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 12 included studies that examined the effect 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. Eight studies evaluated larvivorous fish in localized water bodies (including wells, domestic water containers, fishponds and pools, and riverbed pools created after dam construction), three studies used rice field plots, and two studies used water canals; see Table 2.
Section 1: Localized water bodies
Two studies from India evaluated larviciding in wells (Sitaraman 1976; Menon 1978).
Sitaraman and colleagues introduced fish (100 P. 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 effect of larvivorous fish on the immature An. stephensi population.
Baseline values in the control (four wells) and experimental groups (10 wells) were comparable before fish were introduced (assuming that these are the numerical totals across the 10 intervention and four control wells; Table 1A). In the experimental wells, immature mosquito numbers decreased rapidly after 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 and 40 larvae at 24 and 28 days' post-intervention, respectively. 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 also 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 a 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 average density still 60% lower than baseline after 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 reappear at lower levels from day 24 onwards. With lower stocking levels, a partial effect was noted for two weeks only, with rebound.
Table 1A. Sitaraman 1976 : An. stephensi immature numbers before and after introduction of fish (100 guppies per well)
|Intervention||Immature stages||Pre-intervention||Follow-up (days)|
|Control (four wells)|
L1 + L2
L3 + L4
|Intervention (10 wells)|
L1 + L2
L3 + L4
Table 2A. Sitaraman 1976 : An. stephensi immature numbers before and after introduction of fish (50 guppies per well)
|Intervention||Immature stages||Pre-intervention||Follow-up (days)|
|Control (five wells)|
L1 + L2
L3 + L4
|Intervention (12 wells)|
L1 + L2
L3 + L4
In a second study from India, Menon and colleagues introduced Gambusia or Aplocheilus fish to 3438 wells but kept 317 wells as controls. In experimental 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 experimental 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 experimental arm (< 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 appears to provide evidence of a larvicidal effect of fish in wells using relatively high stocking levels.
Table 3A. Menon 1978 : percentage of wells with An. stephensi larvae in wells immediately before and after introduction of fish
(b) Domestic water containers
Two studies examined larviciding in domestic water containers (Fletcher 1992; Sabatinelli 1991). In Ethiopia, Fletcher and colleagues introduced fish to wells, barrels, cisterns, and washbasins. On the Comoro Islands, located off the south-east coast of Africa, Sabatinelli and colleagues introduced fish to ablution basins and tanks.
Fletcher and colleagues introduced Aphanius dispar to 60 domestic water containers and kept 51 water containers as controls. They measured the An. culicifacies adanensis larval population using a standard dipping procedure pre-intervention and then either every two weeks (May to August 1987) or monthly for a total of 11 months. Control and experimental 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.
Table 4A. Fletcher 1992 : percentage of sites with An. culicifacies adanensis larvae before and after introduction of fish
(percentage of sites)
Sabatinelli and colleagues 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 experimental and control groups four times during the 11 months' follow-up. Control and experimental 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 5A).
This study appears to show fish that reduce the number of domestic wash basins with larvae when added to these sites for up to 11 months.
Table 5A. Sabatinelli 1991 : percentage of sites with An. gambiae larvae before and after introduction of fish
(percentage of sites)
(c) Fishponds and pools
Two studies based in Kenya examined 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 effect, 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.
Trends in the graph: The authors provide 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 experimental 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 experimental ponds.
Extracting data from the graph: We took fixed time points before and after the intervention. Table 6A 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 supports evidence of reduction in the immature An. gambiae population in the first experimental but not in the second experimental pond.
Table 6A. Howard 2007 : An. gambiae immatures in three ponds before and after the introduction of fish
|Intervention||Pre-intervention (months)||Follow-up (months)|
|First experimental pond1||3||7||0||0|
|Second experimental pond2||2||4||2||2|
1Referred to as Pond C within Howard 2007 study.
2Referred to as Pond D within Howard 2007 study.
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 experimental pond 1 and 94.1% for experimental pond 2; and similar high reductions for An. funestus (98.3% in experimental pond 1, 97.5% in experimental 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. So one interpretation of these data is that fish are effective; the other is that these large effects are the result of particular ecological changes happening in the control pond.
This study appears to provide limited evidence of a possible larvicidal effect of fish in ponds.
For the Imbahale 2011a study, refer to the water canals section below.
(d) 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 m2 at baseline (day before fish were introduced) and up to 120 days' follow-up. Control and experimental groups had similar baseline values. At follow-up, the experimental 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 7A).
This study appears to provide evidence of a larvicidal effect of fish in riverbed pools below dams sustained up to four months.
Table 7A. Kusumawathie 2008a : average percentage of pools with Anopheles larvae, mean number of larvae per pool, and mean number of larvae per 100 dips before and after introduction of larvivorous fish
|Percentage of pools with Anopheles larvae|
|Mean number of larvae per pool|
|Mean number of larvae per 100 dips|
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 m2 for one year pre-intervention and one year post-intervention. Baseline values at control and experimental 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 8A).
This study indicates a partial effect of fish on presence and density of larvae in riverbed pools below dams for up to a year.
Table 8A. Kusumawathie 2008b : average percentage of pools with Anopheles larvae, mean number of larvae per 100 pools, and mean number of larvae per 100 dips before and after introduction of larvivorous fish
|Percentage of pools with Anopheles larvae|
|Mean number of larvae per 100 pools|
|Mean number of larvae per 100 dips|
Section 2: Rice field plots
Three studies, one in Central Java (Nalim 1988) and two in South Korea (Kim 2002; Yu 1989), evaluated fish introduced to rice fields; .
In Central Java, Nalim and colleagues stocked 23.9 hectares of rice fields with P. reticulata and C. carpio fish. They did not specify the size of the control area that they used or the total number of control and experimental field plots. Using 80 emergence traps randomly located in the treated and control areas, they reported the numbers of An. aconitus, An. barbirostris, and An. annularis newly emerged adult mosquitoes collected/m2/day (trap area = 0.25 m2) over six years. Effects were mixed, with some evidence of an impact of fish on An. aconitus and An. annularis, but not on An. barbirostris (Table 9A).
This study indicates a partial effect 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.
Table 9A. Nalim 1988 : average number of adult mosquitoes collected per m2 per day
| An. aconitus 1|
| An. barbirostris 1|
| An. annularis 1|
1We discarded two years of data (1982, 1983), as the study authors reported that the control area was sprayed with fenitrothion at the end of 1982.
In the South Korean study, Kim and colleagues introduced three slightly different interventions to three rice field plots measuring about 300 m2 to 600 m2. They compared these with a control area of three rice field plots of similar size. They introduced either Tilapia mossambicus and A. 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 average number of An. sinensis larvae.
We extracted data for specific time points before and after 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 provide a robust controlled before-and-after study (Treatments B and C), with four time points in the control period (Table 10A). 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. After 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 affords 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 11A). The number of An. sinensis larvae increased between one week and five weeks' follow-up at both control and experimental sites. However, the number of larvae decreased by 13 weeks' follow-up at both control and experimental sites. This shows an average difference in larvae density between control and intervention over the entire period of observation. However, these data are weaker, as no baseline density was noted in the intervention arm, and any difference from the control could be due to chance.
This study appears to provide limited evidence of a possible larvicidal effect of fish on An. sinensis larvae in rice paddy plots.
Table 10A. Kim 2002: An. sinensis larvae at control (three plots) and experimental sites (two plots) before and after introduction of fish
|Intervention||Pre-intervention (weeks)||Follow-up (weeks)|
Table 11A. Kim 2002: An. sinensis larvae at control plots (three plots) and at an experimental plot (one plot) after introduction of fish
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. The researchers selected six plots, 45 m2 in size and 0.3 m in depth, located within a confined rice field of 1000 m2. 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 12A).
In the next two intervention plots, they introduced two fish species, and follow-up at four weeks 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 12A).
This study provides some evidence that larvivorous fish can constrain the rapid increases in larvae populations seen in untreated ponds.
Table 12A. Yu 1989: average number of An. sinensis larvae in ponds before intervention and after introduction of fish
| One-fish||4.19||7.00||Bacteria introduced|
1We recalculated the average pre-intervention values that the study authors reported in control and intervention groups, as the study authors incorrectly reported these values.
Section 3: Water canals
Two studies introduced fish to irrigation canals — one in Kenya (Imbahale 2011a) and one in Sudan (Mahmoud 1985).
In Kenya, Imbahale and colleagues compared the effects of G. affinis introduced to ponds or water canals versus control sites. The water sources were discrete; 18 ponds were 1 m2 in size and 1 m depth, and 12 canals were 15 m2 in size and 0.3 m in depth. For ponds, the authors evaluated the effects of single stocking and multiple stocking of fish by measuring An. gambiae s. l. larvae twice a week for 13 weeks; and for canals, they compared controls with a single stocking of fish. The study authors divided outcomes by younger larvae (L1 and L2) and older larvae (L3 and L4), and reported estimated marginal mean values. No difference was demonstrated between control and experimental groups at follow-up, apart from the fact that the numbers of older larvae were smaller in the canal intervention group (Table 13A).
This study provides some evidence of an effect of larvivorous fish up to 13 weeks in water canals but not in ponds.
Table 13A: Imbahale 2011a: estimated marginal mean values of immature anopheline numbers after introduction of fish
|Younger larvae (L1 and L2) 1||Older larvae (L3 and L4) 1|
| Ponds||Control||2.667 (2.217 to 3.117)||0.758 (0.551 to 0.964)|
| ||Fish (stocked once)||2.667 (2.217 to 3.117)||0.964 (0.757 to 1.170)|
| ||Fish (multiple stocking)||3.067 (2.604 to 3.505)||0.903 (0.697 to 1.109)|
| Canal||Control||3.417 (2.896 to 3.937)||1.177 (0.974 to 1.380)|
| ||Fish (stocked once)||1.906 (1.386 to 2.427)||0.547 (0.344 to 0.750)|
1The study authors reported the estimated marginal mean ± 95% confidence interval (CI).
In Sudan, Mahmoud and colleagues introduced G. affinis 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 experimental group and five canals in the control group. In experimental canals, they released fish at 1 km intervals. They measured the density of a late larval stage of An. arabiensis (L4) larvae in both experimental and control canals by performing larval dips at two spots per kilometre in each canal, reporting averages by month from weekly dipping of 10 dips per spot for 14 months.
No baseline was provided, but An. arabiensis density was less in intervention canals for two months (five months' and six months' post-intervention) just before and at the beginning of the dry season (Table 14A). Larval densities dropped in both intervention and control groups in the dry season (seven months' post-intervention) and at the end of the rainy season (13 months' post-intervention). Fish numbers failed to increase after the rainy season and during the last six 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. Also, during the rainy season, rainwater pools act as suitable breeding sites for An. arabiensis.
Introducing larvivorous fish appears to partly constrain An. arabiensis larval density increases at the beginning of the dry season.
Table 14A. Mahmoud 1985:density of An. arabiensis L4 larvae after introduction of fish
| Control canals||42||153||7||125|
| Experimental canals||25||24||1||124|