Background: The role of larval predators in regulating the malaria vectors population remains relatively unknown. This study aimed to investigate the common predators that co-exist with Anopheles funestus group larvae and evaluate factors that influence their abundance in rural south-eastern Tanzania.

Methods: Mosquito larvae and predators were sampled concurrently using standard dipper (350 ml) or 10 L bucket in previously identified aquatic habitats in selected villages in southern Tanzania. Predators were identified using standard identification keys. All positive habitats were geo-located and their physical features characterized. Water physicochemical parameters such as dissolved oxygen (DO), pH, electrical conductivity (EC), total dissolved solids (TDS) and temperature were also recorded.

Results: A total of 85 previously identified An. funestus aquatic habitats in nine villages were sampled for larvae and potential predators. A total of 8,295 predators were sampled. Of these Coenagrionidae 57.7% (n=4785), Corixidae 12.8% (n=1,060), Notonectidae 9.9% (n=822), Aeshnidae 4.9% (n=405), Amphibian 4.5% (n=370), Dytiscidae 3.8% (n=313) were common.

A total of 5,260 mosquito larvae were sampled, whereby Anopheles funestus group were 60.3% (n= 3,170), Culex spp. 24.3% (n= 1,279), An. gambie s.l. 8.3% (n= 438) and other anophelines 7.1% (n= 373). Permanent and aquatic habitats larger than 100m2 were positively associated with An. funestus group larvae (P<0.05) and predator abundance (P<0.05). Habitats with submerged vegetation were negative associated with An. funestus group larvae (P<0.05). Only dissolved oxygen (DO) was positively and significantly affect the abundance of An. funestus group larvae (P<0.05). While predators’ abundance was not impacted by all physicochemical parameters.

Conclusion: Six potential predator families were common in aquatic habitats of An. funestus group larvae. Additional studies are needed to demonstrate the efficacy of different predators on larval density and adult fitness traits. Interventions leveraging the interaction between mosquitoes and predators can be established to disrupt the transmission potential and survival of the An. funestus mosquitoes.

Study area

A cross-sectional survey was conducted, between March and May 2022, in nine villages in south-eastern Tanzania, namely Chikuti (-8.6028°, 36.7288°), Mzelezi (-8.8934°, 36.7343°), Chirombola (-8.93041°, 36.75753°), Ebuyu (-8.9719°, 36.7608°), Mwaya (-8.91022°, 36.823139°) and Tulizamoyo (-8.35447°, 36.70546°) in Ulanga district and Ikwambi (-7.97927°, 36.81630°), Kisawasawa (-7.89657°, 36.88058°) and Mpofu (-8.17220°, 36.21651°) in Kilombero district (Fig 1). In this area An. funestus is responsible for more than 85% of overall malaria transmission [17]. The residents in these villages practise extensive rice farming, which creates suitable habitat for mosquito breeding. Common aquatic habitats for An. funestus in the villages are well known and have been previously characterized [24]. Eighty-five known habitats from the nine villages were sampled for both mosquito larvae and potential predators.

Fig1: Map of Kilombero and Ulanga districts showing the nine study villages

Sampling and morphological identification of mosquito larvae and aquatic predators

Mosquito larvae and predators were sampled using standard dippers (350 ml) or 10 L buckets, as previously described [8,15,24]. A minimum of 3 dips and a maximum of 20 dips were taken depending on the size and depth of the habitat. 

In a previous study mosquito larva from the same villages were taken to the laboratory in Ifakara, allowed to emerge and eventually identified to species by PCR. Of those identified 53% were An. funestus s.s. whilst 28% were An. rivulorum and 12% were An. leesoni [24]. All three species were found to occupy the same habitats. A similar approach was followed with samples of fourth instar larvae during the present study but identification to species level was not performed. Earlier stage larvae were identified based on their predominant characteristics as done in a previous study and separated into An. funestus group, An. gambiae s.l. or Anopheles sp. following the identification key by Gilles and Coetzee [25,26]. Culicines were identified to genera only. Predators were morphologically identified to family level using the keys of the Stroud Water Research Center [27] and Gerber and Gabriel [28]. Mosquito larvae and predators that were sampled by each dipper or bucket were counted and recorded. Additionally, geographical locations of the surveyed habitats were recorded at access points using a hand-held GPS device (Garmin eTrex 20x Handheld GPS Receiver).

Aquatic habitats characterization

Only positive aquatic habitats for An. funestus group larvae were sampled for mosquito larvae and predators. Their overall physical characteristics were recorded and physicochemical parameters of the water (pH, temperature, electrical conductivity (EC), total dissolved solids (TDS) were measured using a portable water quality meter (ZJ practical 4 in 1 Water Tester). A Trans Instruments Dissolved Oxygen Meter (HD3030) was used to measure dissolved oxygen (DO), using standard recording procedures. Habitats were classified as being either: swamp, stream, river, rice-field, stream-pool, ground-pool, ditch, spring-fed pool, puddle, hoof-print, man-made wells, brick or sand pit. Water colour was categorized as being clear (transparent and odourless) or coloured (cloudy, not transparent, turbid or with a film of oil).

The source of water was also classified as rainwater or others (non-rainwater). Algal quantities in the habitats were classified as none, moderate, or abundant. Algal type was classified as filamentous, green, blue-green or brown. Water was also classified as being stagnant, slow or fast moving. The land use surrounding the aquatic habitats was classified as scrub, cattle grazing or cultivated field. Shade over the habitats was classified as none, partial or heavy. Habitat size was measured using tape and classified as being less than 100 m2 or more than 100 m2. Vegetation quantity and vegetation type were also classified as (none, moderate or abundant) and (emergent, or submerged) respectively. Water bodies known to have existed for three months or more were considered to be permanent whilst other collections of water were considered to be ‘temporary’. Water depth was classified as being less than 50 cm or more than 50 cm deep.  The distance from aquatic habitats to the nearest houses were estimated visually and classified as being less than 100 m or more than 100 m.

 Statistical analysis

Analysis was done using open source software R version 4.2.1. [29]. Generalised linear mixed effects models (GLMM)  using template model builder (TMB) with zero-inflated negative binomial implemented under the glmmTMB package [30] were used to (i) assess the associations between water physicochemical parameters and the abundance of aquatic predators ii) assess the associations between water physicochemical parameters and the abundance of  An. funestus group larvae (iii)  assess which habitat characteristics contributed to the abundance of predators and An. funestus group larvae and (iv) assess the impact of each predator family on the abundance of An. funestus group larvae. All variables (i-iv) were assessed individually and later combined in the final model.

Due to a large number of dips with zero larvae the negative binomial with zero inflated models were used. In all models, the study villages in which the aquatic habitats were identified and habitat ID were used as random terms to capture unexplained variations between villages and habitats. The best fitting models were selected using Akaike Information Criterion (AIC) and results presented as risk ratios (RR) at 95% CI and statistical significance was considered when the P-value < 0.05.

Ethical considerations

Research proposal was presented to the Nelson Mandela Institute of Science and Technology and approval for this study was obtained from the institutional review board of Ifakara Health Institute (Ref: IHI/IRB/No: 13-2022) and from the Medical Research Coordinating Committee (MRCC) at the National Institutes of Medical Research (NIMR) (Ref: NIMR/HQ/R.8a/Vol. IX/3353).  The consent of publication this manuscript was obtained from the National Institute for Medical Research (NIMR) (Ref. No: NIMR/HQ/P.12 VOL XXXV/61). Written permission to conduct study was obtained from local leaders in each village whereby the purpose, procedure and benefits of the study were clearly explained. Verbal and written informed consents were obtained from community members who assisted to sample aquatic predators.