The worldwide incidence, severity, and mortality rate of diseases caused by arboviruses – particularly dengue fever – have grown dramatically in recent decades (Anonymous, 2019). One recent estimate indicates 390 million infections/year, mostly in Central and South America, Africa and Asia (Bhatt et al., 2013). In the USA, 26 states are infested by
The main strategies to combat
For larval control, greater focus has been placed on chemical and microbial larvicides, insect growth-regulators and predatory fish, copepods and Toxorhynchite larvae. Nonetheless, field trials have brought mixed reports of the prospect of these approaches to prevent or curb dengue fever outbreaks (Horstick and Runge-Ranzinger, 2018; Achee et al., 2019).
Recent reviews on biological control of mosquitoes (Benelli et al., 2016; Huang et al., 2017) have not even mentioned entomopathogenic nematodes (EPNs) as potential agents. Nonetheless, a range of
Most studies on the efficacy of EPNs against mosquito larvae have been conducted only in the laboratory, with a variety of methodological procedures that limits generalizations (for a review see Cardoso et al., 2015). Recent assays still have not examined key parameters that may determine the efficacy and viability of EPNs for biocontrol of mosquitoes (Chaudhary et al., 2017; Ulvedal et al., 2017; Toksoz and Saruhan, 2018; Dilipkumar et al., 2019).
In a follow-up study, the efficacy of
Urban populations of
Hence, the goal of this work was to assess the efficacy of
Larvae of the wax moth (
Floor drain types 1 through 4 were used (Fig. 1A–D). Type 1 have a bottom internal surface area of 34.5 cm2. In type 1, the water permanently retained in the drain’s bottom (Fig. 1E) is about 40 ml. In types 2 through 4, the bottom internal surface areas are 47.7, 83.3, and 188.5 cm², respectively, and the amounts of water retained are 110, 270, and 1,120 ml, respectively.
(A–D) floor drain types (1–4); (E) water permanently retained in the bottom of all drain types; (F) medium size pot and saucer; (G) 65 liter water barrels; (H) ridges and grooves on the surface of pot saucers.
For each drain type, 10 drains received 10 L3/L4 and 1,000 IJs. The blank control consisted of 10 drains in which L3/L4 only were applied. The drains were placed in a growth chamber, in the dark, at 25°C. A voile fabric was attached to the drain to avoid releasing adult mosquitoes into the environment. The evaluation was carried out 7 to 8 d later, when adult mosquitoes had emerged in the blank control. Dead larvae and pupae were counted and inspected for the presence of nematodes in their body. This assay was repeated twice.
A second set of floor drain assays was conducted as described before, with the exception of using the adjustable dose of 25 IJs/cm2 of drain bottom internal surface. This assay was repeated once.
Small, medium (Fig. 1F), and large pots with saucers were used. When tap water was poured in the pot, it leaked through the bottom holes and created the saucer’s standing water. For the small, medium, and large saucers, the water volumes were 20, 65, and 368 ml, respectively. These volumes were retained mainly around the pot, in areas of 13.3, 87.9, and 245 cm², respectively.
For each saucer size, the assay procedures were as described before, except that the pots and saucers were maintained on laboratory benches, at 25°C. The assays, using either 1,000 IJs/saucer or the adjustable IJ dose, were conducted twice.
65 liter barrels were used, with a bottom internal surface of 961 cm2. Sixty liters of tap water was added, with the water column reaching 50 cm high. Ten barrels received 10 L3/L4 and 25 IJs/cm2 (24,000 IJs), and 10 barrels served as blank controls, receiving 10 L3/L4 only. The assay procedures were as described before, except that the barrels were kept closed with their own lids on the laboratory floor, at 25°C. This assay was repeated once.
The assays’ raw data are publicly available at:
For all assays, the data on the dead mosquito larvae were tested for homogeneity of variances (Cochran and Bartlett tests) and for normality of errors (Lilliefors test), at 5% probability (Ribeiro, 2001). Since the assumptions were satisfied, ANOVA was conducted considering time as one of the factors. No statistical significance was found for the floor drain assays, so their data were pooled. Time was a significant factor for assays in pot saucers and water barrels, so the data were analyzed separately for each assay. The treatments’ mean numbers of dead larvae were compared through the Tukey test at 5% probability, and expressed in the tables as mortality rate (%).
Mortality rate (%) of
Types (bottom internal surface area, in cm2) | ||||
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Treatments | 1 (34.5) | 2 (47.7) | 3 (83.3) | 4 (188.5) |
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Treated | 74a | 67a | 64a | 45a |
Control | 23b | 23b | 10b | 0b |
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Treated | 82a | 78a | 76a | 56a |
Control | 3.5b | 3.5b | 5b | 1b |
Notes: Values are means of three assays, each with 10 drains/type, and 10 larvae/drain. In the columns, values followed by different letters are statistically different according to the Tukey test at 5%.
Parthenogenetic female of
This suggests that each mosquito larva browses a certain bottom area of the oviposition site, and that at 25 IJs/cm2 most larvae ingest enough nematodes to get infected and killed. Nonetheless, it remains unclear why nematode efficacy declined in the largest drains, since the IJ dose was the same in all sizes. It is possible that with more nematodes applied in the largest drains, they formed more and larger clumps, as typically occur with some
In pot saucers, applying 10 IJs/mosquito larva resulted in mortality rates of 46 to 53% in the smallest saucers, decreasing to 35% in the largest ones (Table 2). The IJ dose of 25 IJs/cm2 did not improve the efficacy, even when the IJs/mosquito larva rate was about 600. The bottom internal surface of pot saucers, marked with grooves and ridges (Fig. 1H), may have hindered the encounter of mosquito larvae and nematodes. Finney and Harding (1981) reported a steep decline in the efficacy of
Mortality rate (%) of
Sizes (bottom internal surface area, in cm2) | |||
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Treatments | Small (13.3) | Medium (87.9) | Large (245) |
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Assay 1 | |||
Treated | 46a | 29a | 35a |
Control | 3b | 11b | 14b |
Assay 2 | |||
Treated | 53a | 53a | 35a |
Control | 18b | 13b | 8b |
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|||
Assay 1 | |||
Treated | 31a | 44a | 38a |
Control | 0b | 0b | 0b |
Assay 2 | |||
Treated | 28a | 37a | 30a |
Control | 0b | 0b | 0b |
Notes: Values are means of 10 pot saucers/size, and 10 larvae/pot saucer. In the columns, values followed by different letters are statistically different according to the Tukey test at 5%.
Grooves and ridges were also present in the bottom internal surface of the water barrels. The dose of 25 IJs/cm2 resulted in 24,000 IJs applied/barrel, but the efficacy was unacceptably low: mortality rates of 0.1 and 1.7% in assays 1 and 2, respectively. This clearly suggests that a much higher dose would be needed to treat water barrels, and that EPNs would not be feasible to treat abandoned swimming pools, which are also important domiciliary oviposition sites.
Since Welch (1960) (cited by Finney and Harding, 1981), EPNs have been investigated for biocontrol of various mosquito species. In some
In this study,
Although EPNs seem promising to control