Aphagy and vestigial stylets in first-instar nymphs of Aradidae (Hemiptera, Heteroptera).

Aradidae are known for their remarkably long stylets, coiled at rest in the anterior part of the head. However, previous reports indicated that at least some species lacked stylets during the first nymphal instar. A more detailed examination of Aradus betulae 1st-instar nymphs showed that their mandibular and maxillary stylets are abnormally short, not coiled, improperly interlocked, and clearly non-functional. The anteclypeus is relatively small and its internal diverticulum, which accommodates the stylet coil in the older stages, is vestigial. In contrast, the labium, labrum, food canal, and associated structures and muscles, including protractors and retractors of the stylets, are all normally developed. First-instar nymphs of Aradidae are the first known Heteroptera with non-functional mouthparts. To explain this phenomenon, a hypothesis is proposed which links previously unexplained records of non-feeding (but endowed with regular, functional mouthparts) 1st-instar nymphs of various pentatomomorphan families with the special role of that stage in acquiring microbial gut symbionts. A presumed loss of symbionts in the ancestors of Aradidae may have led to reduction of the now useless stylets in the first instar, which retained aphagy, despite a spectacular elongation of stylets in the older, feeding instars.

The mouthparts of the Aradidae (Insecta: Hemiptera: Heteroptera).

The flat bugs, Aradidae, have exceptionally long piercing-sucking stylets coiled up at rest in the anterior part of the head. Previous studies suggested that the majority of aradids can be divided into two groups by the direction of stylet coiling, clockwise or anticlockwise. Detailed reconstruction of the head skeleton and musculature from series of polished sections, examined in SEM, of epon-embedded specimens of three species has shown that these groups represent two disparate modifications of the head groundplan. In Aradus betulae (L.), the stylet coil is accommodated inside the greatly enlarged anteclypeus within an expansible membranous diverticulum of its epipharyngeal cuticle. In contrast, in Isodermus planus Erichson and Carventus brachypterus Kormilev, the coil lies freely underneath the anteclypeus between the extended maxillary lobes (in I. planus fused with the extended gular lobe). The intraclypeal coils occur in the subfamilies Aradinae, Calisiinae, and Chinamyersiinae and the subclypeal coils in Isoderminae, Carventinae, Mezirinae, Aneurinae, Prosympiestinae, and possibly in the closely related family Termitaphididae. Each method of stylet coiling is associated with a suite of divergently specialized structural traits, suggesting that the two groups have independently evolved from ancestors endowed with regular stylets. Functional mechanics of the coiled stylet bundles are discussed.

Pronymphs, hatching, and proboscis assembly in leafhoppers and froghoppers (Hemiptera, Cicadellidae and Aphrophoridae).

Pharate 1st instar nymphs enclosed in the embryonic cuticle, referred to as pronymphs, were studied in a froghopper Aphrophora pectoralis Mats. (Aphrophoridae) and the leafhoppers Oncopsis flavicollis (L.), Populicerus populi (L.), Alebra wahlbergi (Boh.), Igutettix oculatus (Lindb.), and Scenergates viridis (Vilb.) (Cicadellidae). The species vary in the relative length of the pronymphal antennae and details of sculpturing of the cephalic region. No egg bursting structures were observed, except small denticles on the crown region of S. viridis pronymphs. Rudimentary mandibular and maxillary stylets of a pronymph are external, short, tubular appendages containing tips of the corresponding nymphal stylets, whose more basal parts develop inside of the head. Casting off of the embryonic cuticle results in the nymphal stylets being passively pulled out and assuming a close-set parallel orientation. Once the sheaths of unsclerotized cuticle secreted by the peripodial epithelium and enveloping each developing stylet have been cast off with the exuviae, the bare stylets become squeezed and interlocked into a functional bundle. The roles of the maxillary plates, clypeus, labrum, and labium in the stylet bundle assembly are discussed. The process repeats after each molt.

Brochosomins and other novel proteins from brochosomes of leafhoppers (Insecta, Hemiptera, Cicadellidae).

Brochosomes (BS) are secretory granules resembling buckyballs, produced intracellularly in specialized glandular segments of the Malpighian tubules and forming superhydrophobic coatings on the integuments of leafhoppers (Hemiptera, Cicadellidae). Their composition is poorly known. Using a combination of SDS-PAGE, LC-MS/MS, next-generation sequencing (RNAseq) and bioinformatics we demonstrate that the major structural component of BS of the leafhopper Graphocephala fennahi Young is a novel family of 21-40-kDa secretory proteins, referred to herein as brochosomins (BSM), apparently cross-linked by disulfide bonds. At least 28 paralogous BSM were identified in a transcriptome assembly of this species, most of which were detected in BS. Multiple additional BS-associated proteins (BSAP), possibly loosely attached to the outer and inner surfaces of BS, were also identified; some of these were glycine-, tyrosine- and proline-rich. BSM and BSAP together accounted for half of the 100 most expressed transcripts in the Malpighian tubules of G. fennahi. Except for several minor BSAP possibly related to cyclases, BSM and BSAP had no homologs among known proteins, thus representing taxonomically restricted gene families (orphans). Searching in 50 whole-body transcriptome assemblies of Hemiptera found homologs of BSM in representatives of all five families of the superfamily Membracoidea (Cicadellidae, Myerslopiidae, Aetalionidae, Membracidae, and Melizoderidae), but not in other lineages. Among the identified proteins only BSM were shared in common between all 17 surveyed leafhoppers known to produce BS. Combined CHN elemental and aminoacid analyses estimated the total protein content of BS from the integument of G. fennahi to be 60-70%.

The Oncometopia orbona species group (Hemiptera, Cicadellidae, Proconiini).

The Oncometopia orbona group of species is defined based on the characteristic structure of the male aedeagus. A key to males and females of 9 species is included, among which 5 are newly described from the U.S.A., Mexico, and Central America: O. orbona (F.), O. nigricans (Walker), O. obtusa (F.), O. clarior (Walker) (= Proconia badia Walker syn. n., Proconia scutellata Walker synonymy reinstated), O. hamiltoni sp. n., O. acicularis sp. n., O. lepida sp. n., O. pelvica sp. n., and O. unispina sp. n. The systematics of the group presents some unusual cases. In particular, O. lepida sp. n. and O. pelvica sp. n. differ from one another in the shape of the female genital chamber sclerites, but not in the male genitalia. Oncometopia nigricans is almost identical in the genitalic characters of both sexes to O. hamiltoni sp. n., from which it is dissimilar externally, as confirmed by morphometric analysis, but is externally similar to O. orbona, from which it strongly differs in both the male and female genitalia. The rare intermediate forms from northern Florida and southern Georgia are interpreted as orbona x nigricans hybrids. Oncometopia clarior is treated as a complex of species which at present cannot be separated.

First karyotype data on the family Myerslopiidae (Hemiptera, Auchenorrhyncha, Cicadomorpha).

In the first cytogenetic study of the recently proposed family Myerslopiidae the male karyotype of Mapucheachilensis (Nielson, 1996) was analyzed using conventional chromosome staining, AgNOR- and C-bandings, and fluorescence in situ hybridization (FISH) with 18S rDNA and (TTAGG) n telomeric probes. A karyotype of 2n = 16 + XY, NOR on a medium-sized pair of autosomes, subterminal location of C-heterochromatin, and presence of (TTAGG) n telomeric sequence were determined. Additionally, the male internal reproductive system was studied.

Brochosomes protect leafhoppers (Insecta, Hemiptera, Cicadellidae) from sticky exudates.

Leafhoppers (Insecta, Hemiptera, Cicadellidae) actively coat their integuments with buckyball-shaped submicron proteinaceous secretory particles, called brochosomes. Here, we demonstrate that brochosomal coats, recently shown to be superhydrophobic, act as non-stick coatings and protect leafhoppers from contamination with their own sticky exudates--filtered plant sap. We exposed 137 wings of Alnetoidia alneti (Dahlbom), from half of which brochosomes were removed, to the rain of exudates under a colony of live A. alneti. One hundred and fifty-two droplets became stuck to the bared wings and only three to the intact wings. Inspection of the wings with a scanning electron microscope confirmed that the droplets that had hit the intact wings had rolled or bounced off the brochosomal coats. This is the first experimental study that tested a biological function of the brochosomal coats of leafhopper integuments. We argue that the production of brochosomes in leafhoppers and production of epidermal wax blooms in other sap-sucking hemipterans are alternative solutions, both serving to protect these insects from entrapment by their exudates.

Brochosomal coats turn leafhopper (Insecta, Hemiptera, Cicadellidae) integument to superhydrophobic state.

Leafhoppers (Insecta, Hemiptera, Cicadellidae) actively coat their integuments with brochosomes, hollow proteinaceous spheres of usually 200-700 nm in diameter, with honeycombed walls. The coats have been previously suggested to act as a water-repellent and anti-adhesive protective barrier against the insect's own exudates. We estimated their wettability through contact angle (CA) measurements of water, diiodomethane, ethylene glycol and ethanol on detached wings of the leafhoppers Alnetoidia alneti, Athysanus argentarius and Cicadella viridis. Intact brochosome-coated integuments were repellent to all test liquids, except ethanol, and exhibited superhydrophobicity, with the average water CAs of 165-172°, and the apparent surface free energy (SFE) estimates not exceeding 0.74 mN m(-1). By contrast, the integuments from which brochosomes were removed with a peeling technique using fluid polyvinylsiloxane displayed water CAs of only 103-129° and SFEs above 20 mN m(-1). Observations of water-sprayed wings in a cryo-scanning electron microscope confirmed that brochosomal coats prevented water from contacting the integument. Their superhydrophobic properties appear to result from fractal roughness, which dramatically reduces the area of contact with high-surface-tension liquids, including, presumably, leafhopper exudates.

Egg parasitoids (Hymenoptera: Mymaridae and Trichogrammatidae) of the gall-making leafhopper Scenergates viridis (Hemiptera: Cicadellidae) from Uzbekistan, with taxonomic notes on the Palaearctic species of Aphelinoidea.

A new species of the Aphelinoidea Girault (Hymenoptera: Trichogrammatidae), A. (Aphelinoidea) sariq Triapitsyn & Rakitov sp. n., is described from Uzbekistan. Both sexes were reared from eggs of the only known truly gall-making leafhopper, Scenergates viridis (Vilbaste), laid inside its galls on camelthorn, Alhagi maurorum Medikus; additional females were found dead inside the galls. Aphelinoidea sariq is the only known species of the nominate subgenus of Aphelinoidea whose body color is predominantly yellow. Taxonomic notes on other Palaearctic species of Aphelinoidea are provided; A. scythica Fursov, syn. n. is synonymized underA. (Aphelinoidea) turanica S. Trjapitzin. Another trichogrammatid, Par-acentrobia (Paracentrobia) sp., was reared from eggs of S. viridis in much smaller numbers. Also described from the same locality and host is Gonatocerus (Lymaenon) mitjaevi Triapitsyn & Rakitov sp. n. (Hymenoptera: Mymaridae).

Decoding of superimposed traces produced by direct sequencing of heterozygous indels.

Direct Sanger sequencing of a diploid template containing a heterozygous insertion or deletion results in a difficult-to-interpret mixed trace formed by two allelic traces superimposed onto each other. Existing computational methods for deconvolution of such traces require knowledge of a reference sequence or the availability of both direct and reverse mixed sequences of the same template. We describe a simple yet accurate method, which uses dynamic programming optimization to predict superimposed allelic sequences solely from a string of letters representing peaks within an individual mixed trace. We used the method to decode 104 human traces (mean length 294 bp) containing heterozygous indels 5 to 30 bp with a mean of 99.1% bases per allelic sequence reconstructed correctly and unambiguously. Simulations with artificial sequences have demonstrated that the method yields accurate reconstructions when (1) the allelic sequences forming the mixed trace are sufficiently similar, (2) the analyzed fragment is significantly longer than the indel, and (3) multiple indels, if present, are well-spaced. Because these conditions occur in most encountered DNA sequences, the method is widely applicable. It is available as a free Web application Indelligent at http://ctap.inhs.uiuc.edu/dmitriev/indel.asp.