Effect of fluoride on the learning and memory ability of larvae of Zaprionus indianus

Many pesticides contain fluoride that enters the food chain and affect the non-target organisms. Fluoride is a known neurotoxin and may cause neurobehavioral defects. A study was conducted to see the effect of fluoride on the learning and memory ability of larvae of Zaprionus indianus. The learning and memory ability of 2nd instar larvae of normal (control) and sodium fluoride (NaF)-treated Zaprionus indianus was compared. Sublethal concentration of NaF for Z. indianus was found to be 0.8 ppm. Olfactory assay results showed that the larvae of normal (control) Z. indianus had better learning and memory ability in comparison to NaF-treated larvae. This study indicates that the insects exposed to pesticides containing fluoride may have difficulty in locating food sources and carrying out pollination.


Background
Exposure to fluoride can occur through dietary intake, respiration, and water. Fluoride enters the environment through volcanic eruptions, rock dissolution, and numerous human activities (coal burning, ore processing, production and use of fertilizers, and industrial plants). Many pesticides, insecticides, and weedicides contain fluoride in high concentrations, and the overuse of such chemicals paves way for fluoride to enter the system of non-targeted organisms such as human beings and other animals and cause derogatory effects. Acute pesticide poisoning occurs frequently in children worldwide, and subclinical pesticide toxicity is also widespread (Grandjean & Landrigan, 2014). Clinical data suggest that acute pesticide poisoning during childhood might lead to lasting neurobehavioral deficits (Kofman, Berger, Massarwa, Friedman, & Jaffar, 2006;London et al., 2012). Thus, there is a need to study the neurotoxic effect of fluoride.
Fruit flies and mammals share many genes suggesting that the molecular mechanisms of behavioral plasticity might also be shared (Rubin et al. 2000). Short life span, large number of offspring produced, a well-known anatomy, and occurrence of a wide variety of mutants are convenient characteristics of fruit flies as a model organism (Jeibman and Paulus, 2009).
Olfaction in fruit fly is crucial for a variety of behaviors, including associative learning (Quinn et al., 1974, Tully andQuinn, 1985) courtship (Gailley et al. 1986), foraging (Shaver et al. 1998;Frye and Dickinson, 2004), and flight (Schneiderman and Trimarchi, 1995). Fruit flies can learn to associate olfactory or visual cues with rewarding or punishing reinforcement. Fruit fly memory persists for hours or days, depending on the training protocol. Multiple spaced training trials form long-term memory that can persist for days (Keene and Waddell, 2007). Fruit fly larvae can be used as model organisms to study the neurotoxic effect of fluoride.
Zaprionus indianus (Gupta, 1970) is an arthropod belonging to the fruit fly family Drosophilidae. Z. indianus is abundantly found around fruit trees such as guava and mango and may help the trees in carrying out the process of pollination. Due to this, Z. indianus is at a risk of being exposed to insecticides that contain fluoride that is a potential neurotoxin. This may result in the organism losing track of its trail, and if that happens, the fly will ultimately die because it will not be able to find its food, and the trees dependent on the fly for the dispersal of pollen may suffer too. A study on this aspect has not been conducted so far. This paper presents the effect of sublethal level of sodium fluoride (NaF) on the learning and memory ability of 2nd instar larvae of Z. indianus (Gupta, 1970).

Methods
The assessment of memory ability of Z. indianus was done using its 2nd instar larvae. Z. indianus flies were trapped using fruit baits and cultured in the laboratory on cornmeal medium. Single line culture of flies was maintained by transferring a gravid fly in separate cornmeal medium containing bottles. The larvae obtained were assessed for their learning and memory ability with the help of olfactory assay following Scherer, Stocker, and Bertram Gerber (2003). Four sets of 100 ml cornmeal medium were prepared to be poured in sixteen glass bottles. Each set contained four bottles. Out of all the four sets, one set was used as control, and the rest three were experimental set up.
Thereafter, 1000 ppm NaF stock solution was prepared by adding 2.21 g of NaF into 1000 ml distilled water. NaF of the concentrations 0.8 ppm, 1.0 ppm, and 1.5 ppm was taken to treat the flies. Three sets of cornmeal medium were prepared. Each set contained four bottles.
The first set had 0.8 ppm NaF, the second set had 1.0 ppm NaF, and the third set had 1.5 ppm NaF containing cornmeal medium. Zaprionus indianus flies from single line stock culture were transferred into each of these bottles such that each bottle contained at least one gravid fly.

Olfactory assay
Plain agar petri plates were taken. A filter paper was taken on which a circle and two vertical lines were drawn in the center, and two diametrically opposite points were marked close to the periphery of the petri plates and were termed as C 1 and C 2 .

Procedure for olfactory assay Control test
Agar plate was divided into two halves, and one drop of distilled water was placed on each side with the help of a dropper. Sixty 2nd instar larvae were introduced at the center, covered with black box and left for 2 min. After 2 min, larvae were counted on both sides (C 1 and C 2 ), and Olfactory Response Index (ORI) was calculated.

Experimental test
10 −2 concentration of ACV was used as attractant on one side and distilled water on the other side of petri plate with plain agar. Same set of 2nd instar larvae was introduced at the center covered with black box and left   for 2 min. After 2 min, larvae were counted on both sides, and ORI was calculated.
Formula for calculating ORI ORI = C 1 − C 2 /C 1 + C 2 Here, C 1 = no. of larvae on side one (ACV) C 2 = no. of larvae on side second (distilled water)

Avoidance test
Next, agar plate was neatly cut into half, and half of it was removed and replaced with agar containing 20 mM NaCl. NaCl played the role of irritant. ACV was placed on the side containing NaCl, and on the other side distilled water was put. Same set of larvae was introduced in the center, and assay was performed as during experimental test. ORI was calculated.

Confirmatory test
Next, same set of larvae was again placed on plain agar petri plate containing ACV on one side and distilled water on the other, and olfactory assay performed as during experimental test. ORI is calculated.

Experimental set up
Similarly, larvae treated with sublethal level of NaF were also introduced on plain agar petri plates and control test, experimental test, avoidance test, and confirmatory test were performed.

Statistical analysis
Students t-test was performed to compare the mean ORI of control vs NaF-treated flies, and P < 0.05 was considered as statistically significant.

Results
The ORI of Z. indianus larvae towards IAA and ACV is shown in Table 1. The larvae were not found to be attracted towards IAA. However, they showed attraction towards ACV. 10 −2 was the favored concentration. So, 10 −2 concentration of ACV was taken for further olfactory assay. Sublethal concentration of NaF for Z. indianus was found to be 0.8 ppm. This concentration was used for performing olfactory assay because flies were found to survive and reproduce in this concentration (Table 2). On the other hand, 1.0 ppm and 1.5 ppm NaF concentrations were found to be lethal for the flies as at this concentration the flies were unable to reproduce and grow in number (Tables 3 and 4).
Larvae reared on normal cornmeal medium were taken as control and were assessed for their learning and memory ability by performing olfactory assay with 10 −2 concentration of ACV (Table 5). Z. indianus larvae reared on 0.8 ppm concentration of NaF were assessed for their learning and memory ability by performing olfactory assay with 10 −2 concentration of apple cider vinegar (Table 6). A statistically significant difference was found in the means of ORI of normal vs NaF-treated larvae during the confirmatory test (t, 4.3; df = 4; P < 0.05) ( Table 7).

Discussion
Olfactory assay of the larvae of native Z. indianus has been conducted for the first time in the present study. Khurana and Siddiqui (2013) studied the response of 3rd instar Drosophila larvae towards 53 odorants. Such   elaborate studies on response profile of Drosophila larvae were very valuable while performing olfactory assay. Tabassum, Kumari, Singh, and Yasmin (2017) studied a comparative account of the olfactory behavior of pureline Drosophila melanogaster (inbred up to 10 generations) and CsBz with that of native Drosophila melanogaster by using iso-amyl acetate odourant.
Zaprionus indianus larvae did not show appreciable response towards iso-amyl acetate. So, based on experiments done by Joshi et al. (2014) at Pennsylvania, ACV was used as attractant. Based on the ORI values obtained, it was observed that Z. indianus larvae showed maximum attraction at 10 −2 concentration apple cider vinegar.
In the experiment, it was found that 2nd instar larvae of Zaprionus indianus showed abnormalities on treatment with NaF. At concentration of NaF, more than 0.8 ppm (i.e., 1.0 ppm and 1.5 ppm) Z. indianus flies did not lay eggs, and flies died in a few days. Due to the effect of fluoride, the learning and memory ability of Zaprionus larvae was hampered, which became evident with the ORI results obtained (positive value of confirmatory test), as opposed to the ORI results of normal larvae (not exposed to NaF), where ORI value was negative during confirmatory test. The abnormalities displayed by NaF-treated Zaprionus indianus larvae can be said to be because of NaF reacting with the brain of the larvae. Fluoride is a known neurotoxin (Spittle, 2011). F toxicity may also result in low IQ children (Yasmin et al. 2013).
Though, the killing action of fluoride can be very helpful in insecticides (Metcalf, 1966), the aspect of fluoride affecting the nervous system cannot be dealt leniently (Grandjean and Landrigan, 2014). Z. indianus has been considered as a pest in many countries such as Veracruz in Mexico (Lasa and Tadeo, 2015). But the fact that it is found in the orchards indicates that it may be helping in the process of pollination (Landolt et al., 2012). This makes the fly a significant component of the natural ecosystem. If all such flies and other insects are treated with pesticides containing fluoride, it can lead to their death or reduced efficiency in carrying out pollination. In either case, the whole system of symbiotic association between trees and the insects will be disrupted. This will ultimately lead to reduced productivity of the trees.

Conclusions
The study showed that NaF-treated larvae suffered some neurological disorder that affected their learning and memory ability. Pesticides containing fluoride can cause death of non-target insect populations or can reduce their efficiency in carrying out pollination by affecting their learning and memory aspects.