Compared nymphal development of Tingis americana ( Hemiptera , Tingidae ) in two Handroanthus species ( Bignoniaceae ) and reproductive parameters in seedlings of Handroanthus heptaphyllus

Nymphs and adults of Tingis americana Drake, 1922 were found feeding on leaves of Handroanthus heptaphyllus (Vell.) Mattos and Handroanthus chrysotrichus (Mart. ex A. DC.) Mattos in the Botanic Garden, Porto Alegre, state of Rio Grande do Sul, Brazil. This is the first record of T. americana on these host plants and in the southern Brazil. We aimed to compare the nymphal development on both hosts and to analyze the reproductive parameters on H. heptaphyllus (25 ± 1°C; 60 ± 10% RH; 16 h photophase). The mean nymphal period (days) was shorter in individuals reared on H. heptaphyllus (12.69 ± 0.076) than on H. chrysotrichus (19.11 ± 0.208) (P < 0.0001), however, nymph viability was similar. On H. heptaphyllus, the mean embryonic period lasted 12.32 ± 0.274 days and the egg viability was 92%. The mean total and daily fecundity were 310.0 ± 19.40 eggs/female and 7.46 ± 0.302 eggs/female/day, respectively. Paired males and females showed similar longevity (P = 0.0691), while unpaired females lived longer than unpaired males (P = 0.0460).

Most lace bugs feed on a single or on closely related host plant species, usually on the abaxial side of Angiospermae leaves (Schuh & Slater, 1995).Their feeding habits may result in puncture marks, white spots, and/or leaf abscission (Stonedahl et al., 1992;Neal & Schaefer, 2000).The oviposition site is reported as highly species-specific and determines the mode of oviposition (Livingstone & Yacoob, 1987).Eggs can be laid singly or in clusters, in any part or position relative to the hostplant, as horizontally to the leaf surface ("lamina oviposition"), or perpendicularly along midrib and veins, and also on different parts of flowers and stems (Livingstone & Yacoob, 1987).
Tingis americana is recorded in Brazil, Paraguay and Argentina and so far its only host plant record is Compared nymphal development of Tingis americana (Hemiptera, Tingidae)... Tecoma sp.(Bignoniaceae) (Drake & Ruhoff, 1965).Nymphs and adults of this species were found feeding on two Bignoniaceae, Handroanthus heptaphyllus (Vell.)Mattos (purple trumpet tree) and H. chrysotrichus (Mart.ex DC) Mattos (yellow trumpet tree) in the Porto Alegre Botanic Garden, causing seedling death.Biological data of T. americana are still lacking.In this paper we compared the nymphal development of this lace bug on both H. heptaphyllus and H. chrysotrichus, analysing its reproductive parameters on H. heptaphyllus.Additionally, this is the first record of T. americana on these host plants and in southern Brazil.

MATERIAL AND METHODS
Tingis americana colony rearing.Individuals were collected in the Botanic Garden in March 2011, from seedlings of H. heptaphyllus.Adults were kept at 25 ± 1°C; 60 ± 10% RH; 16 h photophase, in seedlings obtained from the same place.Adults were maintained on the same seedlings for egg obtaining.Seedlings were replaced by fresh ones whenever damaged.
Nymphal development.Newly emerged nymphs were individualized in Petri dishes with a leaflet of H. heptaphyllus (n = 117 individuals) or H. chrysotrichus (n = 110) inserted in agar and water, plus antifungical (nipagin) and antimicrobian (streptomycin sulfate) (adapted from Foelckel et al., 2009).The dishes were kept under the same conditions as the colony.Previously, the leaflets were cleaned in a 1-2% sodium hipochlorid solution during one to two minutes, and then dried with paper towel.Leaflets were replaced every three days.Exuviae and dead individuals were daily recorded under stereomicroscope.Sex ratio was calculated based on the emerged insects.
Longevity and reproductive parameters.Unpaired, newly emerged adults (60 females and 41 males) obtained from the colony were individualized in Petri dishes with a leaflet of H. heptaphyllus, which was replaced every three or four days.These individuals were observed until their death.Paired specimens (16 couples) were maintained under the same conditions in order to register longevity, fecundity, fertility and, pre-, post-and oviposition periods.Handroanthus heptaphyllus leaflets were used as food and oviposition substrate as well.Couples were daily observed and transferred to a new leaflet, until their death.Fecundity was evaluated by daily computing the number of eggs laid in each leaflet, observed under a stereomicroscope provided with transmitted light.Fertility was measured through the number of hatched insects.
Statistical analyses.Data were tested for normality by D'Agostino test and, according to their distribution, compared by t-Student, Mann-Whitney, ANOVA or Kruskal-Wallis tests.Sex ratio (sr) was calculated by [sr = number of females/(number of females + males)], and nymphal viability (%) was compared by Qui-square test (heterogeneity and adherence).The relation between age of females and number of laid eggs was measured by Pearson correlation.We adopted the significance level of 0.05 in all the tests performed.For these analyses, we used the softwares Microsoft ® Excel 2010 and Bioestat ® 5.0 (Ayres et al., 2007).
The lace bug specimens were identified by Dr. Luiz Costa (Museu Nacional -MNRJ, Rio de Janeiro, Brazil).Vouchers are deposited in the entomological collection of the Museu de Ciências Naturais -MCNZ, Fundação Zoobotânica do Rio Grande do Sul, Porto Alegre, Brazil (40 specimens, catalog numbers 181.841 to 181.880).

Plant
Longevity and reproductive parameters on H. heptaphyllus.Tingis americana oviposited anywhere on the leaves, but usually near primary or secondary veins, on abaxial side.Eggs, laid singly or in small groups, were partially or deeply inserted into vegetal tissue, characterizing an endophytic oviposition.However, the operculum was always apparent just above foliar epidermis.
The mean total fecundity was 310.0 ± 19.40 eggs/ female, while the mean daily fecundity was 7.46 ± 0.302.The maximum number of eggs was 21/female during a 24h period, but the most frequent pattern (85 of 718 occasions) was one egg a day, followed by clusters of eight, nine and seven eggs, respectively.The oviposition period represented about 84.5% of the female total longevity (Tab.II).As the female age increased, the mean daily fecundity decreased (r = -0.7171;P < 0.0001) (Fig. 1).
Considering the total of 4,960 eggs laid, the mean number of fertile eggs per female was 286.25 ± 18.920.The egg viability was 92% and the mean embryonic period was 12.32 ± 0.274 days.

DISCUSSION
Despite the longer developmental period on H. chrysotrichus, the nymphal viability of T. americana was similar on both host plants.The higher amount of tricomes on the leaflets of H. chrysotrichus could explain the difference.Tricomes are usuallly efficient barriers to immature or small insects, hampering their movements or their feeding activities (Schoonhoven et al., 2005).This may result in a quantitative or qualitative nutritional deficit, reflecting on development, fecundity, and/or longevity (Parra, 1991).However, Ribeiro et al. (1994) demonstrated that the congeneric T. tecomae did not show preference by leaves of Tabebuia ochracea [= Handroanthus ochraceous (Cham.)Mattos] with less density of tricomes.Instead, that species preferred larger leaves.Other hypotheses for our results include differences on chemical composition and nutritional value among the host plant species.The influence of these factors should be also investigated for the T. americana -H.chrysotrichus and H. heptaphyllus interaction.
Oviposition pattern in T. americana shows common traits with T. cardui, which oviposits singly on the abaxial side of leaves, usually at either side of veins (Eguagie, 1974), and also to T. beesoni, whose eggs are laid in small groups, spread on foliar surface (Mathur, 1979).The mean embryonic period varies among Tingis species.In T. beesoni, this period lasts for two to six days (Mathur, 1979).For T. cardui, Eguagie (1974) reported a mean of 18 days, longer than T. ampliata (8.4 days) (Eguagie, 1972).However, comparisons should be done cautiously, as many of these studies were carried out under uncontrolled conditions and based on few specimens.The high egg viability showed by T. americana was similar to the obtained for T. ampliata at 25°C (93.7%) (Eguagie, 1972), while the mean total fecundity was higher to that recorded for T. cardui (65 eggs/female) (Eguagie, 1974).The oviposition period in T. americana represented about 84.5% of the total female lifespan.
The sex ratio in T. americana, on both hosts, was similar to the observed for T. cardui (Eguagie, 1974).The shortest longevity showed by unpaired individuals could be explained by the distinct treatment, because these lace bugs were provided with leaflets replaced every three or four days.Conversely, paired individuals were fed on fresh, daily replaced leaflets in order to obtain eggs.Further experiments standardizing this procedure could be carried out to test this hypothesis.
Based on pre-oviposition, egg and nymphal periods, under these conditions, we could obtain a new generation of T. americana in about every 28 days.Handroanthus heptaphyllus, in the state of Rio Grande do Sul, retains its foliage from October to April (Longhi, 1995).Supposedly, it would allow the development of about seven or eight generations of this lace bug.Further studies about T. americana on adult trees, under natural conditions, are needed to have a clearer picture of its biology, including the possible occurrence of diapause at this latitude, when leaves are not available.Another aspect we should take into account is that Tingis may be a paraphyletic genus, composed by unrelated species.This could explain the differences found regarding their biology.