{'fraction_non_alphanumeric': 0.026670156071564522, 'fraction_numerical': 0.014822040350209365, 'mean_word_length': 5.025301032110092, 'pattern_counts': {'":': 0, '<': 1, '<?xml version=': 0, '>': 0, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 7, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Larvivorous fish for preventing malaria transmission

Abstract Background Adult female Anopheles mosquitoes can transmit Plasmodium parasites that cause malaria. Some fish species eat mosquito larvae and pupae. In disease control policy documents, the World Health Organization (WHO) includes biological control of malaria vectors by stocking ponds, rivers, and water collections near where people live with larvivorous fish to reduce Plasmodium parasite transmission. In the past, the Global Fund has financed larvivorous fish programmes in some countries, and, with increasing efforts in eradication of malaria, policymakers may return to this option. Therefore, we assessed the evidence base for larvivorous fish programmes in malaria control. Objectives To evaluate whether introducing larvivorous fish to anopheline larval habitats impacts Plasmodium parasite transmission. We also sought to summarize studies that evaluated whether introducing larvivorous fish influences the density and presence of Anopheles larvae and pupae in water sources. Search methods We searched the Cochrane Infectious Diseases Group Specialized Register; the Cochrane Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library; MEDLINE (PubMed); Embase (Ovid); CABS Abstracts; LILACS; and the metaRegister of Controlled Trials (mRCT) up to 6 July 2017. We checked the reference lists of all studies identified by the search. We examined references listed in review articles and previously compiled bibliographies to look for eligible studies. Also we contacted researchers in the field and the authors of studies that met the inclusion criteria for additional information regarding potential studies for inclusion and ongoing studies. This is an update of a Cochrane Review published in 2013. Selection criteria Randomized controlled trials (RCTs) and non‐RCTs, including controlled before‐and‐after studies, controlled time series, and controlled interrupted time series studies from malaria‐endemic regions that introduced fish as a larvicide and reported on malaria in the community or the density of the adult anopheline population. In the absence of direct evidence of an effect on transmission, we performed a secondary analysis on studies that evaluated the effect of introducing larvivorous fish on the density or presence of immature anopheline mosquitoes (larvae and pupae forms) in water sources to determine whether this intervention has any potential that may justify further research in the control of malaria vectors. Data collection and analysis Two review authors independently screened each article by title and abstract, and examined potentially relevant studies for inclusion using an eligibility form. At least two review authors independently extracted data and assessed risk of bias of included studies. If relevant data were unclear or were not reported, we contacted the study authors for clarification. We presented data in tables, and we summarized studies that evaluated the effects of introducing fish on anopheline immature density or presence, or both. We used the GRADE approach to summarize the certainty of the evidence. We also examined whether the included studies reported any possible adverse impact of introducing larvivorous fish on non‐target native species. Main results We identified no studies that reported the effects of introducing larvivorous fish on the primary outcomes of this review: malaria infection in nearby communities, entomological inoculation rate, or on adult Anopheles density. For the secondary analysis, we examined the effects of introducing larvivorous fish on the density and presence of anopheline larvae and pupae in community water sources, and found 15 small studies with a follow‐up period between 22 days and five years. These studies were undertaken in Sri Lanka (two studies), India (three studies), Ethiopia (one study), Kenya (two studies), Sudan (one study), Grande Comore Island (one study), Korea (two studies), Indonesia (one study), and Tajikistan (two studies). These studies were conducted in a variety of settings, including localized water bodies (such as wells, domestic water containers, fishponds, and pools (seven studies); riverbed pools below dams (two studies)); rice field plots (five studies); and water canals (two studies). All included studies were at high risk of bias. The research was insufficient to determine whether larvivorous fish reduce the density of Anopheles larvae and pupae (12 studies, unpooled data, very low certainty evidence). Some studies with high stocking levels of fish seemed to arrest the increase in immature anopheline populations, or to reduce the number of immature anopheline mosquitoes, compared with controls. However, this finding was not consistent, and in studies that showed a decrease in immature anopheline populations, the effect was not always consistently sustained. In contrast, some studies reported larvivorous fish reduced the number of water sources withAnopheles larvae and pupae (five studies, unpooled data, low certainty evidence). None of the included studies reported effects of larvivorous fish on local native fish populations or other species. Authors' conclusions We do not know whether introducing larvivorous fish reduces malaria transmission or the density of adult anopheline mosquito populations. In research studies that examined the effects on immature anopheline stages of introducing fish to potential malaria vector larval habitats, high stocking levels of fish may reduce the density or presence of immature anopheline mosquitoes in the short term. We do not know whether this translates into impact on malaria transmission. Our interpretation of the current evidence is that countries should not invest in fish stocking as a stand alone or supplementary larval control measure in any malaria transmission areas outside the context of research using carefully controlled field studies or quasi‐experimental designs. Such research should examine the effects on native fish and other non‐target species. 12 April 2019 Up to date All studies incorporated from most recent search All eligible published studies found in the last search (6 Jul, 2017) were included


A B S T R A C T Background
Adult female Anopheles mosquitoes can transmit Plasmodium parasites that cause malaria. Some fish species eat mosquito larvae and pupae. In disease control policy documents, the World Health Organization (WHO) includes biological control of malaria vectors by stocking ponds, rivers, and water collections near where people live with larvivorous fish to reduce Plasmodium parasite transmission. In the past, the Global Fund has financed larvivorous fish programmes in some countries, and, with increasing e orts in eradication of malaria, policymakers may return to this option. Therefore, we assessed the evidence base for larvivorous fish programmes in malaria control.

Objectives
To evaluate whether introducing larvivorous fish to anopheline larval habitats impacts Plasmodium parasite transmission. We also sought to summarize studies that evaluated whether introducing larvivorous fish influences the density and presence of Anopheles larvae and pupae in water sources.

Search methods
We searched the Cochrane Infectious Diseases Group Specialized Register; the Cochrane Central Register of Controlled Trials (CENTRAL), published in the Cochrane Library; MEDLINE (PubMed); Embase (Ovid); CABS Abstracts; LILACS; and the metaRegister of Controlled Trials (mRCT) up to 6 July 2017. We checked the reference lists of all studies identified by the search. We examined references listed in review articles and previously compiled bibliographies to look for eligible studies. Also we contacted researchers in the field and the authors of studies that met the inclusion criteria for additional information regarding potential studies for inclusion and ongoing studies. This is an update of a Cochrane Review published in 2013.

Selection criteria
Randomized controlled trials (RCTs) and non-RCTs, including controlled before-and-a er studies, controlled time series, and controlled interrupted time series studies from malaria-endemic regions that introduced fish as a larvicide and reported on malaria in the community or the density of the adult anopheline population. In the absence of direct evidence of an e ect on transmission, we performed a secondary analysis on studies that evaluated the e ect of introducing larvivorous fish on the density or presence of immature anopheline mosquitoes (larvae and pupae forms) in water sources to determine whether this intervention has any potential that may justify further research in the control of malaria vectors.
For the secondary analysis, we examined the e ects of introducing larvivorous fish on the density and presence of anopheline larvae and pupae in community water sources, and found 15 small studies with a follow-up period between 22 days and five years. These studies were undertaken in Sri Lanka (two studies), India (three studies), Ethiopia (one study), Kenya (two studies), Sudan (one study), Grande Comore Island (one study), Korea (two studies), Indonesia (one study), and Tajikistan (two studies). These studies were conducted in a variety of settings, including localized water bodies (such as wells, domestic water containers, fishponds, and pools (seven studies); riverbed pools below dams (two studies)); rice field plots (five studies); and water canals (two studies). All included studies were at high risk of bias. The research was insu icient to determine whether larvivorous fish reduce the density of Anopheles larvae and pupae (12 studies, unpooled data, very low certainty evidence). Some studies with high stocking levels of fish seemed to arrest the increase in immature anopheline populations, or to reduce the number of immature anopheline mosquitoes, compared with controls. However, this finding was not consistent, and in studies that showed a decrease in immature anopheline populations, the e ect was not always consistently sustained. In contrast, some studies reported larvivorous fish reduced the number of water sources withAnopheles larvae and pupae (five studies, unpooled data, low certainty evidence).
None of the included studies reported e ects of larvivorous fish on local native fish populations or other species.

Authors' conclusions
We do not know whether introducing larvivorous fish reduces malaria transmission or the density of adult anopheline mosquito populations.
In research studies that examined the e ects on immature anopheline stages of introducing fish to potential malaria vector larval habitats, high stocking levels of fish may reduce the density or presence of immature anopheline mosquitoes in the short term. We do not know whether this translates into impact on malaria transmission. Our interpretation of the current evidence is that countries should not invest in fish stocking as a stand alone or supplementary larval control measure in any malaria transmission areas outside the context of research using carefully controlled field studies or quasi-experimental designs. Such research should examine the e ects on native fish and other non-target species.

April 2019
Up to date All studies incorporated from most recent search All eligible published studies found in the last search (6 Jul, 2017) were included

P L A I N L A N G U A G E S U M M A R Y Fish that feed on mosquito larvae for preventing malaria transmission
What is the aim of this review?
Adult female Anopheles mosquitoes transmit the Plasmodium parasites that cause malaria. The aim of this Cochrane Review was to evaluate whether introducing fish that eat mosquito larvae and pupae (early life stages of mosquitoes) into water sources near where people live will decrease the adult Anopheles mosquito population and thus the number of people infected with Plasmodium parasites. Effects on larvae at potential mosquito larval sites

Density of immature vector stages in water bodies
Quasi-experimental studies --Not pooled.
Variable effects reported.

Larval sites positive for immature vector stages
Quasi-experimental studies --Not pooled Positive effects reported 5 ⊕⊕⊝⊝ Low 1,2,10-12 Larvivorous fish may reduce the number of larval sites positive for immature anopheline mosquitoes.

Library
Trusted evidence. Informed decisions. Better health.
Cochrane Database of Systematic Reviews

Description of the condition
Malaria is the most common vector-borne disease worldwide and is endemic in 91 countries. At the start of 2016, almost half the world's population was at risk of malaria. The World Health Organization (WHO) reported an estimated 212 million new cases of malaria worldwide (range 148 million to 304 million) and 429,000 deaths (range 235,000 to 639,000) from malaria in 2015. People living in sub-Saharan Africa continue to be at highest risk of contracting the disease; the WHO African Region accounted for an estimated 90% of malaria cases and 92% of malaria deaths in 2015 (WHO 2016). Plasmodium spp. parasites cause malaria in humans and are transmitted by female mosquitoes of the genus Anopheles. Of approximately 430 Anopheles species, between 30 and 50 species act as dominant vectors.
The main strategies for preventing and controlling malaria include the following: • prevention through vector control, mainly using long-lasting insecticidal nets ( LLINs and IRS were developed against the most e ective vectors, which share the attributes of feeding late at night and being anthropophilic (preferring to feed on humans), endophagic (preferring to feed indoors), and endophilic (preferring to rest indoors) (Lengeler 2004;Tanser 2007). However, many vectors, particularly in Asia and South America (but also in Africa), prefer animals to humans for their blood meals (are zoophilic) or feed early in the evening or outside of houses, where they will be less likely to encounter LLINs or IRS. The two main vector control strategies may be less e ective in regions where vectors have these behavioural attributes. These factors have led some agencies and governments to propose other strategies for vector control, and interest in larviciding as a potential means of malaria control has been renewed (Ejov 2014;NVBDCP 2017;WHO-EURO 2006;WHO-GMP 2012;WHO 2017).

Description of the intervention
Larviciding attempts to control malaria by seeking to reduce the size of the immature vector population. Strategies include the following.
A separate Cochrane Review summarizes larviciding for strategies (1) and (2) (Tusting 2013). The review authors examined clusterrandomized controlled trials, controlled before-and-a er trials with at least one year of baseline data, and randomized crossover trials that compared larval source management (LSM) with no LSM for malaria control. The review authors found some large e ects in some studies but not in others. They concluded that when larval habitats were not too extensive, and when a su icient proportion of these habitats could be targeted, LSM probably reduces the number of people who develop malaria and probably reduces the proportion of the population infected with the Plasmodium parasite at any one time (moderate certainty evidence). In the included studies, the intervention appeared to be e ective in reducing malaria transmission in a range of countries where larviciding was implemented at a wide variety of sites. In one study from The Gambia, where mosquito larval habitats were large swamps and rice paddies, spraying of swamps with larvicide by ground teams did not lead to any benefit. A separate Cochrane Review, which focuses on larviciding alone, is in preparation (Choi 2017b). In this review, we evaluated the most common strategy for biological control: the use of fish that attack mosquito larvae and pupae.
The potential of the larvivorous fish Gambusia (Gambusia a inis and Gambusia holbrooki; Pyke 2005) to ingest large numbers of mosquito larvae led to a series of laboratory-based studies on mosquito larval prey preferences and the optimization of systems to propagate these fish. Subsequently, field evaluations of Gambusia were undertaken to assess their impact on larval prevalence and density in mosquito larval habitats.G. a inis and G. holbrooki are native to the south-eastern USA but have been transported and released in multiple countries globally, so that these species are now collectively the most widely geographically dispersed freshwater fishes in the world (Pyke 2008).
Gambusia may adversely a ect native fishes and other organisms besides mosquitoes when introduced into new areas. Specialists are now examining the use of native fish species for larval control. Approximately 315 larvivorous fish species belonging to 32 genera under seven families are used for mosquito control, and the family Cyprinodontidae contribute the highest number of genera (15) and species (300) (Goutam 2013). Other promising species for mosquito control belong to the genera Aphanius, Valencia,Aplocheilus,Oryzias, Epiplatys,Aphyosemion,Rolo ia,Nothobranchius,Pachypanchax,Rivulus,Fundulu and Cynolebias (Walton 2007).

How the intervention might work
As adult female Anopheles mosquitoes transmit malaria, the intensity of transmission is partly dependent on the following: • whether Anopheles are infected with the Plasmodium sporozoite stage; and • how many Anopheles feed on humans during the transmission season or year.
The percentage of infected mosquitoes multiplied by the biting rate is a common parameter by which to estimate the force of infection, and is called the entomological inoculation rate (EIR).
Anopheles mosquitoes lay their eggs in water sources in which they develop into larvae and then pupae. Anopheles larvae are Cochrane Database of Systematic Reviews found in a wide range of habitats, including fresh-or salt-water marshes; rice fields; mangrove swamps; edges of streams and rivers; grassy ditches; and small, temporary rain pools. Most species prefer clean, unpolluted water. Some mosquitoes may prefer specific sites in which to lay eggs, whilst others use a wide variety of larval habitats (such as temporary ground water pools, including footprints and ditches, as well as more permanent water sources, such as swamps and wells). The abundance of adult mosquitoes depends on a variety of factors, including the number and size of suitable oviposition sites and the density of the immature mosquito stages at these sites. Several other ecological and environmental factors may influence the adult anopheline population, such as temperature, rainfall patterns, and availability of bloodmeal sources.
The larger the mosquito population, the greater the potential number of bites by vectors on humans, unless people take measures to avoid mosquito bites, such as sleeping under an LLIN. For a given sporozoite rate, increases in human-biting rate or mosquito density, or both, will result in higher inoculation rates and greater malaria transmission. If the size of the vector population is limited by interventions that reduce the number of larval habitats or the density of vector larvae per larval habitat, then malaria transmission to humans (with all other factors remaining the same) might potentially be reduced ( Figure 1). However, reducing the density of anopheline immature mosquitoes at a larval habitat might have little or no e ect on adult numbers because adult numbers may be determined largely or entirely by other factors. Reductions in the density of immature vectors could result in larger, more robust, longer-lived adults through reduced competition between immature Anopheles for resources (densitydependent e ects), thereby minimizing the potential reduction in malaria transmission. However, Bond 2005 demonstrated that Anopheles pseudopunctipennis larvae had significantly prolonged developmental times in the presence of Poecilia sphenops fish and emerged as smaller adults. Smaller adult females can have reduced host-seeking responses (Takken 1998), and may produce smaller egg batches (Lyimo 1993).

Why it is important to do this review
The WHO recommendations from 2012 state that antilarval measures are likely to be cost-e ective for control of malaria in areas where the larval habitats are limited in number, permanent, and easily found (that is, they are "fixed, finite and findable") (WHO-GMP 2012). The WHO has stated that environmental factors that increase the likelihood that larval control will be e ective include a short transmission season, cool temperatures that extend for the duration of the immature stages, and larval habitats that are manmade and homogeneous in nature. In Africa, larviciding is thought to have the best potential to be e ective in urban and arid areas and possibly in the East African highlands (WHO-GMP 2012). Indeed, the Cochrane Review of mosquito LSM indicated that the intervention o en appeared to impact transmission when implemented in areas where it was feasible to do so (Tusting 2013).
Whether larvivorous fish are an option for LSM is the subject of this Cochrane Review. Since the 1970s, the WHO has promoted the use of larvivorous fish as an environmentally friendly alternative to insecticide-based interventions for malaria control. A WHOsponsored interregional conference on malaria control in 1974 reported that "the utilization of larvivorous fish, mainly Gambusia or suitable local species, is the only practical measure that can be recommended where applicable, as in lakes, ponds, pools, wells, rice fields" (WHO 1974). A 2001 regional meeting in Kazakhstan recommended that more studies on larger numbers of local larvivorous and phytophagous fish be undertaken in di erent ecoepidemiological settings in that region, and that the search for e ective larvivorous fish should continue (WHO-EURO 2001).
More recently, momentum has gathered in e orts to eliminate malaria, resulting in the 2006-2015 WHO-EURO regional strategy (WHO-EURO 2006) and the 2014-2020 WHO-EURO regional strategy (Ejov 2014), which includes larval control by introduction of larvivorous fish preferentially over other forms of larviciding. However, in its current framework for malaria elimination, the WHO does not include larvivorous fish among the recommended vector control strategies for elimination of malaria (WHO 2017).
WHO recommendations for larviciding as a general strategy are guarded and conditional, but the use of fish is o en included in listings of options, alongside clearly established e ective measures such as LLINs. For example, the WHO integrated vector management plan to control malaria includes the "e ective use of biologically-based agents such as bacterial larvicides and larvivorous fish" (HELI 2005). Fish were one of the traditional means of malaria control in the ex-Soviet Republics of Central Asia, where their use continues (Kondrashin 2017;RTDC 2008;Zvantsov 2008). For example, the Global Fund provided funds for implementation of larvivorous fish against malaria in Tajikistan, although this investment appears modest (UNDP 2013).

Library
Trusted evidence. Informed decisions. Better health.

Cochrane Database of Systematic Reviews
Thus, there appear to be di ering views on whether introducing larvivorous fish is an e ective larvicidal approach; some are strong advocates, whilst others question whether su icient evidence exists to demonstrate its e ectiveness, and whether the strategy can achieve the large reductions in larval numbers required to impact the size of the adult population. In addition, problems are associated with finding and treating all anopheline mosquito larval habitats within a specific area, and some larval habitats may be unsuitable for treatment. Dissemination of larvivorous fish as a control strategy for malaria has the potential for adverse e ects on local ecosystems by reducing or eliminating indigenous fish, amphibians, and invertebrates (Walton 2007).
Therefore, we carried out a systematic review of reliable research examining whether evidence shows that this form of larviciding has an impact on malaria. We also sought evidence of the potential to a ect transmission, by summarizing studies on the e ects of introducing fish on the density and presence of immature anopheline mosquitoes at potential larval habitats. This is an update of a Cochrane Review published in 2013 (Walshe 2013).

O B J E C T I V E S
To evaluate whether introducing larvivorous fish to anopheline larval habitats impacts Plasmodium parasite transmission. We also sought to summarize studies that evaluated whether introducing larvivorous fish influences the density and presence of Anopheles larvae and pupae in water sources.

M E T H O D S
Criteria for considering studies for this review

Types of studies
We included randomized controlled trials (RCTs) and non-RCTs, including before-and-a er controlled studies, controlled time series, and controlled interrupted time series designs ( Figure 2). Comparison groups were geographically defined areas, and thus for RCTs, cluster-randomized designs were used. To be included, intervention and control groups needed to have the following: • equivalent accompanying antimalarial interventions; • baseline information; • contemporaneous data collection; • same locality (within the same regional area of the country); • comparable resident populations in relation to ethnic groups, housing, and wealth, based on baseline data provided within the study; • similar intensities of malaria transmission, based on baseline data provided within the study; and • su icient geographic size to minimize masking of the impact of the intervention by immigrating vectors.
In studies of malaria transmission, we specified that intervention and control sites were at least 1 km apart with a human population sample size adequate to detect greater than 25% reduction in Plasmodium parasite-positive people.

Types of participants
Children and adults living in rural and urban malaria-endemic areas.

Interventions
Introduction of larvivorous fish of any species, either adults or juveniles, into anopheline mosquito larval habitats. This may have been done as a single intervention or as part of a more comprehensive vector control programme that included access to and use of LLINs, IRS, larvicides (including microbial larvicides and insect growth regulators), polystyrene beads, and environmental management.
Due to seasonal, climatic, and random variations at both immature (larvae and pupae) and adult stages, we included studies that monitored for one or more full years before fish were introduced and those that monitored at one or more time points at least Library Trusted evidence. Informed decisions. Better health.
Cochrane Database of Systematic Reviews 12 months a er fish were introduced into intervention areas. For studies of immature anopheline mosquito populations, we included only studies with a follow-up period longer than three weeks, so that several generations of immature anophelines were monitored.

Controls
No larvivorous fish were introduced into control areas. All other vector control measures were the same in intervention and control arms. Thus, for example, we excluded studies that examined introduction of larvivorous fish combined with IRS and those that did not use IRS in the control arm.

Primary outcomes
• Number of confirmed episodes of malaria among community members: defined as malaria infections as laboratoryconfirmed cases of malaria (Plasmodium parasitaemia detected by microscopy or by rapid diagnostic tests in active or passive case detection). • Entomological inoculation rate (EIR): defined as the estimated number of bites by infectious mosquitoes per person per unit of time (the product of the number of bites per person per day during the transmission season or per year by vector mosquitoes (the "human-biting rate") and the fraction of vector mosquitoes that are infectious (the "sporozoite rate")). • Density of adult vector mosquitoes: included measures in which sampling techniques appropriate for these vectors were used, including counting adult anopheline mosquitoes that either landed on exposed body parts of humans acting as bait or were collected resting inside buildings with the use of knockdown spray catches.

Secondary outcomes
• Density of immature vector stages at larval sites, as measured by larval dipping (Silver 2008). • Percentage of larval sites positive for immature anopheline mosquitoes.
In any studies that met the inclusion criteria, we checked whether the study authors reports on native fish populations or other e ects on the local ecosystem.

Search methods for identification of studies
We searched for all relevant studies regardless of language or publication status (published, unpublished, in press, or ongoing).

Electronic searches
We examined the following databases up to 6 July 2017 using the search terms detailed in Appendix 1: the Cochrane Infectious Diseases Group Specialized Register; the Cochrane Central Register of Controlled Trials (CENTRAL); MEDLINE (PubMed); Embase (Ovid); CABS Abstracts; and LILACS. We searched the metaRegister of Controlled Trials (mRCT) using 'malaria' and 'larvicide* or fish' as search terms; and the literature database of the Armed Forces Pest Management Board using the search terms ('frogs' and 'fish') and 'malaria'.

Reference lists
We checked the reference lists of all studies identified by the above methods, references listed in review articles (Beltran 1973;Chandra 2008;Pyke 2008;Walker 2007), and previously compiled bibliographies (Gerberich 1968) to identify potential studies.

Researchers
We contacted researchers in the field and the authors of studies that met the inclusion criteria for additional information regarding potential studies for inclusion and ongoing studies.

Selection of studies
Two review authors screened the abstract of each title obtained from the search for potentially relevant studies. We retrieved the corresponding full articles of these identified studies, and two review authors assessed inclusion by using an eligibility form. We independently screened each search result, assessed each article, and resolved any discrepancies between eligibility results through discussion. If studies did not meet the methods specified, we did not scrutinize further, and if eligibility was unclear, we sought clarification from the study authors.

Data extraction and management
Two review authors independently extracted data from each study report onto a predesigned data extraction form. We discussed any discrepancies with a third review author.
For the secondary analysis of the e ect of introducing larvivorous fish on immature anopheline mosquitoes in water sources, we extracted information on study characteristics and study methods, including setting, comparability between sites, details of the fish intervention, and outcomes, and we examined how study authors measured these. We extracted descriptions of the epidemiology and intensity of transmission from each study, using the terms used by the study authors; co-interventions and whether both control and intervention arms experienced the same co-interventions; and, when study authors presented outcome data in graph or table format, the raw data when possible.

Design quality
We assessed the study design quality of each included study by examining whether study authors also reported on four specific factors.
• Whether other larvivorous species were present. • Whether vegetation was cleared or removed from the sites.

Assessment of risk of bias in included studies
For trials that examined the e ects of larvivorous fish on malaria transmission, we planned to evaluate the risk of bias using standard Cochrane 'Risk of bias' criteria.

Library
Trusted evidence. Informed decisions. Better health.

Cochrane Database of Systematic Reviews
For studies that examined e ects on larvae, we assessed risk of bias on the basis of six factors: study design; site selection; site allocation; blinding of assessors; baseline values comparable between sites; and the number of sites. In Table 1, we have shown the exact criteria that we used to assess the risk of bias. Two review authors (DPW and PG for Walshe 2013; DPW and either AAA or TB for this review update) independently assessed the risk of bias for each included study, and resolved any discrepancies by discussion with a third review author.

Data synthesis
We performed individual critical appraisal of each included study on the possible e ects of introduction of larvivorous fish on immature mosquitoes. The large variation in study design, outcomes, and reporting precluded any data synthesis. We tried to draw patterns of e ect by grouping studies by habitat as follows.
• Localized water bodies, including wells, domestic water containers, fishponds and man-made pools, and pools in a riverbed below a dam. • Rice field plots.
We described each study in a short narrative and presented the outcome results in table format. We reported results at baseline and at pre-specified time points at follow-up, and used the GRADE approach to assess the certainty of the evidence.

Description of studies
We have provided descriptions of the included and excluded studies in the Characteristics of included studies and Characteristics of excluded studies tables.

Results of the search
In the previous version of this Cochrane Review, we identified 1286 titles and abstracts from the electronic search of databases and 12 additional articles a er contacting researchers and screening reference lists (Walshe 2013). A er we removed duplicates, 915 records remained. Of these, we obtained 117 potentially eligible articles. We identified no studies that fulfilled the selection criteria and reported on primary outcomes. Of the 117 potentially eligible articles, we identified 12 studies that fulfilled the selection criteria for the secondary outcomes only and 105 studies that did not meet the eligibility criteria.
For this review update, we identified 214 titles and abstract from electronic searches of databases and seven additional articles through other sources. There were 220 articles a er we removed duplicates. Of these, there were 14 potentially eligible articles. None of these articles fulfilled the inclusion criteria and reported on the primary outcomes. Three new studies (four articles) met the inclusion criteria and reported only on the secondary outcomes (Haq 2013;RTDC 2008;Zvantsov 2008).
We excluded 10 studies a er full-text assessment with reasons (Characteristics of excluded studies table). Figure 3, a study flow diagram, illustrates the study selection process, Library Trusted evidence. Informed decisions. Better health.
Cochrane Database of Systematic Reviews None of the included studies reported on cases of malaria, EIR, or density of adult vector mosquitoes. Thus, there was no direct evidence this intervention impacts malaria transmission. Therefore, our analysis focuses only on the e ects of fish stocking on the secondary outcomes: the presence or density of immature mosquitoes in water sources.

Sites
We summarized the sites by type of water sources stocked, number of sites stocked, and site size (Table 2). Ecological sites included the following.
• Localized water bodies such as wells (Menon 1978;Sitaraman 1976 The number and size of habitat sites chosen by the trial authors varied (see Table 2). For example, Fletcher 1992 introduced fish to 68 habitat sites and maintained 60 habitat sites as controls. Haq 2013 introduced fish to 295 water storage containers in one village, including underground water tanks (127), kothi (big mud pots) and barrels (167), and no fish to the control village; and monitored 30 containers in the intervention village and 25 in the control village. Menon 1978 stocked fish in 3438 wells and le 317 wells without fish as controls. However, Howard 2007 used two fishponds as intervention sites and one fishpond as a control. Habitat sizes ranged from small, 1 m × 1 m × 1 m man-made ponds (Howard 2007)  Cochrane Database of Systematic Reviews to 24.8 ha plots of land (Nalim 1988). Notably, Nalim 1988 recorded the number of adult mosquitoes collected in emergence traps, and we used these data to determine the e ects of larvivorous fish on the immature mosquito population.

Intervention
We summarized in Table 3 the key details of the fish intervention provided for each included study.

Design quality
We evaluated the following study design quality factors of the included studies and summarized the results in Table 4.

Excluded studies
In the previous version of this review, Walshe 2013, we excluded 105 studies because they did not meet the eligibility criteria, or they did not report any outcome of interest, or both. We have given the reasons for exclusion in the Characteristics of excluded studies table: Anopheles and Culex populations were not monitored separately (seven studies); studies were not fish studies (29 studies); no primary outcomes were reported (20 studies); no secondary outcomes were reported (eight studies); multiple interventions were introduced, meaning that the e ect of fish alone could not be determined (eight studies); study was laboratorybased, not field-based (four studies); inappropriate study design was applied (54 studies); or the outcome data were already presented in another paper (four studies). In several cases, we excluded a study for more than one reason.
One study had an unclear study design (de Buen 1930). Table 1 shows the criteria we used to assess the risk of bias in included studies and we have presented our findings in the 'Risk of bias' tables in the Characteristics of included studies section. We have summarized the risk of bias results in Figure 4 and Figure 5.

Library
Trusted evidence. Informed decisions. Better health.
Cochrane Database of Systematic Reviews

Study design
None of the studies included randomized comparisons, and therefore all were at high risk of bias.

Site selection
Eight studies did not state how they selected sites (Fletcher 1992;Kim 2002;Kusumawathie 2008a;Mahmoud 1985;Nalim 1988;Sabatinelli 1991;Sitaraman 1976;Zvantsov 2008), and were at unclear risk of bias. Six studies stated clearly how the sites were selected within the study area and were at low risk of bias (Haq 2013; Howard 2007; Imbahale 2011a; Kusumawathie 2008b; Menon 1978; Yu 1989). One study was at high risk of bias regarding site selection as the intervention and control areas each included one district with malaria cases and one district without malaria cases; the study authors provided no indication regarding how the sites for intervention and control areas were allocated (RTDC 2008).

Site allocation
Study authors did not give information about how they chose the comparator sites in 13 studies (Haq 2013;Howard 2007;Imbahale 2011a;Kim 2002;Kusumawathie 2008a;Kusumawathie 2008b;Mahmoud 1985;Menon 1978;Nalim 1988;RTDC 2008;Sabatinelli 1991;Sitaraman 1976;Yu 1989), and the studies were at unclear risk of bias. One study was at high risk of bias as sites were allocated to treatment by alternation (Fletcher 1992). One study was at low risk of bias as the study authors stated that allocation of treatment within a checkerboard pattern was random (Zvantsov 2008).

Baseline values comparable between sites
In three studies, baseline values before the intervention was introduced were not comparable between control and intervention sites, and the studies were classified as having high risk of bias (Kusumawathie 2008b;Menon 1978;Sitaraman 1976

Number of sites
Five studies were at low risk of bias, as they had an adequate number of sites (20 or more) per group (Fletcher 1992;Haq 2013;Kusumawathie 2008a;Menon 1978;Sabatinelli 1991

E ects of interventions
See: Summary of findings for the main comparison 'Summary of findings' table 1

Primary analysis
We identified no studies that reported the primary outcomes (number of confirmed episodes of malaria among community members, EIR, or density of adult vector mosquitoes). Thus, there is no direct evidence that indicates this intervention impacts malaria transmission.

Library
Trusted evidence. Informed decisions. Better health.

Secondary analysis
For the secondary analysis of whether introduction of larvivorous fish impacts immature anopheline mosquitoes, all studies were at high risk of bias and provided only indirect evidence of the potential e ectiveness of this intervention. As the methods of the included studied varied, we have given a full critical appraisal of each study in Appendix 2 and a summary in Table 5. Fi een studies met the inclusion criteria, which were conducted in localized water bodies, including wells, domestic water containers, and fishponds and pools (seven studies); pools in a riverbed below a dam (two studies); rice field plots (four studies); or water canals (two studies Of the 15 included studies, 12 studies reported on the density of immature Anopheles mosquito stages in water bodies, and we do not know if larvivorous fish reduce the density (12 studies, unpooled data, very low certainty evidence). Some evidence from studies that ranged in size suggested that larvivorous fish could sometimes prevent increases in immature anopheline mosquito densities compared with control sites, and some studies provided evidence of sustained reductions in immature anopheline numbers up to 13 months of follow-up, but these findings were not consistent. Despite stratification by site and careful critical analysis of each individual study, clear patterns were not evident, although stocking density seemed to have some impact on whether introducing larvivorous fish influenced immature anopheline density.
Of the 15 included studies, five studies reported on larval sites positive for immature vector stages. All reported a reduction in the number of sites positive for Anopheles immatures or "prevention of an increase" in the number of sites positive for Anopheles immatures. Larvivorous fish may reduce the number of larval sites positive for immature anopheline mosquitoes (five studies, unpooled data, low certainty evidence).
None of the studies reported on other ecosystem e ects, including densities of endogenous fish.

Summary of main results
We identified no RCTs or quasi-experimental studies that examined the direct impact of larvivorous fish on malaria in people living in malaria-endemic communities; or on outcomes related to transmission, including EIR and the density of adult vector mosquitoes. Therefore, we do not know whether larvivorous fish have an e ect on adult anopheline mosquito populations or on malaria transmission in endemic communities.
In addition, we examined whether any evidence suggested that this form of vector control had any potential for an e ect on malaria. We examined the e ect of larvivorous fish stocking on two secondary outcomes: density of immature vector stages and percentage of larval sites positive for immature anopheline mosquitoes compared with controls. Fi een small-scale studies met the inclusion criteria of this review and reported on these secondary outcomes only. These studies ranged from three weeks up to five years. They were undertaken in a variety of settings, including localized water bodies (wells, domestic water containers, fishponds or pools, and riverbed pools below dams; nine studies), rice field plots (four studies), and water canals (two studies). Evidence of an e ect of larvivorous fish on the density of immature vector stages in water bodies was variable. We do not know from the available evidence whether larvivorous fish reduce the density of immature anopheline stages (12 studies, unpooled data,very low certainty evidence). Larvivorous fish may cause a reduction in the percentage of larval sites positive for immature vector stages (five studies, unpooled data, low certainty evidence).
Based on the current evidence base and due to the poor quality of the included studies and the absence of any consistent e ect, this is not an intervention that could sensibly be used in malaria vector control. Whether these data can guide future research on which larvivorous fish species should be evaluated and which categories of larval sites should be tested is also not entirely clear. Some reports describe almost 100% reduction of the immature Anopheles population (Fletcher 1992;Haq 2013;Kusumawathie 2008a;Menon 1978;RTDC 2008;Sitaraman 1976). E ects of the fish intervention on immature anopheline populations were mainly reported in studies that used high stocking densities of fish in localized water bodies with short follow-up periods (less than four months), although one study suggested that increasing larval numbers were inhibited for the 11 months' follow-up in domestic water sources (Fletcher 1992).
Notably, monitoring of the immature mosquito population did not appear to influence decisions regarding implementation, such as fish restocking or increase in fish stocking density, in the included studies. None of the included studies examined the impact, if any, of larvivorous fish introduction on the environment or on native species present apart from the target Anopheles mosquito species.

Overall completeness and applicability of evidence
This review demonstrates that there is currently insu icient evidence regarding whether larviciding with fish impacts cases of human malaria or malaria transmission. In some circumstances, the intervention may lead to a reduction in immature mosquitoes in the water sources stocked with fish. This does not show an e ect on malaria transmission but simply shows that the intervention may have a potential benefit worthy of further research.

Quality of the evidence
We found no evidence for the primary outcome of examining the e ects of introducing larvivorous fish on malaria transmission. The certainty of the evidence exploring the larvicidal e ect of fish was low or very low, and overall study design was poor.

Potential biases in the review process
Our search strategy was comprehensive, and it was not limited by language or publication status. Many of the older studies contained anecdotal evidence, and in many studies, fish were combined with other antimalarial interventions in uncontrolled designs, so attribution of an e ect was not possible. We contacted study authors where information was missing or unclear.

Library
Trusted evidence. Informed decisions. Better health.

Agreements and disagreements with other studies or reviews
There is a Cochrane Review of larvicides that excluded fish (Tusting 2013). This review indicated that larviciding could be e ective for preventing malaria transmission, but raised questions about whether it was feasible to undertake this in many areas of Africa.
The current WHO regional strategy for the WHO European Region 2014-2020 recommends the introduction of Gambusia fish "into all sites where Anopheles breed" in areas with "high receptivity and vulnerability" (Ejov 2014). This endorses the same guidelines of the WHO regional strategy for the WHO European Region 2006-2015, which recommended the use of larvivorous fish "in all existing or potential reservoirs where Anopheles species breed with particular attention to rice fields" (WHO-EURO 2006). However, the WHO does not currently recommend this intervention as a vector control strategy for elimination of malaria in its framework for malaria elimination (WHO 2017). The use of larvivorous fish as part of an integrated programme to control malaria has been advocated, subject to further vector biology studies to ensure that the actual vector is targeted (Ghosh 2007). However, further high certainty evidence is required before these recommendations can be supported. Although this Cochrane Review update demonstrated that use of larvivorous fish can cause a significant reduction in the number of immature mosquitoes, particularly in fixed larval sites as opposed to temporary larval sites, a direct correlation between reduction of immature mosquito numbers and reduction of the adult vector population or the number of cases of malaria in people needs to be demonstrated.

Implications for practice
There is no reliable research evidence that introducing larvivorous fish has any e ect on outcomes of transmission of human malaria. Whilst sometimes presented as biologically friendly compared with chemical larvicides, some authors have raised the possibility that larvivorous fish may harm indigenous species, including frogs and other fish species.

Implications for research
This Cochrane Review provides limited research evidence that larvivorous fish can decrease immature mosquito populations in defined water bodies. This is hardly surprising as we know fish eat larvae, and in itself insu icient evidence to support investing in the intervention as a policy without further reliable research. What is unclear is whether this question is worth pursuing. Fish stocking is always going to be expensive, and the e ects almost inevitably will be marginal given the large numbers of water bodies usually present in areas where malaria-transmitting Anopheles lay eggs.
If researchers judge that this is a potentially e ective intervention, then well-designed experimental studies to examine the e ects on malaria in humans or, at the very least, on the EIR or the density of adult vector mosquitoes are required. It is important to note that researchers should carefully consider the design of the studies and should randomly allocate interventions to sites to minimize the risk of bias. In addition, researchers should undertake power calculations to decide the size of the study.
These studies should consider in the design any factors that could influence or bias the results (study design, baseline values, number of sites, pupae numbers reported, distance between sites, other larvivorous species present, vegetation cleared). Several e ect modifiers had dramatic e ects on immature forms, both within and between studies. This includes the ecological zones and settings, fish species, stocking density, and Anopheles species.
This research needs to be undertaken in a variety of ecological zones and settings, including household water sources, ponds, water canals, riverbed pools below dams, and rice fields, and should take into account the seasonality of malaria transmission in these study areas. Notably, testing the impact of fish in the absence of either LLINs or IRS would be unethical and against WHOrecommended policy. Therefore, the fish intervention would need tested in combination with the interventions of LLINs or IRS.
Research is required before larvivorous fish are used in malaria control, to be used either alone or in combination with other vector control methods. Furthermore, research studies should assess the environmental impact of larvivorous fish, particularly non-native introduced species, on the habitats into which they are released.
Apart from e icacy, questions remain regarding whether it is practical to deliver this method with the requisite quality and completeness of coverage on a larger scale than in experimental settings, whether it is cost-e ective, whether it should be delivered as a stand-alone intervention or as an addition to IRS or LLINs, and whether this can be sustained for years.

A C K N O W L E D G E M E N T S
Hellen Gelband was the academic editor for this review.
We are grateful to David Sinclair for his help with the GRADE assessment in the original review (Walshe 2013).
We are grateful to our a iliated institutions and organizations, and we thank the referees and editors for their constructive comments.
In addition, we are grateful to Dr Caroline Je ery for translating French articles, and to Dr Charles V Hoyle and Dr Lena Lantsova for assistance with the translation of Russian articles.

WHO-EURO 2006
World Health Organization. Regional O ice for Europe.

Wurtsbaugh 1980
Wurtsbaugh WA, Cech JJ, Compton J. E ect of fish size on prey selection in Gambusia a inis. Proceedings and papers of the forty-eighth annual Conference of the California Mosquito and Vector Control Association, January 20-23. 1980; Vol. 48:48-51.

Bias Authors' judgement Support for judgement
Study design High risk Quasi-RCT: "In every other house or mosque, fish were stocked in all wells and water storage containers." Site selection Unclear risk "A total of 54 households were selected by systematic sampling. All six mosques were also included in the study. Seven households were excluded because they had only jerrycans and buckets for water storage. They were replaced by seven other households selected by lottery system."

Fletcher 1992
Larvivorous fish for preventing malaria transmission ( (167), in Pithai village. 30 containers in Pithai (intervention) and 25 in Anara (control) village monitored. Only cement tanks were included in longitudinal monitoring because of declining fish populations in other containers due to frequent replenishment Restocked: no. Fish were released once during the 1-year study period, with 10 to 25 fish/tank or per container, depending on container size.

Co-interventions: "routine intervention"
Outcomes Density of immature An. stephensi stages (larvae instars I and II; III, IV and pupae) at weekly intervals for 4 weeks, then every 2 weeks. Only total % reduction in III/IV instar and pupae shown.
Method: standard larval dipper method using the mean of 3 dips. Reduction in III and IV instar larvae and pupae was calculated as per the formula: % reduction = 100 -[(C1 × T2)/C2 × T1)] × 100 where: C1 = pre-release larval density in control tanks; C2 = post-release larval density in control tanks; T1 = pre-release larval density in fish tanks; and T2 = post-release larval density in fish tanks.
Source of funding Sardar Sarowar Narmadad Nigam Limited (SSNL), Gujarat Cochrane Database of Systematic Reviews Notes Correspondence with study author: "The same person/team counting the larval density were counting the fish density in tanks. The arbitrary presence of fishes was recorded in each tank and with the help of torch in under ground tanks". The study author was unable to provide raw data on number of fish or immature Anopheles.

Bias Authors' judgement Support for judgement
Study design High risk Controlled time series study Site selection Low risk The trial authors selected 2 villages, Anara and Pithai, from 15 villages surveyed in Kheda district due to "the similar conditions in respect of type of domestic tanks, water supply and water storage practices." "Randomly, one of the Village Pithai was selected for introduction of Aphanius fish in all the tanks and water containers."

Site allocation Unclear risk
The study introduced fish to 295 water storage containers, such as cement tanks including underground tanks (127), kothi (big mud pots), and barrels (167), in Pithai village. "The survival of the fish and mosquito larval was monitored in 30 containers in the experimental village and 25 in the control village." However, it is unclear how the trial selected which containers to monitor.
Blinding of outcome assessment (detection bias) All outcomes

High risk
The assessors were not blinded to treatment. "The survival of the fish and mosquito breeding was monitored...presence of fish was monitored with the help on a bright light torch." The study author stated via email that: "The same person/team counting the larval density were counting the fish density in tanks. The arbitrary presence of fishes was recorded in each tank and with the help of torch in under ground tanks. It was observed that 2-3 fishes were able to control the larval breeding may be because of the absence of alternate food in the domestic tanks filled with tap water." Baseline values Unclear risk Baseline values for houses positive for mosquito larvae were comparable, but a higher number of containers were positive for mosquito in Anara (control) than in Pithai (Intervention) during baseline monitoring in July 2010 (container index 83.2 (Anara) versus 47.84 (Pithai)). It is unclear how comparable baseline values were before introduction of fish in the 2 villages in November/December 2010. We were unable to obtain further data from the corresponding study author due to "transfer from Nadiad to New Delhi HQ in 2012." Number of sites Low risk Adequate numbers of sites in control and Intervention groups. Study started with Pond B included, but as it was destroyed during the study period, the authors were unable to collect data for it for the requisite time period.

Bias Authors' judgement Support for judgement
Study design High risk Controlled before-and-after study.
Site selection Unclear risk "Sixty isolated riverbed pools...were selected and labeled." Site allocation Unclear risk "P. reticulata was stocked in 29 randomly selected pools". Method of randomization not described.

Cochrane Database of Systematic Reviews
Blinding of outcome assessment (detection bias) All outcomes High risk "Visual counts of P. reticulata were made in each pool, monthly. Visual counts were possible, as the pools were small (not exceeding 1 m surface area), shallow (< 1 m depth) and contained clean water." Baseline values Low risk Comparable between control and intervention sites.
Number of sites Low risk Adequate numbers of sites in control (31 site) and intervention groups (29 sites).

Methods
Study design: controlled before-and-after study Cochrane Database of Systematic Reviews Notes Flow of water from large branch canals was controlled by gates opened at certain times; this system deprived the Gambusia of free movement into the smaller canals, which usually are richer in mosquito larvae than the larger ones, where the fish had originally been stocked. Method: authors used a standard net of 20 cm diameter. The net was immersed in water and held to 0.5 m in 1 direction, then taken in the opposite direction. The net contents were rinsed and the number of fish, and mosquito larvae and pupae counted. Five such samples gave the number of fish and the immature mosquitoes/m .

Notes
No environmental data reported

Bias Authors' judgement Support for judgement
Study design High risk Controlled time series.
Site selection High risk Intervention and control areas each included 1 district with malaria cases and 1 district without malaria cases (no indication how sites for intervention and control areas were allocated).

Site allocation Unclear risk
The study authors did not state how treatment was allocated to study sites.
Blinding of outcome assessment (detection bias) All outcomes Unclear risk Not possible to blind (presence of fish should be obvious); unclear if those who stocked the fish also sampled for larvae.

Baseline values
Unclear risk Study authors did not provide values for the interventions sites prior to introduction of fish.

Number of sites
High risk There were 2 sites in the intervention group and 2 in the control group.

Bias Authors' judgement Support for judgement
Study design High risk Controlled interrupted time series study.
Site selection Low risk "A confined field plot of ca 1,000 m ...the rice paddy was composed of 6 similar sized (10 × 15 × 0.3 m) plots." Site allocation Unclear risk "2 random selection of paddies was made for each group." Method of random selection not specified.
Blinding of outcome assessment (detection bias) All outcomes Populated sites: in each paddy field, 9 rice field checks (3 m x 3 m) were used: 3 filled with P. reticulata, 3 with G. affinis, and 3 served as controls. Checks with the different species of fish were arranged in a chequer board pattern.
(A rice check is a square or rectangular area of a paddy field created by low, narrow banks of earth (dykes) that serve to divide the paddy field into manageable areas and to control the flow of water.) Restocked: yes, but unclear whether for P. reticulata alone due to poor survival or both P. reticulata and G. affinis. Graphs indicated that fish were introduced twice, but text stated "Because of the problem of using guppies as larviphages, related to their much worse survival rate in the native conditions in Tajikistan than the survival rate of gambezi (which can safely be regarded as a representative of the local ichthyofauna), it was necessary to re-release guppies into the rice checks to reduce the number of larvae."

Co-interventions: not stated
Outcomes Outcome: density of either younger or older Anopheles larvae/m Method of measurement: a 20 cm diameter net, or a photographic cuvette, was immersed in the water to half-way down the rim, swept for 1 m to 1 side, trawling the superficial layers, then turned sharply and swept the other way for 1 m to trawl the bottom layers. Net contents were rinsed into a cuvette and the numbers of fish and mosquito larvae and pupae counted. 5 such samples will give the number of fish and pre-imago mosquitoes per m .

Source of funding No information provided
Notes Article in Russian. Data were estimated from graphs In intervention checks G. affinis multiplied successfully despite the presence of predators dragonfly larvae, water bugs, water beetles, marsh frogs. P. reticulata had lower survival in the field than G. affinis.

Bias Authors' judgement Support for judgement
Study design High risk Controlled time series.
Site selection Unclear risk Study authors did not state how they selected sites.
Site allocation Low risk Checks with Gambusia or Poecilia or control were arranged in a chequer board pattern. Study authors stated that allocation was random.
Blinding of outcome assessment (detection bias) All outcomes Unclear risk Presence of fish was obvious, but it was not clear whether observers of larval densities were blinded.       At high fish stocking levels, larvae were eliminated in the first 4 days in wells but reappeared at lower levels from day 24 onwards.
With lower fish stocking levels, there was a partial effect for 2 weeks only, with rebound.  In the control group and with treatments B and C, the number of An. sinensis larvae was higher at 2 weeks' pre-intervention than at 6 weeks' pre-intervention. At 2 weeks' follow-up, the An. sinensis larval population in the control group was the same as at 2 weeks' pre-intervention, but decreased at 6 weeks' follow-up. Larvae were clearly reduced at the 2 sites where fish were introduced.
For treatment A, the number of An. sinensis larvae increased between one week' and five weeks' follow-up at both control and intervention sites. However, the number of larvae decreased by 13 weeks' follow-up at both control and intervention sites. This shows a mean difference in larvae density between control and intervention over the entire period of observation. However, these data were weaker, as no baseline density was noted in the intervention arm, and any difference from the control could be due to chance.

Rice field plots
Nalim  At 4 weeks, larvae had increased against baseline in both control and intervention plots, but the size of the increase was lower in the 2 plots treated with 1 species.
Follow-up at 4 weeks and 7 weeks showed considerably lower values in the 2 plots treated with 2 species than in the control.  An. arabiensis density was lower in intervention canals for 2 months (5 months' and 6 months' post-intervention) just before and at the beginning of the dry season. Larval densities dropped in both intervention and control groups in the dry season (7 months' post-intervention) and at the end of the rainy season (13 months' post-intervention). Fish numbers did not increase after the rainy season and during the last 6 months of the study. According to the authors, control of the flow of water from large to branch canals by gates deprived the fish of free movement. In addition, during the rainy season, rainwater pools act as suitable larval habitats for An. arabiensis. Cochrane Database of Systematic Reviews

Appendix 2. Descriptive analysis of included studies
None of the included studies reported on cases of malaria, EIR, or the density of adult vector mosquitoes. Therefore, we did not find any direct evidence that this intervention impacts malaria transmission. We performed a descriptive analysis of the 14 included studies that examined the e ect of fish stocking on immature anopheline mosquito presence or density, or both. We analysed the studies by the habitat type that study authors introduced for the larvivorous fish. Nine studies evaluated larvivorous fish in localized water bodies (including wells, domestic water containers, fishponds and pools, and riverbed pools created a er dam construction), four studies used rice field plots, and two studies used water canals; see Table 2.
Sitaraman and colleagues introduced fish (100 Poecilia reticulata) to 10 wells and maintained four wells as controls. The authors measured An. stephensi larval and pupal densities by taking five dips per well every four days until 28 days' post-intervention. They measured baseline values immediately before the introduction of larvivorous fish to the 10 wells. We examined the raw data reported by the authors for evidence of an e ect of larvivorous fish on the immature An. stephensi population.
Baseline values in the control (four wells) and intervention groups (10 wells) were comparable before fish were introduced (assuming that these were the numerical totals across the 10 intervention and four control wells; Table 1a). In the intervention wells, immature mosquito numbers decreased rapidly a er fish were introduced. This decrease in immature mosquito numbers was greater than in the control group.
The study authors did not detect any immature mosquitoes in the 10 wells at four days' follow-up. They measured only 15 larvae at 24 days' post-intervention and 40 larvae at 28 days' post-intervention. At 28 days, the immature mosquito numbers (L1 to L4 stages) increased, and the study authors introduced fish into the control wells.
Sitaraman and colleagues released 50 fish per well into 12 wells, with five wells in the same ward serving as controls, and followed immature mosquito numbers for 22 days (Table 2a). A dramatic drop in larvae from daily dips (50 per well) was seen early, with a 69% reduction in larvae and an 82% reduction in pupae by day 2; no such change was seen in the control wells. However, recovery of relatively immature larvae (L1 and L2 instars) was relatively rapid and baseline values were restored by day 10; although recovery of mature larvae (L3 and L4) was slower and less complete, with mean density still 60% lower than baseline a er three weeks ( Table 1, page 317 of the paper).
With high fish stocking levels, larvae are eliminated in the first four days in wells but reappeared at lower levels from day 24 onwards. With lower stocking levels, there was a partial e ect for two weeks only, with rebound. Control (4 wells  In a second study from India, Menon and colleagues introduced Gambusia or Aplocheilus fish to 3438 wells but kept 317 wells as controls.
In intervention sites, if they found mosquito larvae, they stocked with 50 fish per well; if no larvae were present, they stocked with 20 fish per well. They measured An. stephensi larval density at baseline and monthly for four months.
The proportion of wells with larvae was greater in the intervention group (32.8%) than in the control group (7.7%) at baseline (Table 3a).
At follow-up, the proportion of wells with larvae dropped markedly in the intervention arm (less than 1%) but not in the control arm. In the control group, percentage of wells with larvae increased to a maximum of 9.6% during follow-up.
This study appeared to provide evidence of a larvicidal e ect of fish in wells using relatively high stocking levels.

Domestic water containers
Three studies examined larviciding in domestic water containers (Fletcher 1992;Haq 2013;Sabatinelli 1991). In Ethiopia, Fletcher and colleagues introduced fish to wells, barrels, cisterns, and washbasins. In Gujarat State, India, Haq and colleagues added fish to water storage containers, such as cement tanks including underground tanks, kothi (big mud pots), and barrels. On the Comoro Islands, located o the south-east coast of Africa, Sabatinelli and colleagues introduced fish to ablution basins and tanks.
Fletcher 1992 introduced Aphanius dispar to 60 domestic water containers and kept 51 water containers as controls. They measured the Anopheles culicifacies adanensis larval population using a standard dipping procedure pre-intervention and then either every two weeks (May to August 1987) or monthly for 11 months. Control and intervention values were identical at baseline (0%). Sites allocated to the fish intervention had fewer An. culicifacies adanensis larvae at one year post-intervention compared with control sites (see Table 4a).
Fish introduction appears to prevent an increase in the number of domestic water container sites with larvae compared with controls up to 11 months' follow-up. Haq 2013 added A. dispar to 295 water containers in the intervention village and monitored 30 of these containers, and monitored 25 containers in the control village. The study authors measured the An. stephensi larval and pupal population from trial initiation in December 2010 for 12 months (up to November 2011) using a standard dipping procedure taking the mean of three dips at weekly intervals for four weeks followed by fortnightly. The study authors only reported data as % reduction in immature density of L3 and L4 larvae plus pupae of An. stephensi at day 0, 7, 15, and every 15 days therea er. The percentage reduction was greater than 60% for all time points, even up to one year post-intervention (see Table 5a). Sabatinelli 1991 introduced P. reticulata to domestic water containers in Hantsambou village (59 ablution basins sites in November 1987, total number of sites not specified) and kept 20 ablution basins in Bandamadji village as control sites. They measured the percentage of containers positive for An. gambiae larvae by examining the surface and bottom of containers (at least 15 cm in diameter) in both intervention and control groups four times during the 11 months' follow-up. Control and intervention values were identical at baseline. At follow-up, the proportion of sites positive for An. gambiae larvae decreased at fish-treated sites but not at control sites (see Table 6a).
This study appeared to show fish that reduce the number of domestic wash basins with larvae when added to these sites for up to 11 months.

Fishponds and pools
Two studies based in Kenya examined the use of larvivorous fish in ponds (Howard 2007; Imbahale 2011a).
Howard and colleagues compared two intervention ponds and one control pond, all located within 150 m of each other. They measured the number of immature An. gambiae and An. funestus mosquitoes by taking larval dips five to seven days per week. We explored the evidence for an e ect, if any, in three ways: we made a simple description of trends in the graph; we extracted data carefully from the graph; and we examined the authors' analysis.

Library
Trusted evidence. Informed decisions. Better health.

Cochrane Database of Systematic Reviews
Trends in the graph: the authors provided a detailed graph showing An. gambiae immature populations over time in the three ponds. They used a 15-week baseline period before the fish were introduced into two of the three ponds. The control pond had much lower densities of An. gambiae immatures in the baseline period, with none present in the first 1.5 months; then followed a gradual increase in density month by month over the intervention period, with wide week-by-week and, at certain time points day-by-day, variations. At six months' post-intervention, larvae numbers peaked and the authors introduced fish to the control pond.
For the first intervention pond, densities were much higher than for the control pond at baseline. When fish were introduced, the density remained low, or possibly attenuated. For the second intervention pond, the intervention did not appear to be associated with any substantive visual pattern of reduction in density, although it could be argued that some attenuation was evident in the first five months. Thus, critical appraisal of Figure 2 in Howard 2007 indicated increasing immatures in the control pond but did not provide convincing evidence of substantial and sustained decline in the two intervention ponds.
Extracting data from the graph: we took fixed time points before and a er the intervention. Table 7a shows these data, which we estimated using a ruler against the y axis. We chose the one-and three-month time points as standard normal values. We did not include the end time point of the experiment -when the study authors introduced fish to the control pond -as this will introduce bias as it is defined by an increase in larvae. Our analysis below supported evidence of reduction in the immature An. gambiae population in the first intervention pond but not in the second intervention pond. Authors' analysis: the authors used Mulla's formula to calculate percentage reduction in An. gambiae and An. funestus immatures, with estimates of 95.8% reduction in An. gambiae immatures in intervention pond 1 and 94.1% for intervention pond 2; and similar high reductions for An. funestus (98.3% in intervention pond 1, 97.5% in intervention pond 2). However, Mulla's formula depends on rates in the control arm, in which an increase in immature numbers was clearly seen over time. Therefore, one interpretation of these data is that fish are e ective; the other is that these large e ects are the result of particular ecological changes happening in the control pond.
This study appeared to provide limited evidence of a possible larvicidal e ect of fish in ponds.
For the Imbahale 2011a study, refer to the water canals section below.

Riverbed pools below dams
Two studies in Sri Lanka evaluated fish introduced to riverbeds pools located below dams (Kusumawathie 2008a; Kusumawathie 2008b).
In the Kusumawathie 2008a study, authors introduced P. reticulata to 29 riverbed pools below Kotmale dam and used 31 pools as controls. They measured the number of immature Anopheles using a 100 mL larval dipper at a frequency of six dips per m at baseline (day before fish were introduced) and up to 120 days' follow-up. Control and intervention groups had similar baseline values. At follow-up, the intervention group had greater reductions than the control group for the outcomes of percentage of pools with Anopheles larvae, mean number of larvae per pool, and mean number of larvae per 100 dips (Table 8a).
This study appears to provide evidence of a larvicidal e ect of fish in riverbed pools below dams sustained up to four months. In the second study (Kusumawathie 2008b), Kusumawathie and colleagues introduced P. reticulata to all riverbed pools in Laxapana and Kotmale 1 study sites. They used riverbed pools in Kotmale 2 and Nilambe as control sites. They measured immature Anopheles densities using a 100 mL larval dipper at a frequency of six dips per m for one year pre-intervention and one year post-intervention. Baseline values at control and intervention sites were similar for the outcomes percentage pools with Anopheles larvae and mean number of larvae per 100 dips, but not for mean number of larvae per 100 pools. At follow-up, the riverbed pools stocked with fish had larger reductions in terms of presence and density of larvae (Table 9a).
This study indicated a partial e ect of fish on presence and density of larvae in riverbed pools below dams for up to one year.
In Central Java, Nalim and colleagues stocked 23.9 ha of rice fields with P. reticulata and Cyprinus carpio fish. They did not specify the size of the control area or the total number of control and intervention field plots. Using 80 emergence traps randomly located in the treated and control areas, they reported the numbers of Anopheles aconitus, Anopheles barbirostris, and Anopheles annularis newly emerged adult mosquitoes collected/m /day (trap area = 0.25 m ) over six years. E ects were mixed, with some evidence of an impact of fish on An. aconitus and An. annularis, but not on An. barbirostris (Table 10a).
This study indicates a partial e ect of fish on the density of newly emerged An. aconitus and An. annularis, but not An. barbirostris, in rice field plots below dams for up to six years. We discarded two years of data (1982,1983), as the study authors reported that the control area was sprayed with fenitrothion (a phosphorothioate (organophosphate) insecticide) at the end of 1982.
In the South Korean study, Kim and colleagues introduced three slightly di erent interventions to three rice field plots measuring about 300 m to 600 m (Kim 2002). They compared these with a control area of three rice field plots of similar size. They introduced either Tilapia mossambicus and Aplocheilus latipes (treatment A) or Aphyocypris chinensis and Tilapia mossambicus (treatment B and treatment C) to rice field plots and took two dips, with between two and four replicates per rice field, every two weeks, to examine the mean number of An. sinensis larvae.
We extracted data for specific time points before and a er the intervention. The study authors used a six-week baseline period for treatments B and C but no baseline for treatment A before the fish were introduced into two plots.
The results provided a robust controlled before-and-a er study (treatments B and C), with four time points in the control period (Table  11a). Baseline measurements appeared similar at control and intervention sites. In the control group and for treatments B and C, the number of An. sinensis larvae was higher at two weeks' pre-intervention than at six weeks' pre-intervention. A er fish were introduced to the intervention sites, the An. sinensis larval population in the control group was the same at two weeks' follow-up but decreased at six weeks' follow-up. Larvae were clearly reduced at the two sites where fish were introduced.
The study also a orded a controlled time series comparison between the control group and a third intervention site, where the fish were introduced at the start of observations (treatment A; Table 12a). The number of An. sinensis larvae increased between one week' and five weeks' follow-up at both control and intervention sites. However, the number of larvae decreased by 13 weeks' follow-up at both control and intervention sites. This shows a mean di erence in larvae density between control and intervention over the entire period of observation. However, these data were weaker, as no baseline density was noted in the intervention arm, and any di erence from the control could be due to chance.
This study appeared to provide limited evidence of a possible larvicidal e ect of fish on An. sinensis larvae in rice field plots.  In South Korea, Yu and colleagues compared ponds treated with two species of fish (A. latipes and Tilapia mossambicus), one species alone (A. latipes), and a control group (Yu 1989). The researchers selected six plots, 45 m in size and 0.3 m in depth, located within a confined rice field of 1000 m . They randomly assigned two plots to each treatment group. They took measurements of the An. sinensis larval population every week, using a 500 mL dipper (two to four dips per rice field plot) or a nylon net (eight to 10 sweepings per sample).
The study authors monitored the An. sinensis larval population for eight weeks before they introduced fish, and pre-intervention values were comparable between sites. In the first two intervention plots, they introduced one fish species: at four weeks, larvae had increased against baseline in both control and intervention ponds, but the size of the increase was smaller in the one-fish intervention pond (7.00 compared with 16.00, 56% lower; Table 13a).
In the next two intervention plots, they introduced two fish species, and follow-up at four and seven weeks showed considerably lower values in the two-fish intervention pond than in the control pond (4.21 compared with 16.13, 74% lower; Table 13a).
This study provided some evidence that larvivorous fish can constrain the rapid increases in larvae populations seen in untreated ponds. We recalculated the mean pre-intervention values that the study authors reported in control and intervention groups, as the study authors incorrectly reported these values.
In Tajikistan, RTDC 2008 compared two rice field sites treated with Gambusia a inis compared with two control sites. The study authors did not state the size of the plots and how treatment was assigned to the sites. They sampled the immature Anopheles population and G. a inis population every 10 or 11 days using a standard net of 20 cm diameter, which was held to 0.5 m in one direction and then taken in the opposite direction. The net contents were rinsed and the number of fish, and mosquito larvae and pupae counted. Five such samples gave the number of fish and the immature mosquitoes/m .
The study authors did not report any baseline data. They monitored the immature Anopheles population up to either 41 (Vakhsh district site Kirov 2) or 40 days (Bokhta district site of Saidov (Table 14a). The number of immature Anopheles mosquitoes were comparable in both control sites over this time period, and a decrease in the number of immature of Anopheles mosquitoes was observed in the intervention sites.
This study appeared to provide limited evidence of a larvicidal e ect of G. a inis fish on immature Anophelesmosquitoes in rice field plots.  The authors of Zvantsov 2008 examined the e ect of introduction of either G. a inis or P. reticulata to rice field plots in two districts with established rice production: Farkhor (Kizilpakhtachi village) and Shaartuz (Birlyash village). In each rice field nine checks were used: three filled with P. reticulata, three filled with G. a inis, and three served as controls. For this study, each rice check measured 3 m × 3 m, and checks treated with the di erent species of fish were arranged in a chequer board pattern. Fish were released into the rice checks at the rate of five pregnant females/m , i.e. 45 females per 3 m × 3 m check at day 0. Fish were restocked at day 38 or 39 in both sites, but it is unclear whether P. reticulata alone was restocked due to poor survival or both P. reticulata and G. a inis were restocked. The study authors reported the mean value of younger larvae and of older larvae per m .
Mean baseline data, measured immediately before introduction of fish, was comparable in the control and intervention checks in Shaartuz (Birlyash village). In Farkhor district (Kizilpakhtachi village), mean baseline values reported were lower in the control checks than in the intervention checks. The study authors monitored the immature Anopheles population up to 62 (Birlyash village) or 65 days (Kizilpakhtachi village) a er introduction of fish. The number of immature Anopheles mosquitoes decreased in both intervention sites using G. a inis and in one site using P. reticulata (Kizilpakhtachi village) ( Table 15a; Table 16a). However, in Shaartuz district (Birlyash village), a partial e ect was noted using P. reticulata with rebound above baseline levels (Table 15a).
This study provided some evidence of a larvicidal e ect of G. a inis fish on immature Anopheles mosquitoes in rice field plots. With P. reticulata, there was some evidence of a larvicidal e ect in one district and a partial e ect in one district with rebound. The study authors reported the estimated marginal mean ± 95% confidence interval (CI).
In Sudan, Mahmoud and colleagues introduced G. a inis to Gezira irrigation canals (4 km to 10 km in length, 2 m in width, 1 m in depth). They used 20 canals in the intervention group and five canals in the control group. In intervention canals, they released fish at 1 km intervals.