Measurements of the host morphologies were applied for comparing the sexual dimorphism, the developmental trajectory in both sexes of the mantids, and the parasitic effect on the mantids hosts, H. formosana (field-collected) and H. patellifera (reared).
2.6.1 Measurement of wing, leg and pronotum characteristics
Wings, legs and pronota of the field-collected H. formosana were removed from specimens and preserved in 75% ethanol solution. The wings were unfolded, flattened and covered by a transparent plastic slide. All dissected parts were photographed using a camera with a scale of 1 mm.
Pronotum and leg lengths were measured according to the description provided by Prete et al. (1990, 2002). The pronotum was measured from the rostral-most to the caudal-most midpoint of the prothoracic tergum. The leg was measured from the coxa to the end of the tarsus (the length of the tarsus was not included in the analysis of raptorial legs). The wings were measured according to sclerotized wing venation, as described by Roy (2005). The length of the forewing was measured from the base of the costal vein to the end of the second radial vein, which is the visually longest wing length. The length of the hindwing was measured from the base of the costal vein to the end of the radial posterior vein. The measurements were performed using the segmented line function in ImageJ 1.47 (Rasband, 1997-2016), and calibrated spatially to the scale included in each picture.
The forewing shape index was computed using the following formula:
(BR−AR)/(BR+AR), where AR is the area above the radial vein and BR is the area below the radial vein. A high shape index indicates the relatively large area below the radial vein. The area of each part was measured using the polygon selection function
in ImageJ 1.47.
Detailed methods is described in Chiu et al. (2015).
2.6.2 Measurement of antennal characteristics
The antennae were collected and preserved in a 75% alcohol solution before being examined. Each molting skin was preserved in a small tube and rinsed by 75%
ethanol solution before being examined.
The comparison of the field-collected H. formosana is to examine the sexual dimorphism and parasitic effect on the field-collected mantids. Three antennal characteristics including 1) density of the grooved basiconic sensillum on antennae, 2) the first segment bearing grooved basiconic sensilla, and 3) the total number of antennal segments were examined using SEM (same method in the sample preparation with Method 2.1–2.2) or a light microscope. The density of grooved basiconic sensilla was calculated using the average number of sensilla per 25 × 25μm2 on each selected antennal flagella. One antennal flagellum was selected for every 10 segments from the 10th to 100th segment; thus, 10 antennal segments were selected from each sample. The narrow median strip of each antennal segment was first divided into 16–96 cells of 25 × 25μm2. The total number of grooved basiconic sensilla in this narrow median strip was counted and then divided by the number of square areas. In addition to the sensillum density, the first segment bearing grooved basiconic sensilla and the total number of flagellum segments were also recorded (samples with incomplete or broken antennae were not included in the analysis of the total number of segments). Detailed methods is described in Chiu et al., (2015).
The comparisons of antennae in different instars of H. patellifera which were free from horsehair worm's infection is to examine the developmental trajectories and sexual differentiation in both sexes of the mantids. All the antennae (adults) and the
antennal part of the molting skin (nymphs) were examined under SEM. For every flagellum segments, we counted number of the large trichoid sensilla, small trichoid sensilla, and groove basiconic sensilla, and measured the length and the wide of each segment. According to these measurements, the total number of large trichoid sensilla, the total number of small trichoid sensilla, the total number of groove basiconic sensilla, the total number of flagellum segment, antennal surface area of each flagellum segment, the first flagellum segment bearing the grooved basiconic sensilla, and the total number of flagellum segment bearing the grooved basiconic sensilla ("the total number of flagellum segment" minus " the first flagellum segment bearing the grooved basiconic sensilla") were compared between the sexes of the adult, the first instar nymph, the 8th instar nymph, and the 9th (last) instar nymph. The antennal surface area ("the length" times "the wide") of each flagellum segment were also compared between the sexes of the adult, the first instar nymph, and the 9th (last) instar nymph. According to the first flagellum segment bearing the grooved basiconic sensilla (BS) and the total number of flagellum segment (TS) in the different sexes, an antenna was separated into three zones: male BS to female BS (Zone1), female BS to female TS (Zone2), and female TS to male TS (Zone3) (see chapter 4.3.1, Table 2).
Total number of the grooved basiconic sensilla on each of these three zones were compared between the sexes of adult mantids. The antennal surface area of the Zone2 was also compared between the sexes of the adult, the first instar nymph, and the 9th (last) instar nymph.
The examination of the antennae on the male adult mantids artificially infected by Chordodes sp. was to investigate the relationship of the host morphological change and horsehair worm's developmental time. The antennae were examined by a light microscope and recorded the first flagellum segment bearing more than three grooved
basiconic sensilla. This value was first compared between the infected mantids (n = 26) and the control mantids (n = 11). The values of the infected male adults were separated into "manipulated group" (n = 13) which is outside of two standard deviations of the control mantids and "non-manipulated group" (n = 10) which is inside of one standard deviation of the control ones. These two categories were coded as 0 (Non-manipulated group) and 1 (Manipulated group) and analyzed by logistic regression model against the horsehair worm's developmental time before the last molting of the host to get the time when 50% and 90% of the infected mantids showed the manipulated antennal characteristic.
The examinations of antennal characteristics in the infected hosts of the adult female and the last instar male were to test the hypothesis of the juvenilization. The antennae of the adult female examined were collected from the female adults artificially infected by C. formosanus. The antennal part of the molting skin of the last instar male examined were collected from the control mantids and the mantids infected by Chordodes sp. All the infected mantids harbored the horsehair worms more than 30 days (when 50% of the infected host start to show the manipulated morphology, see chapter 4.3.2). The first flagellum segment bearing more than three grooved basiconic sensilla were applied to compare the difference between 1) the infected and control host of the last instar male, 2) the infected and control host of the adult female, and 3) the infected adult female and its last instar nymph.