Description of Immature Stages and Life Cycle of the Treehopper, Guayaquila projecta

Immature stages of the membracid Guayaquila projecta (Funkhouser) (Hemiptera: Cicadomorpha: Membracidae), collected in San Salvador de Jujuy, Argentina on Bougainvillea glabra Choisy (Caryophyllales: Nyctaginaceae), are described in detail based on specimens reared in the laboratory. Like other membracids, this species has five nymphal instars, not seven as previously reported. Morphological characters for identifying the different instars of G. projecta, determining the sex of later instars and distinguishing this species from other members of the Guayaquila pugnax group, are discussed. At 19 ±± 4°°C, RH 59 ±± 9%, and a 12:12 L:D photoperiod, the time required for development from egg to adult emergence was 73 ±± 5 days.


Introduction
The family Membracidae Rafinesque (Hemiptera: Cicadomorpha) includes about 3100 species classified into 9 subfamilies and 48 tribes worldwide (McKamey 1998, Cryan et al. 2000, Wallace and Deitz 2004. It is the most diverse in the Neotropical region. Dietrich and Deitz (1991) revised the tribe Aconophorini (Membracinae), species of which are distributed throughout the Neotropics. According to this revision the tribe has 51 species grouped into 3 genera: Guayaquila Goding, Calloconophora Dietrich, and Aconophora Fairmaire. The genus Guayaquila has 22 species that are included in 12 groups. One of them, the Pugnax group, includes G. pugnax (Germar), G. peruviensis (Dietrich), and G. projecta (Funkhouser). The last is the only species recorded from Argentina, in the provinces of Catamarca, Salta, and Tucumán.
Previously Marcus (1950) reared nymphs of G. projecta from Bolivia and reported seven nymphal instars, a departure from the usual five instars in other Membracidae. The aim of this work is to investigate the life cycle of G. projecta in more detail and to analyze the morphological characteristics useful for differentiating its different stages and instars.

Life cycle
Egg-layings were obtained under laboratory conditions. Ten newborn nymphs were put on each of ten freshly cut branches of B. glabra and then placed in breeding chambers at 19 ± 4 º C, RH 59 ± 9%, and 12:12 L:D photoperiod. The branches were periodically renewed. The duration of different nymphal instars and adult longevity were recorded.

Morphology
The morphological characteristics of eggs dissected from the ovaries of gravid females, eggs recently laid, and mature eggs were studied. Morphological characteristics of the first-instar nymph were analyzed in detail, and only the observed changes were added to the descriptions of later instars. Color was described based on examination of living specimens anesthetized with sulfuric ether. Preparations were measured microscopically using a stereo-microscope. Reported lengths were measured on the midline and reported width is the maximum for a given structure. Chaetotaxy is reported for the left half of the body.
Reported measurements are averages for 10 individuals. All the dimensions are given in millimeters. A camera lucida on a Reichert microscope was used to make the drawings.
To corroborate the number of nymphal instars linear regression analyses were carried out for nine measurements: head length and width, rostrum length, interocular distance, ocular diameter, meso and metathorax length, and tibia III maximum width and length. Also, the relationship between the rostrum length and the total length of the body and the growth factor for those characters was calculated as the difference between the average value of an instar and that of the previous one.
The G. projecta studied specimens were kept as part of the collection at the Instituto de Biología de la Altura, and the B. glabra material was kept at the herbarium of the School of Agrarian Sciences-UNJu (National University of Jujuy) under number 2469 (JUA).

Life cycle duration
Individuals required an average of 75 days to go from egg to adult, with each instar requiring 7-10 days on average ( Table 1). The longevity of the adults varied to a maximum of 60 days.
Description of the immature stages (Figure 1) Egg. The mature ovarian eggs were 0.95 ± 0.03 mm long and 0.35 ± 0.03 mm wide. When newly deposited they were white and 0.99 ± 0.05 mm long and 0.38 ± 0.02 mm wide. They were subcylindrical with the basal end sharp and the distal end rounded ( Figure  1.1). The chorion was translucent and smooth. The average egg mass length was 8 mm and its width was 3.5 mm. The average number of eggs laid was 45, with a range of 24 to 72.  of sensory elements formed by two setae, a middle one and a sensory cone next to base of flagellum and 5 ring shaped rows of sensory receptors. Flagellum tapered with apparent segmentation formed by various ring-shaped sensory receptors; 1.7 times length of the first and second antennules together.
Thorax (  Journal of Insect Science | www.insectscience.org 5 First two segments weakly sclerotised. Segments 3 to 8 with similar chaetotaxy: 3 dorsal, 1 lateral and 2 ventro-lateral setae (Figure 2 I dorsal-ventral). Length of segments subequal except 9 th which is 1.7 times longer than previous segment and with three pairs of setae, dorsal, lateral and ventral, each pair with one long and one short seta. 10 th uromere cylindrical; length 1.4 times width, retracted within 9 th segment (telescopic), with eight-seta posterior ring. Spiracles present on 3 rd to 8 th abdominal segments.
Second instar Nymph (Figure 1 C). Color pattern of the first instar nymph maintained, with ventral portion of abdomen reddish orange on sides and middle part yellow.
General proportions of body maintained. Rostrum extended to posterior extreme of fourth abdominal segment. Coxae with 1 to 3 long setae.
Chaetotaxy as in first instar.
Third instar nymph (Figure 1 D). Thorax lateral margins with white wax band that extends along metathorax posterior margin to sublateral region and dorsally towards head, reaching prothorax posterior edge. Adjacent area to midline lighter forming yellowish band. Legs with body of femur, tibia proximal extreme, and tarsi dark brown. Distal part of femur and tibiae yellowish.
Rostrum not extended beyond third abdominal segment.
Pronotum with anterior horn present as button shaped enlargement at anterior third. Posterior margin sharpened into posterior process, extended to middle of mesonotum. Mesonotum with lateral extensions (wing pads).
Coxae with strong setae on external lateral surface.
General pilosity better developed, obscuring original chaetotaxy Fourth instar nymph (Figure 1 E). Wing pad apices waxen white. Rest of body color similar to that of third instar.
Body with general pilosity abundant on prontum and regularly distributed. Horn, in dorsal view, reaching pronotum anterior edge. Posterior process covering entire mesonotum.
Wing pads extended to second abdominal segment.
Genital plates evident between the eighth and ninth abdominal segments.
Fifth instar nymph (Figure 1 F). Horn brownish. Posterior pronotal process with reddish extreme extended to first abdominal segment.
Horn length sexually dimorphic, shorter in males and not exceeding head anterior edge.
Wing pads extended to fifth abdominal segment. Wing veins evident. The tibial spines of third pair of legs evident. Genital plates larger but more complex structures not evident

Morphometric relationships and growth factor.
Linear regressions show a highly significant adjustment for a linear function in all analyzed measurements. The adjusted R 2 values are shown in Table 2 in which an average growth factor is also evident. These values were highly stable throughout the nymphal development of G. projecta.

Discussion
Morphological characters distinguishing the first two instars of G. projecta are: the dorsal color, the general length of the body, the placement of the apical rostrum segment in relation to the body, and the chaetotaxy. Beginning with the third stage, the relative development of the wing pads and pronotum become more important. The length of the pronotum increases progressively because of the formation of the horn and the posterior process that, in the adults, may cover the whole abdomen.
In the fifth instar nymph, the sexes may be distinguished by the horn length.
The brood obtained under controlled laboratory conditions has clarified details of the life cycle: 73 ± 5 days (egg-adult) duration, 44 % of which corresponds to the egg state and, as in other membracids, five nymphal instars. The results of this study contrast with those of Marcus (1950), who reported seven instars for G. projecta (=Aconophora projecta, Funkhouser) based on differences in size.
The use of linear regression analysis helped to corroborate the five instars. Other studies of the immature stages of Membracoidea have used similar analyses. To identify the nymphal instars of Sextius virescens (Fairmaire) (Cicadellidae), Kitching (1974) used the proportion of the head width to the total width of the nymph; Page (1979) used the growth factor of Austroasca viridigrisea (Paoli) (Cicadellidae) to characterize this species in relation to the size increase of the cephalic capsule. Both kinds of analysis were carried out for G. projecta and the growth factor was very similar between instars in many of the analyzed characteristics such as rostrum length/total length, rostrum length, and head width. Thus, as also seems to be true for many other Hemiptera (Page 1979;Segnini and Montagne 1986;Nixon and Thompson 1987;Klingenberg and Zimmerman 1992;Viscarret and Botto,1997;Rodrigues et al 2005;Setamou and Jones 2005;Rodriguez and Peck 2007), nymphal growth in G. projecta appears to obey Dyar's rule which was originally proposed for Lepidoptera (Page 1979).
Guayaquila projecta is found in Bolivia in Cochabamba (Marcus 1950), La Paz, Sorata; in Brazil in Paraná; in Colombia in La Cumboc; in Paraguay in San Pedro; and in Argentina in Tucumán, Salta, and Catamarca (Dietrich and Deitz 1991). In this study the distribution is extended to the province of Jujuy (Argentina). Dietrich and Deitz (1991) record Mimosa lorentzii (Fabaceae) as a host plant and this study adds a new host record: Bougainvillea glabra.