Wireworm assays

Wireworms (larvae) were collected for testing during November 2016–March 2017 and November 2017–March 2018. Wireworms were collected by digging under Florida sugarcane stools where they are aggregated (Cherry 2007). Collection was made in commercial sugarcane fields after harvest for easy access to fields for collection. After collection, wireworms were stored in moist soil with sliced carrots for food at 18°C until used in tests.

Wireworm attraction/repellency tests were conducted in clear-glass rectangular containers measuring 86 × 10 × 12 cm with a clear-glass top. These containers have proved useful in previous wireworm orientation tests (Cherry and Nuessly 2010). Glass containers were used because glass is odorless, reducing possible odor contamination. Moist (3% soil moisture) sandy soil was sieved to remove plant material and insects and then placed 4 cm deep throughout a container. Containers were covered on their outside bottom and sides with aluminum foil. Thus, light only entered from a clear-glass top to simulate field conditions. Containers also were marked to designate four, equal-sized zones within each container (Fig. 1A). Zone 1 was at one end where the ethanol was placed followed by Zones 2, 3, and then 4 at the other end of the container. This zone sequence was used in order to determine if any gradation in wireworm response to or from Zone 1 occurred. During tests, containers were held at 26°C and a 12:12 h L:D photo regime in an insectary.

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The first test (Test 1) was conducted to determine the distribution of fed wireworms over time in the glass container in the absence of ethanol. Five fed (carrots previously available) wireworms were placed on top of the soil in the center of each of the four zones. These wireworms averaged 0.07 g/wireworm and were previously weighed and selected so that there was no significant difference in wireworm weight among zones in a container. Wireworms quickly dug into the soil, a normal behavior. Containers were held 48 h, then opened, and live wireworms in each zone counted. Live wireworms were defined as active and/or moving when prodded. Each container was one replicate with a total of eight replicates.

The second test (Test 2) was conducted to determine the distribution of fed wireworms over time in the presence of a low concentration of ethanol in the soil. Low ethanol concentration is defined here as 2.5 ml ethanol/860 cm³ soil. This test was essentially a repeat of methods of Test 1 except that ethanol was added to Zone 1. Preliminary data obtained in preliminary ethanol solvent tests indicated that 2.5 ml pure ethanol mixed in the soil of a zone should be attractive to wireworms. Hence, 2.5 ml of pure ethanol was mixed with the soil in Zone 1. The other three zones were also mixed, but contained no ethanol. Each replicate was again stopped after 48 h, and wireworms in each zone counted.

The third test (Test 3) was a repeat of Test 2 except that a higher concentration of ethanol was tested. The high ethanol concentration is defined here as 10 ml ethanol/860 cm³ soil. This test was terminated after four replications because some wireworms recovered in Zones 1–3 were comatose (e.g., larvae appearing alive, but not moving). The percentages of comatose wireworms in Zones 1–4 were 89, 92, 9, and 0%, respectively. Being comatose obviously made it impossible to measure wireworm movement to or from different zones. After being held 48 h in fresh soil, 90% of comatose wireworms regained movement.

The fourth test (Test 4) was conducted to determine the distribution of starved wireworms in the glass container in the absence of ethanol. Wireworms used in this test were held without carrots for 2 weeks previous to testing. Methods were simply a repeat of Test 1 except that starved, rather than fed, wireworms were used in this test.

The fifth test (Test 5) was conducted to determine the distribution of starved wireworms in the presence of the low ethanol concentration. Methods were a repeat of Test 2 except that starved, rather than fed, wireworms were used in this test.

White grub assays

Grubs (third instar larvae) were collected and stored until testing as described previously for wireworms. Like M. communis wireworms, C. parallela grubs are also found aggregated under Florida sugarcane stools (Cherry 1984).

Test 6 was conducted to determine the distribution of the fed grubs in the glass container in the absence of ethanol. Methods were essentially the same as Test 1 for wireworms except that the grubs are larger than the wireworms, averaging 0.5 g/grub versus 0.07 g/wireworm. Each test of one glass container was a replication and eight replications were conducted.

Test 7 was conducted to determine the distribution of fed grubs in the presence of the low concentration of ethanol in the soil. This test originally was intended to correspond with the wireworm Test 2 in the glass container. However, Test 6 showed that, unlike the wireworms, the grubs tended to aggregate at ends of the glass rectangular container. Hence, to eliminate this problem, aluminum circular deep pizza pans were used. Aluminum was used because, like glass, it has no odor. Each pan was 36 cm in diameter and 3 cm deep. Each pan was divided into four, equal-size quadrats (4 zones) labeled 1–4 clockwise (Fig. 1B). This configuration approximated the zonation of the glass container in that one zone (Zone 1) would have ethanol, two zones (Zones 2 and 4) were proximal to Zone 1 (being adjacent to Zone 1), and one zone (Zone 3) would be most distal from Zone 1. Soil was placed 2 cm deep in each pan. Because all four quadrats were exactly the same without corners or ends, testing for grub distribution in the absence of ethanol in a zone was not conducted. Total soil volume in a pan was 60% of the soil volume used in the glass containers. Hence, 1.5 ml of pure ethanol was mixed with the soil in Zone 1, corresponding to the low ethanol concentration used in previous wireworm tests in glass containers. Five grubs were again placed in the middle of each zone. Thereafter, a clear-glass top was placed over each pan to prevent the soil from drying. Pans were held 48 h as previously described. Each test with one pan was a replication and eight replications were used.

Test 8 tested the response of fed grubs to the high ethanol concentration as conducted in Test 3 with the wireworms. In this test, 6 ml of pure ethanol were mixed into Zone 1 of aluminum pans. This high ethanol concentration corresponds to the high ethanol concentration used in the wireworm test (Test 3) in glass containers which had a larger soil volume. This test was stopped after four replications because many grubs, like the smaller wireworms, became comatose, preventing measurement of movement.

Test 9 was conducted to determine the distribution of starved grubs in the glass container in the absence of ethanol. Methods were the same as Test 6 except that grubs starved 2 weeks prior to testing were used rather than fed grubs.

Test 10 was conducted to determine the distribution of starved grubs in the presence of the low concentration of ethanol. Test 9 showed that, like fed grubs, starved grubs also aggregated at the ends of the glass rectangular container with no ethanol present. Hence, Test 10 was essentially a repeat of Test 7, using circular aluminum pans, with the exception that starved grubs were used rather than fed grubs.

Means analysis in all tests was conducted using the least significant difference (LSD) test at the alpha = 0.05 level of significance.

Results and Discussion

Numbers of wireworms in different zones of the glass rectangular containers are shown in Table 1. There were no significant differences (P > 0.05) in numbers of fed or starved wireworms recovered in the four zones without ethanol after 48 h. Recovery of wireworms after 48 h was greater than 95%, and these wireworms were alive and active. The even distribution of the wireworms in the containers was not due to the even placement of wireworms at the test start and their inactivity in zones. This statement is corroborated by noting that wireworms recovered were active, and there was a large range of 2–9 wireworms/zone recovered that showed clear movement among zones.

Table 1 Mean (±SD) numbers of wireworms in different zones at 48 h of exposure to soil with and without ethanol.

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The distribution of the wireworms in the containers changed when exposed to the low ethanol concentration in zone 1. Significantly (P > 0.05) more fed and starved wireworms were found in Zone 1 than other zones. These wireworms were alive and active and not comatose, as observed at the high ethanol concentration of Test 3. There was a gradient of response in that Zone 1 had the most wireworms, intermediate Zones 2 and 3 had intermediate numbers of wireworms, and the mostdistant zone from the ethanol (Zone 4) had the fewest wireworms. Starvation did not increase the wireworm response to ethanol, although we suspected it might. Overall, these data are very consistent in showing that both fed and starved wireworms were attracted to the low concentration of ethanol in the soil, as we had previously observed in preliminary testing (Table 1).

White grubs in different zones of glass and aluminum containers are shown in Table 2. Interestingly, there were behavioral differences in grubs from wireworms in how the grubs distributed themselves in the glass rectangular containers after 48 h. In contrast to wireworms, there were significant (P > 0.05) differences in number of fed and starved grubs recovered in the four zones without ethanol after 48 h. Quite simply, grubs had a tendency to aggregate at the ends of the rectangular container (Zones 1 and 4) with significantly fewer in the middle Zones 2 and 3. Recovery of grubs was greater than 95% being alive and active. Because of significant differences in grubs among zones, it is obvious that the grubs were active and mobile among zones. Reasons for behavioral differences between the wireworms and grubs resulting in differences in their spatial distribution in the rectangular containers without ethanol are not known.

Table 2 Mean (±SD) number of white grubs in different zones at 48 h after exposure to soil with or without ethanol in glass and aluminum test arenas.

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White grub testing with ethanol exposure was switched to circular aluminum pans to avoid grub aggregation at container ends. Both fed and starved grubs were found in higher numbers in Zone 1, containing the low concentration of ethanol, than in the other three zones. These grubs were alive and active and not comatose as observed at the higher ethanol concentration of Test 8. For both fed and starved grubs, significantly more grubs were found in Zone 1 than one other zone, but not the other two zones. As with wireworms, starvation did not increase the response to ethanol.

In comparing data from wireworms in Table 1 and white grubs in Table 2, two differences are apparent. The spatial distributions of the wireworms versus white grubs in the glass container with no alcohol differed. Also, the attraction to ethanol of the wireworms versus white grubs differed. In Table 1, the mean number of wireworms in Zone 1 with the low concentration of ethanol was consistently and significantly greater than the other three zones. In Table 2, more white grubs were found in Zone 1 with the low ethanol concentration than the other three zones, but Zone 1 had significantly greater numbers than the other zones only twice. In Table 1, 43% of all wireworms in the ethanol tests were found in Zone 1. In Table 2, 33% of all white grubs in the ethanol test were found in Zone 1. Lastly, the coefficients of variation (standard deviation [SD]/mean) were 0.12 and 0.13 for fed and starved wireworms, respectively, in Zone 1 with ethanol. In contrast, the coefficients of variation were 0.48 and 0.45 for fed and starved grubs, respectively, in Zone 1 with ethanol. The lower coefficients of variation for the wireworms showed the wireworms were more consistent and less variable in their responses to ethanol versus the grubs, which showed more variability among replications. In all, our data show that white grubs were attracted to the low concentration of ethanol in the soil but not as strongly as were the wireworms.

Root-feeding insects are key components in many terrestrial ecosystems. Like shoot-feeding insect herbivores, they exploit a range of chemical cues to locate host plants. Respiratory emission of carbon dioxide from the roots is widely reported as the main attractant; however, there is conflicting evidence about its exact role. At least 74 other compounds elicit behavioral responses in root-feeding insects (Johnson and Nielsen 2012).

More specifically, Villani and Gould (1985) conducted screening of crude plant extracts as feeding deterrents against M. communis wireworms (larvae). And, most recently, Cherry and Nuessly (2010) reported on repellency of the biopesticide azadirachtin to M. communis wireworms. We know of no reports on the behavior of C. parallela grubs in response to chemicals in the soil. This is the first report to show that larvae of M. communis and C. parallela are attracted to ethanol in soil.

Finally, while studying aquatic plant compounds extracted with ethanol, we discovered that the ethanol itself was attractive to M. communis. Those earlier unpublished observations provided the stimulus for this study. The use of plant material extracted with ethanol to test insects is common as shown by recent studies (Alim et al. 2017; Rijal and Bergh 2016; Wagan et al. 2017). Our study shows that when using plant material extracted with ethanol, attention must be paid to possible behavior effects of the ethanol itself on insects being tested.