Comparison of the effect of bacterial stimulation on the global epigenetic landscape and transcription of immune genes in primarily zoophilic members of the Anopheles gambiae complex (Diptera: Culicidae).

Members of the Anopheles gambiae complex vary in their vector competence, and this is often attributed to behavioural differences. Similarly, there are differences in transmission capabilities of the zoophilic members of this complex despite exhibiting similar behaviours. Therefore, behavioural differences alone cannot fully explain vector competence variation within members of the An. gambiae complex. The immune system of mosquitoes plays a key role in determining susceptibility to parasite infection and consequently transmission capacity. This study aimed to examine variations in the immune response of An. arabiensis, An. merus and An. quadriannulatus, a major, minor, and non-vector respectively. The global epigenetic landscape was characterised and the expression of Defensin-1 and Gambicin was assessed in response to Gram-positive (Streptococcus pyogenes) and Gram-negative (Escherichia coli) bacterial infections. The effect of insecticide resistance in An. arabiensis on these aspects was also assessed. The immune system was stimulated by a blood-borne bacterial supplementation. The 5mC, 5hmC, m6A methylation levels and Histone Acetyl Transferase activity were assessed with commercial ELISA kits. The transcript levels of Defensin-1 and Gambicin were assessed by quantitative Real-Time Polymerase Chain Reaction. Species-specific differences in 5mC and m6A methylation existed both constitutively as well as post immune stimulation. The epigenetic patterns observed in the laboratory strains were largely conserved in F1 offspring of wild-caught adults. The methylation patterns in the major vector typically differed from that of the minor/non-vectors. The differences between insecticide susceptible and resistant An. arabiensis were more reflected in the expression of Defensin-1 and Gambicin. The expression of these peptides differed in the strains only after bacterial stimulation. Anopheles merus and An. quadriannulatus expressed significantly higher levels of antimicrobial peptides, both constitutively and after immune stimulation. These findings suggest molecular variations in the immune response of members of the An. gambiae complex.

The Impact of Climatic Factors on Temporal Mosquito Distribution and Population Dynamics in an Area Targeted for Sterile Insect Technique Pilot Trials.

It is widely accepted that climate affects the mosquito life history traits; however, its precise role in determining mosquito distribution and population dynamics is not fully understood. This study aimed to investigate the influence of various climatic factors on the temporal distribution of Anopheles arabiensis populations in Mamfene, South Africa between 2014 and 2019. Time series analysis, wavelet analysis, cross-correlation analysis, and regression model combined with the autoregressive integrated moving average (ARIMA) model were utilized to assess the relationship between climatic factors and An. arabiensis population density. In total 3826 adult An. arabiensis collected was used for the analysis. ARIMA (0, 1, 2) (0, 0, 1)12 models closely described the trends observed in An. arabiensis population density and distribution. The wavelet coherence and time-lagged correlation analysis showed positive correlations between An. arabiensis population density and temperature (r = 0.537 ), humidity (r = 0.495) and rainfall (r = 0.298) whilst wind showed negative correlations (r = -0.466). The regression model showed that temperature (p = 0.00119), rainfall (p = 0.0436), and humidity (p = 0.0441) as significant predictors for forecasting An. arabiensis abundance. The extended ARIMA model (AIC = 102.08) was a better fit for predicting An. arabiensis abundance compared to the basic model. Anopheles arabiensis still remains the predominant malaria vector in the study area and climate variables were found to have varying effects on the distribution and abundance of An. arabiensis. This necessitates other complementary vector control strategies such as the Sterile Insect Technique (SIT) which involves releasing sterile males into the environment to reduce mosquito populations. This requires timely mosquito and climate information to precisely target releases and enhance the effectiveness of the program, consequently reducing the malaria risk.

A review on the progress of sex-separation techniques for sterile insect technique applications against Anopheles arabiensis.

The feasibility of the sterile insect technique (SIT) as a malaria vector control strategy against Anopheles arabiensis has been under investigation over the past decade. One of the critical steps required for the application of this technique to mosquito control is the availability of an efficient and effective sex-separation system. Sex-separation systems eliminate female mosquitoes from the production line prior to irradiation and field release of sterile males. This is necessary because female mosquitoes can transmit pathogens such as malaria and, therefore, their release must be prevented. Sex separation also increases the efficiency of an SIT programme. Various sex-separation strategies have been explored including the exploitation of developmental and behavioural differences between male and female mosquitoes, and genetic approaches. Most of these are however species-specific and are not indicated for the major African malaria vectors such as An. arabiensis. As there is currently no reliable sex-separation method for An. arabiensis, various strategies were explored in an attempt to develop a robust system that can be applied on a mass-rearing scale. The progress and challenges faced during the development of a sexing system for future pilot and/or large-scale SIT release programmes against An. arabiensis are reviewed here. Three methods of sex separation were examined. The first is the use of pupal size for gender prediction. The second is the elimination of blood-feeding adult females through the addition of an endectocide to a blood meal source. The third is the establishment of a genetic sexing strain (GSS) carrying an insecticide resistance selectable marker (dieldrin-resistance rdl gene and/or other GABA receptor antagonists that can be used as alternative insecticides to dieldrin) or a temperature-sensitive lethal marker.

Optimization of Mass-Rearing Methods for Anopheles arabiensis Larval Stages: Effects of Rearing Water Temperature and Larval Density on Mosquito Life-History Traits.

Insect mass-rearing is an essential requirement for the sterile insect technique. Production at a large scale requires the development of standardized rearing procedures to produce good quality males able to compete with wild males to mate with wild females. Three sets of experiments (using trays placed on the table, the whole tray-rack system, and climate-controlled chambers) have been conducted aiming to determine the optimal water temperature and number of eggs to aliquot into each larval rearing tray to achieve the highest production of pupae. No difference was found in time to pupation, sex ratio, or male body size as a result of altering larval density. However, higher larval densities resulted in decreased emergence rate and female body size. A constant water temperature of 22°C delayed hatching and did not allow Anopheles arabiensis to complete larval development. Hatching eggs in water at 22°C and then increasing water temperature to 27°C resulted in decreased pupae production compared to eggs hatched and larvae maintained at a water temperature of 27°C throughout. Water temperature and larval density affected the production parameters of An. arabiensis mosquitoes, which has implications for mass release programs. We conclude that 4,000 eggs per 4 liter and a water temperature of 27°C were the optimal conditions for mass-rearing this mosquito species which yielded 105,000 pupae/larval rearing unit. These results are valuable information in the development of standard operation procedures for the efficient large-scale rearing of An. arabiensis mosquitoes.

Population Dynamics and Plasmodium falciparum (Haemosporida: Plasmodiidae) Infectivity Rates for the Malaria Vector Anopheles arabiensis (Diptera: Culicidae) at Mamfene, KwaZulu-Natal, South Africa.

Anopheles arabiensis (Patton; Diptera: Culicidae) is a major malaria vector in the southern African region. In South Africa, effective control of this species using indoor-based interventions is reduced owing to its tendency to rest outdoors. As South Africa moves towards malaria elimination there is a need for complementary vector control strategies. One of the methods under consideration is the use of the sterile insect technique (SIT). Key to the successful implementation of an SIT programme is prior knowledge of the size and spatial distribution of the target population. Understanding mosquito population dynamics for both males and females is critical for efficient programme implementation. It is thus necessary to use outdoor-based population monitoring tools capable of sampling both sexes of the target population. In this project mosquito surveillance and evaluation of tools capable of collecting both genders were carried out at Mamfene in northern KwaZulu-Natal Province, South Africa, during the period January 2014 to December 2015. Outdoor- and indoor-resting Anopheles mosquitoes were sampled in three sections of Mamfene over the 2-yr sampling period using modified plastic buckets, clay pots and window exit traps. Morphological and molecular techniques were used for species identifications of all samples. Wild-caught adult females were tested for Plasmodium falciparum (Welch; Haemosporida: Plasmodiidae) infectivity. Out of 1,705 mosquitoes collected, 1,259 (73.8%) and 255 (15%) were identified as members of either the Anopheles gambiae complex or Anopheles funestus group respectively. An. arabiensis was the most abundant species contributing 78.8% of identified specimens. Mosquito density was highest in summer and lowest during winter. Clay pots yielded 16.3 mosquitoes per trap compared to 10.5 for modified plastic buckets over the 2-yr sampling period. P. falciparum infection rates for An. arabiensis were 0.7% and 0.5% for 2014 and 2015, respectively. Logistic regression analysis showed an association between An. arabiensis catches with Section and season of collection but not with sex and collection methods. These data confirmed the presence of a perennial An. arabiensis population at Mamfene and constitute the first records of P. falciparum infective An. arabiensis from South Africa, confirming this species as a major vector in the malaria endemic provinces of the country.

Mating competitiveness of sterile genetic sexing strain males (GAMA) under laboratory and semi-field conditions: Steps towards the use of the Sterile Insect Technique to control the major malaria vector Anopheles arabiensis in South Africa.

BACKGROUND: Anopheles arabiensis Patton is primarily responsible for malaria transmission in South Africa after successful suppression of other major vector species using indoor spraying of residual insecticides. Control of An. arabiensis using current insecticide based approaches is proving difficult owing to the development of insecticide resistance, and variable feeding and resting behaviours. The use of the sterile insect technique as an area-wide integrated pest management system to supplement the control of An. arabiensis was proposed for South Africa and is currently under investigation. The success of this technique is dependent on the ability of laboratory-reared sterile males to compete with wild males for mates. As part of the research and development of the SIT technique for use against An. arabiensis in South Africa, radio-sensitivity and mating competitiveness of a local An. arabiensis sexing strain were assessed. METHODS: The optimal irradiation dose inducing male sterility without compromising mating vigour was tested using Cobalt 60 irradiation doses ranging from 70-100 Gy. Relative mating competitiveness of sterile laboratory-reared males (GAMA strain) compared to fertile wild-type males (AMAL strain) for virgin wild-type females (AMAL) was investigated under laboratory and semi-field conditions using large outdoor cages. Three different sterile male to fertile male to wild-type female ratios were evaluated [1:1:1, 5:1:1 and 10:1:1 (sterile males: fertile, wild-type males: fertile, wild-type females)]. RESULTS: Irradiation at the doses tested did not affect adult emergence but had a moderate effect on adult survivorship and mating vigour. A dose of 75 Gy was selected for the competitiveness assays. Mating competitiveness experiments showed that irradiated GAMA male mosquitoes are a third as competitive as their fertile AMAL counterparts under semi-field conditions. However, they were not as competitive under laboratory conditions. An inundative ratio of 10:1 induced the highest sterility in the representative wild-type population, with potential to effectively suppress reproduction. CONCLUSION: Laboratory-reared and sterilised GAMA male An. arabiensis at a release ratio of 3:1 (3 sterile males to 1 wild, fertile male) can successfully compete for insemination of wild-type females. These results will be used to inform subsequent small-scale pilot field releases in South Africa.