Comparing efficacy of a sweep net and a dip method for collection of mosquito larvae in large bodies of water in South Africa

Introduction

Traditionally, the larval dip method, as described in detail below, has been the standard method for the collection and sampling of mosquito larvae (O’Malley, 1995). However, this method of collection has proven unsatisfactory for the collection of large numbers of Anopheles mosquito larvae from large water bodies needed for our mosquito genetics studies. Many species of Anopheles, such as An. funestus (Tuno et al., 2007) and An. coluzzii (Gimonneau et al., 2015), readily dive making them difficult to collect from large bodies of water. Collecting Anopheles larvae therefore often means spending considerable time in the field. Also, the larval dip method requires significant experience in dipping techniques and source analysis skills in order to successfully collect the desired genus of larvae (O’Malley, 1995), presenting challenges for a novice.

This limitation of the dipping method motivated us to evaluate a sweep net system similar to methods used by Trapido & Aitken (1953) and Robert et al. (2002) as an alternative approach for collecting larvae to increase catch numbers, especially of Anopheles, and reduce time spent collecting in the field. The method also had to be simple enough for a novice to successfully use. We named our modified sweep net approach the “sweep net catch (SNC)” method and tested it as described below.

Methods

Data analysis

A relative abundance of mosquito specimens grouped by genus per collection was calculated. The Wilcoxon-Rank-Sum test implemented in the R statistical package version 3.0.0 was used to compare the efficacy of the two collection methods (Figure 1) and to see if experience level of collectors was a contributing factor in the relative proportions of each genus collected using each collection method (Figure 2).

Figure 1. Box plot representations of relative abundance of mosquito specimens grouped by genus per collection.

Each collection constitutes a ten minute long sweep net and a ten minute long dip.

Figure 2. Box plot representations of relative proportions of mosquito specimens grouped by genus captured using the larval sweep net and larval dip by collectors of different experience levels.

Results

As shown in Table 1, a total of 99 Anopheles larvae were collected using the dip method, of which 77 were from Shingwedzi, 4 from Skukuza, and 18 from Lapalala. A total of 605 Anopheles larvae were collected using the SNC method, of which 530 were from Shingwedzi, 20 from Skukuza, and 55 from Lapalala. A total of 61 Culex larvae were collected using the dip method, 22 being from Shingwedzi, 8 from Skukuza, and 31 from Lapalala. A total of 176 Culex larvae were collected using the SNC method, 63 of these from Shingwedzi, 85 from Skukuza, and 28 from Lapalala.

The number of collected larvae were highly variable between trials, reflecting the general larval density variation between sites. To control for this site variation and compare the relative performance of each collection method, a relative proportion of each genus collected per trial was calculated. The Wilcoxon-Rank-Sum tests on the relative abundance data showed (Figure 1) the SNC method (mean relative abundance = 0.51 ± 0.28) performed significantly better than the dip method (mean relative abundance = 0.12 ± 0.13) in the collection of Anopheles larvae (Wilcoxon-Rank-Sum test P = 8.58 × 10^-5). There was no significant difference in the collection of Culex larvae between the SNC method (mean relative abundance = 0.25 ± 0.29) and the dip method (mean relative abundance = 0.12 ± 0.20) (Wilcoxon-Rank-Sum test P = 0.050).

The collector’s experience was not a contributing factor in the relative proportions of each genus collected using either the SNC method or the dip method (Wilcoxon-Rank-Sum test P ≥ 0.21) (Figure 2).

Discussion

Many of the Anopheles larvae collected were reared to adults and identified using a morphological key (Gillies & Coetzee, 1987) and molecular assays (Koekemoer et al., 2002; Lee et al., 2014; Scott et al., 1993) (see Supplementary material for methods). The species collected included An. arabiensis, An. quadriannulatus, An. coustani, An. pretoriensis and An. funestus group (An. parensis, An. rivulorum, An. leesoni and an as yet undetermined species).

The SNC method performed on average five times better than the dip method in the collection of Anopheles larvae. The SNC samples a larger volume of water than the dip method which likely contributes to the higher catches of Anopheles larvae. Many species, such as An. funestus, An. arabiensis (Tuno et al., 2007) and An. coluzzii (Gimonneau et al., 2015), are known to dive and remain in the substrate for long periods of time when there has been a disturbance on the surface or for feeding purposes. The larger net diameter allows the increased capture of these larvae as they begin to submerge. In shallow parts of the water body the SNC also scoops along the substrate and may collect larvae that have dived and rested there. From our observations the SNC method disturbs the surface of the water less than dipping, which possibly reduces diving behavior.

More Anopheles may also have been captured using the SNC because collectors spent more time sieving through the water in the 10 minute period, thus covering a larger area, whereas during the 10 minutes of collecting, the dip method required spending time actively separating out the larvae after each dip. Depending on the water quality and larval density, the final larval separation for the SNC can be a time-consuming task as mud and debris can make it difficult to see the larvae in the tray. However, the overall processing time (collection and sample separation) per specimen for the SNC (1.09 ± 0.97 minutes) was 75% less than the dip method (4.17 ± 3.95 minutes) (Figure 3). To calculate the average time spent collecting larvae with the SNC we added 12 seconds of processing time for each larva collected to the 10 minutes of sweeping for each trial. This time was then divided by the total number of larvae collected in that trial (Data availability). The average time collecting larvae with the dip method was calculated by dividing the 10 minute dipping period by the total number of larvae collected for each trial (Data availability).

Figure 3. Box plot summary of processing time spent collecting larvae between the dip and sweep net collection methods.

Most of the Culex larvae collected were successfully reared to adults and identified using the key in Jupp (1996). The collections consisted of Cx. poicilipes, Cx. simpsoni and Cx. neavei. It has been shown that many species of Culex larvae exhibit significant diving behavior (Workman & Walton, 2003), which suggests the SNC would also have increased efficiency in collecting these larvae as long as the surface was not too disturbed and the collector’s shadow was cast behind them. However, due to the relatively low numbers of Culex larvae collected throughout our study, we suspect there wasn’t a high enough population density of these larvae at any of our trial sites to accurately determine if either method was more efficient for the collection of Culex larvae. We recommend further research be conducted in areas where Culex larvae occur at higher densities to further evaluate whether the SNC performs better than the dip method.

Even though our current data does not show a significant difference in the relative proportion of larvae collected due to the collector’s experience, our personal observations suggest the SNC is a good method for novices to use. The SNC allows the inexperienced handler to easily collect high numbers of mosquito larvae without analyzing their technique or source characteristics as is required to be successful using the dip method (O’Malley, 1995). A larger trial size may illuminate more conclusively if experience level does affect collection performance.

Conclusion

We endorse and encourage the sweep net method as a preferred technique for larval collection that can be easily used in the field setting regardless of experience level. Our SNC method is particularly effective in capturing Anopheles mosquito larvae. The increased sensitivity of SNC towards Anopheles larvae may be due to (1) the sampling of a larger volume of water than the dipping cup, and/or (2) reducing disturbance of the water surface resulting in fewer larval dives. This increased sensitivity of the SNC method makes it an appropriate larval collection tool for studies when more accurate assessments of larval densities are required and when there is less time available to sample for larvae. In addition, the simplicity of the SNC method makes it a recommended choice for novice collectors. Further research is suggested to more rigorously test if a significant correlation between the collector’s experience level and the relative proportion of larvae collected by either method exists.

Data availability

Figshare: Visual representation of both the sweep net catch and dip larval collection methods as performed by Braack and Cornel. 10.6084/m9.figshare.3123274 (Brisco et al., 2016a).

Figshare: Raw larval collection data for all 15 replicates, including the relative abundance of Anopheles and Culex collected and the estimated time taken to collect one larva for each replicate. 10.6084/m9.figshare.3123463 (Brisco et al., 2016b).