DOI: 10.5061/dryad.kwh70rz7z
title: Common predators and factors influencing their abundance in Anopheles funestus aquatic habitats in rural south-eastern Tanzania

description: 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&lt;0.05) and predator
 abundance (P&lt;0.05). Habitats with submerged vegetation were
 negative associated with An. funestus group larvae (P&lt;0.05). Only
 dissolved oxygen (DO) was positively and significantly affect the
 abundance of An. funestus group larvae (P&lt;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.
description: 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.<strong> </strong>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 &lt; 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.