The method used for testing monoterpenoid ovicidal activity was based on a method used by MEC Ltd. [42,43]. The louse eggs used were those o f the Orlando strain o f clothing lice. Although these lice prefer to lay eggs on tufted fabrics such as the cotton cord normally supplied, they will also lay on thick-fibred gauze if provided with no other substrate. Gauze is more suitable than cord for ovicidal assays, as the material is less absorbent, allowing the test substance to be washed off if desired. Also, the squares formed by the gauze fibres provide reference points for counting eggs and nits at the end o f the test. The gauze is left in with the colony for 2-3 days to ensure a good yield o f eggs, but removed at the end of day 3, as eggs laid after this time would be significantly less developed than those laid on day 1. The egg-laden gauze was removed from the incubator and immediately placed in a plastic Petri dish, packaged in bubble wrap and posted to The School o f Pharmacy, where the tests were carried out on day 3 or 4. As soon as they arrived they were placed at 24-31 °C, 50-90 % RH and kept in these conditions before and after treatment with test agents.
Preliminary experiments were carried out to determine suitable test concentrations and duration o f exposure to enable the relative efficacies o f all agents to be assessed. In the assays, prior to immersion in the test solutions, the sheets o f gauze were cut into squares of convenient size (2 cm x 2cm approx.). Terpenoids and essential oils were diluted in
absolute ethanol, or diethyl ether if ethanol-insoluble, and used to fill 20 ml glass bottles. Approximately 300 eggs (200 minimum) were immersed in each test substances for 10 min. After this time, the gauze was removed, blotted with tissue paper and left for 5 min on a clean piece o f tissue laid flat in a fume cupboard, on low setting, to dry off the solvent and leave the fibres o f the gauze impregnated with the test substance. Eggs exposed to solvent
only were included with each batch of tests to enable correction o f results with control mortality data; a control with no treatment was carried out at intervals to ensure that treatment with solvent alone continued to have no significant effect on the background mortality rate.
The eggs from individual treatments were incubated in separate glass Petri dishes at 24-31 °C, 50-90 % RH until all the nymphs in the control batches had hatched and died. The eggs and nits were scored according to the identification key in Figure 2.1. For calculation o f total percentage mortality, aU half-hatched and unhatched nymphs were grouped together; to assess the mechanism of ovicide action, aU the categories in Figure 2.1 were treated separately. In the first stage of screening, 10 % (w /v or v/v) solutions were used, the most effective terpenoids from the 10 % stage were then screened at 5 %, and the most effective from this stage were screened at 2 % and 1%. As the control (solvent alone) mortality values were regularly above 5 %, Abbott’s Correction [1] was used to adjust the results o f % mortality caused by test agents.
Where at least 3 replicates with corresponding control mortalities o f less than 20 % (higher values render the data less reliable) were obtained for a test agent at one test concentration, the data were subjected to statistical analysis: the quantal response measure o f % mortality is not directly amenable to ANalysis O f VAriance (ANOVA), as the scale o f 1-100 % sets upper and lower limits for the distribution o f data; if data are to follow the assumptions for the ANOVA o f normal distribution and equal variance, the scale must be continuous. A common transformation for percentage values, arcsin(^ /lOo), where Y = percent mortality, was used with the intention of correcting the data sufficiently for analysis by ANOVA. Tukey’s post-test was employed following one-way ANOVA to identify pairs of results with significantly different means; both analyses were performed by Graph-Pad Prism computer package.
Category Hatched Appearance Half-hatched Developed
%
UndevelopedEmpty shell (nit) is transparent and shiny. Operculum open or absent. Any louse nearby has no limbs inside the shell.
Interpretation: The agent did not kill either unhatched nymph or hatchling. Operculum partially or completely open or absent. Louse nymph either wholly or partly inside the shell. Interpretation: The agent killed the hatching nymph, but not the unhatched louse. This is most likely because the agent is prevented from having a true ovicidal effect as it cannot penetrate the shell, instead it persists on the fabric and kills the hatching louse when the seal around the operculum is broken.
Operculum Kilïy closed. Correctly positioned black eyespots are present (near the cap). The formed body o f the louse nymph may or may not be present within the egg: in both cases the egg appears translucent but yellow and matte in appearance. Interpretation: The louse embryo reached a stage o f development where a functional nervous system was present, and then ceased to develop further. The agent was only active on a neuronal target.
Operculum closed and egg appears matte yellow. Eyespot is either black but incorrectly positioned, red (correctly or incorrectly positioned) or absent. Often the inside o f the egg has a homogeneous appearance, although a bubbly liquid interior is also common. The egg may appear shrivelled.
Interpretation: The agent arrested development before the nervous system formed. The agent either has a non-neuronal mode o f action or has neuronal and non-neuronal targets.
Figure 2.1 Key to Identifying the stage at which a Pediculus humanus egg is killed