comparison of means. Percentage values for larval and pupal survivorship as well as adult emergence and insemination rates of individual females were checked for normality and transformed where applicable to achieve normal distribution. These were then compared between wild-caught F1 progeny and MALPAN females using Student-t tests whilst for the crosses ANOVA was used to compare differences. Survival curves were analysed using Kaplan Meier survival analysis in XLSTAT® 2009 and Cox's F test was used to compare mean difference in survivorship between male and female cohorts as well as between samples. In all cases a P-value of less than 0.05 was considered to indicate statistical significance.