Description of the female , egg and first instar nymph of the stick insect Paraphasma paulense ( Phasmatodea : Pseudophasmatidae ) from Southeast Brazil

The stick insect Paraphasmapaulense was described based on one male from the vicinity of Sao Paulo city, state of Sao Paulo, Brazil. Here we describe and illustrate the female, egg and first instar nymph of the species, including scanning electron micrographs of the egg. In addition, we give brief behavioural reports and present a few comments on the biology and morphology of P.paulense.


Introduction
Phasmatodea is one of the most poorly studied insect orders in Brazil.Most of the information on stick insects from the country is limited to original descriptions of species, mainly in the works by Gray (1835), Burmeister (1838), Westwood (1859), Bates (1865) and Kirby (1904), in the monograph by Brunner von Wattenwyl (1907) and Redtenbacher (1906Redtenbacher ( , 1908)), and in various papers by Toledo Piza (1936, 1937, 1938, 1944, among others).Recently, only a few studies have addressed Brazilian stick insects, such as: three broad taxonomic revisions that included some Brazilian taxa (Zompro 2001, 2004, Hennemann et al. 2016); the redescription of Tithonophasma tithonus (Gray, 1835) (Lima et al. 2013); and the descriptions of one new species in the genus Cladomorphus Gray, 1835 (Kumagai and Fonseca 2009), and another in Agrostia Redtenbacher, 1906(Heleodoro et al. 2017).Furthermore, few Brazilian species have the egg described in spite of the importance of egg morphology and egg-laying mechanism in Phasmatodea taxonomy and phylogenetics (Sellick 1997, Clark Sellick 1998, Zompro 2004, Goldberg et al. 2015).
Paraphasma Redtenbacher, 1906 is one of the 15 genera belonging to the monotribal subfamily Stratocleinae (Pseudophasmatidae).This subfamily is restricted to the Neotropics and is composed of small-to medium-sized stick insects, usually with body and legs of delicate aspect, antennae frequently surpassing body length, short tegmina, and both sexes with well-developed hind wings.
Paraphasma currently contains ten species of which eight are recorded from Brazil (Zompro 2004, Brock et al. 2017).The de-scription of the genus was based on characters of the femora, tibiae, tegmina and mesosternum (Redtenbacher 1906).No clear synapomorphy is known for Paraphasma, and it is defined by a vague set of diagnostic characters: profemur with anterodorsal carina distinctly raised (especially in female) and ventromedian carina acute; femora and tibiae of second and third legs with indistinct edges; tegmina short and scale-shaped, bearing distinct and often pointed shoulders; and variegated coloration in shades of yellow or brown (Redtenbacher 1906, Zompro 2004).
Paraphasma paulense Rehn, 1918 was described based on a single male collected in Cantareira, near the city of São Paulo, São Paulo state, Southeast Brazil.The author provided a considerably detailed description for the species and regarded it as related to Paraphasma quadratum (Bates, 1865) based on the relatively long tegmina of both species.However, taxonomic status of the species currently assigned to Paraphasma requires revision, and P. paulense will be soon removed from this genus as a result of an ongoing taxonomic study (Oskar Conle, pers. comm.).
Here we present a contribution to the knowledge of Brazilian stick insects, describing and illustrating the egg, first instar nymph and adult female of P. paulense, and also providing some comments on biological and morphological aspects of the species.

Material and methods
Collecting locality.-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.
Procedures.-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, sensu Moscona (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.
Examined material.-Paraphasmapaulense 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.

Morphology
Paraphasma paulense Rehn, 1918: 191-192 + pl. X figs. 3-5. Holotype male, Brazil, São Paulo state, Cantareira, ii.1914, E. Garbe.Deposited at the Academy of Natural Sciences of Philadelphia, type no.5347 (photograph examined in Phasmida Species File Online, Brock et al. 2017).Figs 1-15; Table 1).-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).

Egg (
Internal layers  and pharate first instar nymph inside egg : 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 , 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 .
First instar nymph .-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  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.
Adult female (26)(27)    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   Rehn, 1918 adult female. 19, 20. Habitus, dorsal and ventral views;21. Head;22, 23.End of abdomen, dorsal and ventral views.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.
Biology.-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.
Journal of orthoptera research 2017, 26(2) (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.

Morphological remarks
Apart from a study dealing specifically with color polymorphism in Anisomorpha buprestoides (Stoll, 1813) (Conle et al. 2009), and some very brief morphological observations on the first instar nymph of Tithonophasma tithonus (Lima et al. 2013), to the authors' knowledge the description of a newly hatched nymph in the family Pseudophasmatidae is herein presented for the first time.Even for Phasmatodea as a whole, morphological descriptions of first instar nymphs have been unusual or strongly brief.Nevertheless, more studies and comparisons of first instar nymphs of different stick insect species should allow a better understanding of structures of taxonomic relevance for the order.
With this background, we could only make general comparisons with first instar nymphs of species belonging to families other than Pseudophasmatidae.Remarkable differences, which may be of taxonomic importance for high-level taxonomic categories, were noted on: antennae length; body pubescence; shape of femora and tibiae; proportions between the sizes of body parts; coloration of body and appendages.
First instar nymph of Phryganistria bachmaensis (Ta and Hoang, 2004) (Phasmatidae: Clitumninae) differs from the one of P. paulense by the color of the whole body and body parts and (P.paulense parenthetical): antenna strongly shortened (long, almost body length), 8-segmented (9-segmented).First instar nymph of Phryganistria tamdaoensis Bresseel & Constant, 2014 was only briefly characterized (Bresseel and Constant 2014).It differs from P. paulense in general body color mostly green but with central portion of profemur and proximal part of meso-and metafemora reddish brown, and in the very short antenna.
Probably the most remarkable difference between the first instar nymph and the adult of P. paulense is the tergite of the median segment presenting a distinctly lower degree of fusion to the metanotum in the nymph (although a suture is still easily discernible separating both regions in the adult).Pictures available in Vallotto et al. (2016) indicate that this difference is also observed between first instar nymph and adult of O. kangi, suggesting that a progressive increase in the fusion between metanotum and median segment tergite throughout post-embryonic development may be a widespread condition in Phasmatodea.In P. paulense, the first instar nymph also differs from the adult in general coloration of body, proportions between size of body segments, and (adult parenthetical): ocelli absent (present); antennae 9-segmented, scape cylindrical, distal flagellomere as long as previous four together (antennae with more than 40 segments, scape dorsoventrally compressed, distal flagellomeres much shorter than the median ones); abdomen dorsally with four longitudinal rows of sparse, short setae (absent).
Paraphasma paulense presents weak sexual dimorphism, with both sexes exhibiting well-developed hind wings, and females being only slightly stouter and longer than males.Additionally, the pair of cephalic dorsomedian large spines (Fig. 21), present in most females, was absent in all males examined, as well as in the holotype (image available in Brock et al. 2017).
Based on the present study of the female of P. paulense, as well as on the examination of males also collected at the BBS (listed in the "Examined material" section), it is noticeable that this species presents some clear morphological differences in relation to the diagnostic characters described for Paraphasma (see Introduction).While the meso-and metafemora of Paraphasma typically lack strong carination, in P. paulense they are approximately trapezoidal in cross section, with the edges corresponding to distinct carinae.Additionally, the tegmina of P. paulense are considerably long, reaching the middle of median segment -in contrast to the characteristic scale-shaped tegmina of Paraphasma, which do not surpass the posterior margin of metanotum (see photographs of the lectotype and paralectotypes of the type species of the genus, Paraphasma marginale Redtenbacher, 1906, available in Brock et al. 2017).
Only a few brief descriptions and simple illustrations are available for eggs of Paraphasma, in Clark Sellick (1997) and Zompro (2004).Based on these studies and on the examination of eggs of P. marginale by the first author, it is clear that P. paulense strongly differs in egg morphology from other representatives of the genus.The most remarkable differences regard opercular angle (approximately 45º towards dorsal surface in P. paulense; not inclined in other Paraphasma representatives), size and shape of micropylar plate (larger and much more elongate in P. paulense) and shell ornamentation (small sinuous projections in P. paulense; strongly bristled in other Paraphasma representatives).Remarkably, P. paulense also differs in the egg-laying strategy, gluing the eggs to the substrate, in contrast to the more common strategy among Phasmatodea (including P. marginale -PICM, pers.obs.) of simply dropping the eggs to the ground.
With the aforementioned differences between P. paulense and other representatives of the genus, both in adult and egg morphology, we present evidence in favour of its future removal from Paraphasma.The transference of P. paulense to another genus will be the subject of a taxonomic study in preparation by Oskar Conle (pers. comm.).
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.