BIOASSAY 1. DETERMINATION OF DIFFERENT TEGMINAL COLORATION PATTERNS OF PARENTAL MAHANARVA SPECTABILIS RETRIEVED FROM THE FIELD AND THEIR FIRST GENERATION OFFSPRING

Approximately 2,000 nymphs of M. spectabilis were collected at an Embrapa Dairy Cattle experimental field in the town of Coronel Pacheco, Minas Gerais State, Brazil. Nymphs were transferred to 500 mL pots with elephant grass as a food substrate and kept in the greenhouse of the Embrapa Dairy Cattle. Emerged virgin adults (n = 242 couples) were classified daily with regard to tegminal coloration pattern, namely a straw-yellowish hue with black spots (YB), a reddish hue with black spots (RB), a totally reddish hue (R), and a totally black hue (B), following Auad et al. (2010). The adults were paired to form the following couples: (i) yellowish with black spots (YB) × yellowish with black spots (YB) (n = 30); (ii) reddish with black spots (RB) × reddish with black spots (RB) (n = 11); (iii) totally reddish (R) × totally reddish (R) (n = 30); (iv) totally black (B) × totally black (B) (n = 7); (v) yellowish with black spots (YB) × reddish with black spots (RB) (n = 22); (vi) yellowish with black spots (YB) × totally reddish (R) (n = 54); (vii) yellowish with black spots (YB) × totally black (B) (n = 24); (viii) reddish with black spots (RB) × totally reddish (R) (n = 25); (ix) reddish with black spots (RB) × totally black (B) (n = 19); (x) totally reddish (R) × totally black (B) (n = 20), with a total of 10 treatments. Replications (n) varied for each treatment due to population oscillation of tegminal coloration patterns of specimens which emerged from the nymphs collected in the field.

Couples were individualized and conditioned on the upper part of elephant grass plants within voile cages so that the F1 generation could be obtained. A piece of gauze was placed at the base of each cage as a substrate for egg laying. After the death of the females, eggs were retrieved from the gauze. The gauze was placed on a set of sieves through which running water was jetted, where eggs remained within the 400 mesh sieve. Eggs from each couple were placed singly in 9 cm Petri plates lined with filter paper and stored in BOD-type acclimatized chambers (28 ± 2 °C, a 14:10 h (L:D) photoperiod, and 70% relative humidity) until the embryo stage was close to hatch (S4). Eggs at stage S4 were placed in 1 × 1 cm filter paper sheets and transferred to 500 mL pots, each with an elephant grass plant whose roots were exposed by jetted water. Exposed roots served as the food substrate for the nymphs. Up to 40 eggs were placed on each plant. Pots were sealed with plastic lids and gauze, and maintained in trays in the greenhouse. Emergence of adults was monitored daily to evaluate the tegminal coloration pattern of the first generation.

Prior to analyzing the different crosses between progenitors, Pearson's χ² goodness-of-fit test was employed for testing for the adherence of the frequency distribution for tegminal coloration phenotypes of the F1 (1,484 adults) to the distribution observed for progenitors (484 adults). Next, for each of the 10 crosses established by the progenitors' wing patterns, the occurrence proportions of the 4 tegminal coloration patterns for the F1 were compared. For 65 out of the 242 progenitor couples, at least 8 descendants developed to adulthood. Thus, in order to ensure a minimal reliability of the proportions, progeny data arising from only these couples were employed to evaluate the tegminal coloration patterns of F1 generation. Taking each of the 10 crosses singly as a specific sub-assay regardless of the others, the proportions of each tegminal coloration pattern occurring within the progeny were compared by Analysis of Variance (ANOVA) followed by Tukey's test with significance level of 0.05. Statistical package R vers. 3.1.3 (R Core Team 2015) was used for analysis.

BIOASSAY 2. BIOMETRY OF FIRST GENERATION OF MAHANARVA SPECTABILIS FROM PROGENITOR WITH DIFFERING TEGMINAL COLORATION PATTERNS

Biometric analysis was undertaken with 20 males and 20 females of each tegminal coloration pattern of M. spectabilis: yellowish-black (YB), reddish-black (RB), reddish (R), and black (B), obtained from the first generation retrieved from Bioassay 1. Insects were monitored with a stereoscopic microscope (Novainstruments, Piracicaba, São Paulo, Brazil), and images were transferred to computer. Anatomic parts were measured with the program Screen Calipers for ProScope (LX - ProScope HR Software, Philadelphia, Pennsylvania, USA). Length and width (mm) of head, pronotum, scutellum, and tegmina were recorded.

Biometric evaluation data were subjected to variance analysis and Tukey's test (α = 0.05). The ANOVA included effects of sex, wing patterns, and the interaction between sex and wing pattern on the width and length of head, pronotum, scutellum, and tegminal. When a significant interaction effect was detected, the levels of one factor were compared across levels of the other by means of ANOVA F-tests, followed by Tukey's post-hoc multiple comparisons with the significance level set at 0.05. Data were analyzed with statistical package R vers. 3.1.3 (R Core Team 2015).

BIOASSAY 3. EXTRACTION AND QUANTIFICATION OF DNA FROM MAHANARVA SPECTABILIS OF DIFFERENT TEGMINAL COLORATION PATTERNS

Molecular analysis was performed for 3 M. spectabilis specimens of each tegminal coloration pattern, collected in Coronel Pacheco and preserved in 70% alcohol. An external control consisted of 3 specimens of M. fimbriolata (Stål) collected in Coronel Pacheco, Minas Gerais, Brazil, and 3 specimens of Mahanarva tristis (F.) (Hemiptera: Cercopidae) collected in the state of Pará, Brazil.

Wings, legs, and head of each insect were extracted with a pair of tweezers. The remaining part of each insect was transferred to a separate Eppendorf tube and ground with liquid nitrogen. DNA was extracted from the residual part at the Molecular Genetic Laboratory of Embrapa Dairy Cattle (Juiz de Fora, Minas Gerais, Brazil) following the protocol of Ferreira and Grattapaglia (1995).

Fifteen Random Amplified Polymorphic DNA (RAPD) markers from Operon Technologies Inc. (Alameda, California, USA) (OPA13; OPA16; OPB1; OPB6; OPB7; OPB20; OPD8; OPE4; OPE11; OPE18; OPF1; OPG8; OPG10; OPG11; OPG17) were selected for genotyping the 18 samples. DNA amplification reactions were performed in 35 µL aliquots with 0.4 mM primer, 0.15 mM dNTPs, 1.0 unit of Taq polymerase, 10 mM Tris-HCl (pH 8.0), 2.5 mM MgCl₂, and 50 mM KCl. The following cycles were used for amplification: 40 cycles at 94 °C for 30 s, at 37 °C for 60 s, and at 72 °C for 30 s; followed by 1 cycle at 72 °C for 7 min. Products were separated by electrophoresis in 2% agarose gel, and images were digitized for further analysis using Gel Analyzer 2010 ( www.gelanalyzer.com).

A binary matrix was built according to presence (1) and absence (0) of bands between tegminal coloration patterns of M. spectabilis. Rates were employed for diversity analysis by the Jaccard Index (Jaccard 1901). A similarity matrix also was employed to perform the unweighted pair group method using arithmetic averages, producing a dendrogram with different tegminal coloration patterns of M. spectabilis and control groups (M. fimbriolata and M. tristis). Molecular marker data underwent molecular variance analysis (Analysis of Molecular Variance, AMOVA) to assess distances between tegminal coloration patterns of M. spectabilis.

BIOASSAY 1. DETERMINATION OF DIFFERENT TEGMINAL COLORATION PATTERNS OF PARENTAL MAHANARVA SPECTABILIS RETRIEVED FROM THE FIELD AND THEIR FIRST GENERATION OFFSPRING

Estimated relative occurrence frequencies of tegminal coloration patterns of progenitors collected in the field were 33%, 33%, 18%, and 16% for patterns reddish (R), yellowish-black (YB), reddish-black (RB), and black (B), respectively, regardless of sex (Fig. 1). First generation adults showed mean occurrence percentages of 40%, 37%, 18%, and 5%, respectively, revealing an increase in the occurrence of tegminal coloration pattern reddish (R) and a reduction in pattern black (B), when compared to progenitors (Fig. 1). So, the frequency distribution of offspring differed significantly from that of the progenitors (χ² = 144.5; gl = 3; P < 0.0001).

The evaluation of generation F1 showed that in crosses in which one of the progenitors had tegminal coloration pattern reddish (R), its offspring had a greater chance (more than 50%) of expressing this same phenotypic pattern of coloration than the other patterns (Fig. 2A, B, D), except cross reddish/reddish-black (R/RB) (Fig. 2C). Furthermore, it should be underscored that in the cross between progenitors of the same coloration pattern reddish (R) (Fig. 2A), the least percentage of descendants with the same pattern reddish (R) was 69%, whereas the maximum percentage of all the other coloration patterns was only 15%. Distancing revealed the prevalence of offspring with coloration pattern reddish (R) derived from the cross.

Also, if one progenitor had tegminal coloration pattern yellowish-black (YB) (Fig. 2 E–G), the chance of having progeny with yellowish-black (YB) coloration was greater (> 40%) when compared to the other patterns, except for cross yellowish-black/reddish-black (YB-RB) (Fig. 2F). This observation suggests persistence in this pattern's occurrence when pattern reddish (R) was absent. Furthermore, couples yellowish-black/yellowish-black (YB-YB) significantly generated the highest mean percentage (56%) of offspring with the yellowish-black (YB) pattern, followed by patterns reddish (R) (25%) and reddish-black (RB) (19%). Since pattern black (B) showed the lowest mean occurrence rate (0.5%) (Fig. 2E), yellowish-black/yellowish-black (YB-YB) progenitors were heterozygous; similarly, we are assuming the same inference for cross reddish-reddish (R-R).

Disregarding coloration patterns reddish (R) and yellowish-black (YB), in crosses where one of the M. spectabilis progenitors had tegminal coloration pattern reddish-black (RB) (Fig. 2H, I), the proportion of descendants with the reddish-black (RB) pattern also tended to be higher (> 50%) than that of the black (B) pattern. There was no significant predominance of any tegminal coloration pattern in generation F1, with mean percentages ranging between 20 and 30%, in crosses where both parents showed tegminal coloration pattern black-black (B-B) (Fig. 2J).

Fig. 1.

Relative frequency of 484 adults collected at the Embrapa Dairy Cattle experimental field in the town of Coronel Pacheco, Minas Gerais, Brazil, from which 242 couples were formed, with their 1,484 offspring, with regard to wing color patterns, regardless of sex.

The results of these crosses indicate that different wing patterns vary in their likelihood of occurrence given the same pattern in a parent. They suggest an order of genetic dominance associated with phenotypes reddish > yellowish-black > reddish-black > black (R > YB > RB > B).

BIOASSAY 2. BIOMETRY OF FIRST GENERATION OF MAHANARVA SPECTABILIS FROM PROGENITOR WITH DIFFERING TEGMINAL COLORATION PATTERNS

Based on morphological studies, no significant interaction effects of sex and wing pattern on the length or width of the head or pronotum (Fig. 3A–D) or on width of tegmina (Fig. 3H) were found. Mahanarva spectabilis females are longer and wider at the head, pronotum, scutellum, and tegmina than males of the same species (Fig. 3A–D, H). Length of head and widths of pronotum and tegmina were not different among specimens of different wing patterns (Fig. 3A, D, H). However, width of head and length of the pronotum were smaller in specimens of the totally black (B) wing pattern (Fig. 3B, C).

Interactions between sex and wing pattern were observed for length (Fig. 3E) and width of scutellum (Fig. 3F) and tegminal length (Fig. 3G). Length and width of the scutellum of pattern reddish (R) and the length of the tegmina of pattern reddish (R) and yellowish-black (YB) females were significantly larger than those of other females. Tegminal length and scutellum width in males did not differ for wing patterns. However, the length of the scutellum was smaller in the black (B) hue specimens (Fig. 3 E–G).

BIOASSAY 3. EXTRACTION AND QUANTIFICATION OF DNA FROM MAHANARVA SPECTABILIS OF DIFFERENT TEGMINAL COLORATION PATTERNS

Molecular studies revealed that 101 bands were generated from the 15 markers (Table 1) among the 18 specimens belonging to different tegminal coloration patterns and controls. Twenty-eight exclusive molecular markers were obtained for the 3 species (10 for M. spectabilis, 8 for M. fimbriolata, and 10 for M. tristis) (Table 1).

Fig. 2.

Box plot representation of the distribution of the offspring's tegminal coloration pattern proportions obtained for each type of mating cross, according to parents' wing color pattern, regardless of sex. Each box spans from the first to the third quartile (interquartile range). The segment inside the box and the filled circle indicate median's and mean's locations, respectively. Whiskers above and below the box extend either to the maximum/minimum data value or to the most extreme value falling within the extent equivalent to 1.5 × interquartile range, starting from the box; points exceeding these limits are considered suspected outliers and are marked with unfilled circles. Different letters within each quadrant indicate significantly different mean proportions (Tukey's test, α = 5%). Value in parentheses are, in the order they appear, the number of replicates (i.e., the number of couples from which at least 8 offspring were obtained, that grew to adulthood), the total number of offspring generated from these replicates that grew to adulthood, the ANOVA residual degrees of freedom, and the ANOVA F-test p-value. *Insufficient n for analysis.

When different morphological patterns of M. spectabilis were taken into account, the analysis of molecular variance (AMOVA) detected much greater variation within each tegminal coloration pattern (99%) than among the different patterns (1%) (PhiPt 0.007; P = 0.365; df = 3,11). This finding demonstrated that there was no parallelism between phenotypic and molecular differences (Fig. 4).

The dendrogram gives a better view of the approximation degree between the different wing patterns in M. spectabilis (Fig. 4). In fact, it reveals that the external group (M. tristis) is actually more distant from the other 2 species (M. fimbriolata and M. spectabilis). However, there is no grouping trend in the latter with regard to similar wing patterns.

Fig. 3.

Comparative biometrics of M. spectabilis by sex and wing color patterns. Distributions observed for head length (A) and width (B), pronotum length (C) and width (D), scutellum length (E) and width (F), and tegmen length (G) and width (H). In cases (A) to (D) and (H), sex × wing color pattern interaction was not significant; thus p-values and letters refer to mean comparison between sexes or among wing color patterns, where means followed by different letters were found to be significantly different. In the other cases, (E) to (G), sex × wing color pattern interaction was found to be significant, and its analysis was conducted: p-values and letters refer to mean comparison among wing color patterns within sex, where different letters indicate significant differences, whereas distributions marked with a same symbol (* or +) indicate a significant difference between the sexes for a given wing color pattern. ANOVA followed by Tukey's test at 5% significance probability level was used in all cases.

Table 1.

Exclusive marks in the 3 species of spittlebugs under analysis (M. spectabilis, M.fimbriolata, and M. tristis).