The collections were conducted in the Boraceia Biological Station (BBS) (23º39'14"S, 45º53'21"W), municipality of Salesópolis, São Paulo state, Southeast Brazil. The BBS is a research station maintained by the Museum of Zoology of University of São Paulo (MZSP) within the Serra do Mar State Park, which is the largest Atlantic Rain Forest fragment in Brazil, with 332,000 ha (Fundação Florestal 2017). The vegetation around the collecting area is predominantly composed of palm trees (especially Euterpe edulis Mart.), ferns and bamboos; in the soil abound heliconias and bromeliads.

Specimens were collected using a light trap by the white wall of the BBS building. Adults were maintained in a plastic container of 34×24 cm and 22 cm high, with water-soaked cotton and branches with leaves of at least three plant species at a time. Eggs were fixed in boiling water; nymphs were fixed as soon as they died. Both were preserved in 80% ethanol; adults were pinned and dried.

Identification of specimens was made by Fernando De Domenico (PhD at MZSP) and the first author, through the key available in Zompro (2004), and by comparison of the collected males with the original description (Rehn 1918) and with the image of the holotype in Brock et al. (2017).

Specimens and eggs were examined under a stereomicroscope Leica M125 and measured with a digital calliper or under stereomicroscope with ocular micrometer. Three eggs had their opercula detached and shells destroyed to allow examination and measurement of the operculum and internal micropylar plate. When different measures were obtained, we gave the highest and lowest value (Tables 1–2). Photographs were taken with a camera Canon 5D mark II coupled to a Canon macro lens MP-E 65mm 1–5×, with a camera Zeiss MRc5 coupled to a stereomicroscope Zeiss SteREO Discovery.V12, and with an iPhone 4S.

Scanning Electron Micrographs (SEM) of the egg were made at the MZSP laboratory with a Carl-Zeiss microscope LEO 440. The egg was cleaned manually with a brush in a water and detergent solution. For embryo scanning, the egg was submitted to a critical point dryer, the shell was ripped out and the embryo was gold covered for a first scanning; secondly the membranes enclosing the embryo were ripped out, the embryo was gold covered and scanned.

Descriptive terminology of the eggshell follows Clark Sellick (1997), but using shell, sensuMoscona (1950), instead of capsule. The elucidation and naming of eggshell (= exochorion) and internal layers (endochorion, vitelline membrane and exuvia) is based on Snodgrass (1935), Moscona (1950), Bedford (1970), Furneaux and Mackay (1976) and Gullan and Cranston (2010). Since the scanned embryo was fully formed and enclosed by a membrane identified as the cast embryonic exuvia, we follow Bedford (1970) and refer to it as a pharate first instar nymph.

Paraphasma paulense Rehn, 1918

All first instar nymphs were obtained through eggs laid by females collected in the BBS. The studied material was deposited at MZSP. The list follows below, with eggs and nymphs listed after their progenitor females.

BRAZIL, São Paulo state, Salesópolis municipality, Estação Biológica de Boraceia, 23º39'14"S, 45º53'21"W, 900m: one adult female (pinned with both wings opened) (MZSP 176), 20–21.iv.2012, four eggs (two for SEM), two egg shells (after nymph eclosion) and two first instar nymphs, F.F. Albertoni col.; one adult male (MZSP 178), 19–23.vi.2012, F.F. Albertoni col.; one adult female (pinned with left wings opened) (MZSP 175), 19–23.x.2012, two first instar nymph, two eggs and one egg shell (after nymph eclosion), F.F. Albertoni and M.A. Ulysséa col.; one adult male (pinned with left wings opened) (MZSP 177), 14–18.i.2013, F.F. Albertoni col.; one adult female (parasitized by two Forcipomiya paulista Falaschi, Albertoni and Fusari, 2014) (MZSP 375), 15–20.ii.2013, one egg, F.F. Albertoni col.; one adult female (MZSP 374) and three adult males (MZSP 174, 179, 180), 08–12.iii.2013, one egg, F.F. Albertoni col.

Description: Elongate, sub-cylindrical, almost round in cross-section, dorsal and lateral surfaces gently convex, ventral surface (where the egg is glued to substrate) and polar area slightly flat. Operculum oval and indistinctly convex, strongly inclined dorsally (opercular angle of approximately 45°), almost touching anterior margin of micropylar plate. Surfaces of shell and operculum covered with minute sinuous projections, often hooked (Figs 6–10). Micropylar plate elongate, approximately 3× longer than wide, extending for more than 1/3 of egg length, slightly widened in posterior region, bearing along its entire length elongate and sinuous ridges; ridges posteriorly culminating on a considerably large hump, occasionally bearing small projections similar to those covering shell. Micropylar cup positioned on posterior margin of micropylar plate, continuous to a short median line hardly visualized externally, but very conspicuous on the inner surface of shell. Internal micropylar plate open (Fig. 4).

Internal layers (Figs 10–15) and pharate first instar nymph inside egg (Figs 16–18): Pharate first instar nymph enclosed by three layers within shell (exochorion), identified as endochorion (outermost), vitelline membrane, and cast off embryonic exuvia (innermost). Endochorion smooth (Figs 10–11), attached to exochorion only in the micropylar stalk. Surface of vitelline membrane covered with minute spherical structures (Figs 14–15). Head integument of pharate first instar nymph bearing sparse small setae (Figs 12–14).

Figures 1–7. 

Paraphasma paulense Rehn, 1918 egg. 1–3. Lateral, dorsal and frontal (opercular) view, respectively; 4. Schematic illustration of internal micropylar plate; 5. Eggs detached from plastic container with pharate nymph inside; 6, 7. SEM of egg with open operculum; 6. Lateral view; 7. Dorsolateral view showing endochorion and internal face of operculum.

Figures 8–9. 

Paraphasma paulense Rehn, 1918 egg. SEM images: 8. Frontal view with operculum; 9. Detail of opercular socket and shell surface projections.

Figures 10–15. 

Paraphasma paulense Rehn, 1918 ultrastructure of egg and internal content. 10. Operculum opened and endochorion surface; (11, 12, 13, 14) pharate first instar nymph: 11. Enclosed in vitelline membrane; 12. With part of the head exposed evidencing nymph integument, embryonic exuvia and vitelline membrane; 13. Integument; 14. Detail of Figure 12; 15. Detail of vitelline membrane surface.

Description. Color: When alive, body yellow to pale brown, head laterally with longitudinal dark stripe, eyes similar to head color or slightly darker, with a longitudinal darker stripe medially. General coloration pale cream after fixed.

Head: Surface smooth but with few small and strongly sparse setae; length approximately 1.5× the width across eyes. Eyes prominent, ellipsoid, 0.4× as long as head. Ocelli absent. Antennae 9-segmented, as long as body, homogeneously covered with fine setae, when alive whitened around last ¼ apicad, distal flagellomere as long as previous four together and darker at apex; scape 1.5× longer than pedicel, cylindrical. Frons bearing several very small setae; clypeus, labrum and distal maxillary and labial palpomere with many tiny setae.

Thorax: Dorsally convex; ventrally flat or slightly concave, slightly rigged laterally. Surface smooth, but with several small and sparse setae. Pronotum narrower and 0.7× longer than head, with one longitudinal furrow medially and one transversally nearer anterior margin; openings of the pair of pronotal glands present, anteriorly. Mesonotum more than 2× longer than pronotum or metanotum. Metanotum slightly longer than median segment. Sternal region of thorax indistinct. Wing buds absent. Legs bearing relatively sparse, short and fine setae, but denser than on thorax and abdomen. Femora flat ventrally. Anterior femur slightly curved proximately, as in adult specimens. First tarsomere as long as or longer than remainder of tarsus. Hind leg the longest, almost as long as body. Arolium well developed, gibbous, almost as long as tarsal claws.

Abdomen: Ten visible tergites. Dorsally with four longitudinal rows of sparse short setae, laterally with several setae. Median segment as long as tergite II. Tergite IX the shortest. One pair of small cerci at apex, as long as tergite X, covered by very small setae.

Figures 16–18. 

Paraphasma paulense Rehn, 1918 habitus of first instar nymph inside the egg. 16. Lateral view; 17. Dorsal view; 18. Ventral view.

Figures 19–23. 

Paraphasma paulense Rehn, 1918 adult female. 19, 20. Habitus, dorsal and ventral views; 21. Head; 22, 23. End of abdomen, dorsal and ventral views.

Diagnosis: Relatively slender stick insects; body length ranging around 55–60 mm. Live specimens predominantly olivaceous, densely spotted in pale yellow or light brown, with a pale yellow dorsomedian line extending along head, pro- and mesothorax; body ventrally light-colored, with a dark median line extending along the body except subgenital plate; tegmina and costal region of hind wing dark olive or brownish green with venation vivid light green. Antennae long, nearly as long as body. Head usually bearing a pair of dorsomedian, large black spines, gently curved forward, absent in some specimens. Tegmina reaching intermediate region of median segment, bearing a pronounced, sharp spine; hind wings well-developed. Subgenital plate distinctly short, not reaching posterior margin of tergite IX.

Description. Color: General pattern dark olive or brown. Head, pro- and mesothorax with dorsal pattern of dark shades of brown, olive or grey, densely spotted in lighter color such as pale yellow or bright brown; dorsomedian longitudinal line in the same lighter color. Metanotum and abdominal terga shiny dark brown, except for three or four final tergites, usually in paler greenish brown. Ventral coloration variable, usually in shades of shiny buff; some individuals greenish on head, pro- and mesosternum; a dark brown ventromedian line extending longitudinally along the entire body except subgenital plate. Tegmina dark grey or olive with venation in vivid light green. Costal region of hind wing in the same general pattern, but with some sparse, light pale green regions; anal region translucent with venation brown. Femora and tibiae variegated, with the same colors as pro- and mesonotum; tarsi dark grey or brown. When alive, with the same general coloration, but more vivid, especially in the green and olivaceous areas.

Head: About 1.1× longer than wide, dorsoventrally compressed, sub-rectangular in dorsal view; surface smooth except for one pair of dorsomedian large black spines, gently curved forward, completely absent in some individuals. Eyes strongly prominent, 0.4× as long as head, pale orange to bright russet, with diffuse dark stains. Ocelli small, usually in the same color as eyes. Antennae long, almost reaching tip of abdomen, covered with fine setae; scape dorsoventrally compressed; pedicel and flagellomeres cylindrical; pedicel 0.7× as long as scape; basal and apical regions of flagellum with shortest flagellomeres, median region with longest ones.

Thorax: Dorsally convex; ventrally flat or slightly concave. Surface generally smooth, with few sparse granules close to the lateral margins of mesonotum. Pronotum sub-rectangular, approximately as long as, and slightly narrower than head, 1.5× longer than wide, with two discreet transversal furrows, one near the anterior margin and one medially; openings of the pair of defensive glands very conspicuous on anterolateral corners, crescent-shaped. Mesonotum as wide as, and 1.6–1.8× longer than pronotum, narrowed towards posterior margin; a pair of slightly elevated ridges originating in anterior margin and extending posteriorly and convergently until base of tegmina. Metanotum and median segment combined 1.6–1.9× longer than mesonotum, as wide as posterior end of mesonotum. Metanotum approximately 2.5× longer than median segment; metanotum and median segment smooth. Metaepisternum with a longitudinal ridge accompanying its ventral margin.

Wings: Tegmina elongate, almost 3× longer than wide, reaching intermediate region of median segment; anterior third bearing a pronounced, approximately conical spine, basally broad and apically acute. Hind wing well-developed and elongate, reaching or surpassing posterior margin of abdominal tergite VIII.

Legs: Profemur curved and compressed basally; anterodorsal and posteroventral carinae markedly raised, especially the former. Profemur and protibia bearing weakly raised ventromedian carina. Femur and tibia of mid- and hind legs approximately trapezoidal in cross section, with distinct carinae on the four edges, ventromedian carina absent, ventral surface slightly convex. All tibiae with conspicuous area apicalis. Each femur slightly longer than the respective tibia. Profemur 1.5–1.8× longer than mesofemur and slightly longer than metafemur. Pro- and metabasitarsus slightly longer than the following three tarsomeres combined, mesobasitarsus similar in length to the following three tarsomeres combined.

Abdomen: Dorsal and ventral surfaces smooth. Tergites II–V parallel-sided, of equal length and width. Tergites VI and VII narrowing towards posterior end; tergite VI of similar length in relation to preceding ones; tergite VII 0.8× as long as VI; tergite VIII slightly expanding towards posterior end; 0.6× as long as VII; tergites IX and X of similar length and 0.6× as long as VIII; tergite IX strongly narrowing towards posterior end; tergite X parallel-sided, posterior margin rounded in dorsal view and horseshoe-shaped in caudal view. Supra-anal plate not visible. Tergites VII–X inconspicuously carinated, degree of carination varying among individuals; tergites VII and VIII longitudinally bicarinated, IX and X single-carinated. Sternites II–VI with a dark, thin, longitudinal carina. Preopercular organ inconspicuous, as a discreet, round, bilobed protuberance on posterior region of sternite VII. Subgenital plate short, not reaching posterior margin of tergite IX; widening in anterior two-thirds, posterior region rounded and concave, apex weakly pointed. Gonapophyses prominent, projecting over posterior margin of subgenital plate and surpassing end of tergite IX. Cerci elongate and relatively stout, width constant from base to dull apex. Ventral surface of segments VIII–X, as well as structures originating from them, covered with minute setae.

No food plant could be determined, though many species were offered to adults and nymphs. Although plant species were not previously identified, among them were: Alchornea sp. (Euphorbiaceae), Eugenia uniflora, Psidium cattleianum (Myrtaceae), and two more unidentified species of Myrtaceae, Olyra sp. (Poaceae), Serjana sp. (Sapindaceae) and Schinus terebinthifolius (Anacardiaceae). The only plant species that one female fed on was Humiriastrum sp. (Humiriaceae), which caused it intestinal dysfunction after 12 hours – faeces were strongly soft and long, not typically broken into solid droplets.

Cryptic eggs were laid and strongly glued on the sticks of the plant branches given for feeding purpose. They took from 104 to 126 days to hatch (n=5). First instar nymphs did not feed on any plant offered and were fixed.

Male and female adults secreted a white substance through the prothoracic glands as a result of contact stimulation, as when held or strongly disturbed (Figs 27–28). Secretion was not observed in first instar nymphs. Adults could spray the substance backwards, in the direction of disturbance (Fig. 28), or let it ooze over the surface of the prothorax (Fig. 27).

Dossey et al. (2012) tested a spiroketal substance from the stick insect Asceles glaber Günther, 1938 (Diapheromeridae: Necrosciinae) as a repellent of the ant Solenopsis invicta Buren, 1972 and the mosquito Aedes aegypti Linnaeus, 1762. Tested spiroketal of the A. glaberi defensive spray demonstrated some efficiency only as a contact repellent. However, the quinolone from the defensive spray of Oreophoetes peruana (Saussure, 1868) (Diapheromeridae: Diapheromerinae) did repel Monomorium pharaonis (Linnaeus, 1758) ants, Schizocosa ceratiola (Gertsch & Wallace, 1935) spiders, Periplaneta americana (Linnaeus, 1758) cockroaches, and irritated the skin of frogs (Pseudacris crucifer (Wied-Neuwied, 1838)) (Eisner et al. 1997). Other stick insect species are mentioned as producing defensive substances that can irritate sensitive mucus membrane of the eyes, nose and mouth (Brutlag et al. 2011, Dossey et al. 2012).

Despite these records one female of P. paulense was collected with two females of the midge Forcipomyia paulista Falaschi, Albertoni & Fusari, 2014 attached to it, one on the prothorax and one on the base of right tegmen (Falaschi et al. 2014). The secreted substance of P. paulense was not chemically evaluated, but immediately after being released by the insect, it smelled fresh-sour and had a sharp odour somewhat reminding of camphor. When deeply sniffed (by FFA), the secretion caused a few sneezes or transitory nose irritation, although the smell was not unpleasant. This “experiment” was performed four times with the same result.

Figures 24–28. 

Paraphasma paulense Rehn, 1918 live specimens. 24, 25. First instar nymph, lateral and dorsal views; 26. Adult female resting; 27. Adult female after releasing the defensive substance, with secretion spread over the thorax; 28. Adult male after spraying the defensive substance backwards, with secretion accumulated on the tegmina spines and mesofemur.