Two new cryptic species within Periphyllus koelreuteriae (Hemiptera: Aphididae) feeding on Koelreuteria (Sapindaceae) in China.

Based on integrative taxonomic practices by incorporating morphological, genetic, and ecological data, two new aphid species, Periphyllus blackmani Li Huang sp. nov. and Periphyllus guangxuei Li Huang sp. nov., are proposed from the known species P. koelreuteriae. Apterous and alate viviparous females of the new species are described and differences with their most similar species P. koelreuteriae are presented. These aphids of P. koelreuteriae species complex feed exclusively on Koelreuteria (Sapindaceae), a native tree group and commonly cultivated as ornamental plants in China. A key to the Chinese species of P. koelreuteriae species complex is provided.

Description of a new Myzocallis (Hemiptera Aphididae) living on Valonia oak in Southern Italy with DNA barcoding accounts on allied species-group.

The aphid Myzocallis macrolepidis sp. n. is described from the Apulia region, Italy, where it was collected on Quercus macrolepis. The new taxon has a pale-yellow colour in life as alate viviparous females and paler nymphs. Its life cycle remains unknown at present, as sexual morphs were not yet collected; nevertheless, being the aphid host plant a deciduous oak species, very probably the insect performs a monoecious holocycle. Morphologically most similar aphids are M. occidentalis and M. schreiberi from which the new species can be mainly distinguished by the different ratio of last rostral joint to second hind tarsomer and a few other morphological and ecological features. The molecular analysis also confirms the genetic difference of the new taxon from the other allied congeneric species, and molecular differences are briefly discussed.

Aphis mirabilis sp. n. (Hemiptera, Aphididae, Aphidina), a South American species resembling A. craccivora, and further data on A. tropaeoli.

We examined 1367 apterous viviparae from 151 samples to study Aphis craccivora, A. tropaeoli and a third, possibly new species in Argentina, resembling both. Principal component analysis (PCA), followed by hierarchical clustering of principal components (HCPC), revealed clusters based on 33 morphological characters of 206 selected specimens. The delimitation of possible taxonomic entities was assessed by logistic regression. PCA confirmed that A. tropaeoli is a valid species. HCPC revealed the existence of a new species distinct from A. craccivora. Recently published data on the variability of A. craccivora, mainly regarding Asian populations, are presented and discussed; the general appearance of the sexuals of this species is also presented. The new species is described with the name Aphis mirabilis Nieto Nafra and Ortego sp. n., based on apterous and alate viviparae, oviparae, and males collected in Mendoza and Neuqun (Argentina) on Adesmia volckmannii (Fabaceae), Atriplex nummularia and At. lampa (Amaranthaceae), Bougainvillea spinosa (Nyctaginaceae), and Robinia pseudoacacia (Fabaceae). This new species has also been collected on plants of another 18 species from 8 additional families, although these specimens are not included in the type series. Features to distinguish A. mirabilis Nieto Nafra and Ortego sp. n. from A. craccivora are presented together with a logistic regression model. The males of A. tropaeoli are described. The list of host plants of A. craccivora in Argentina is updated.

Polyphenism in Antennal Sensilla Among Different Adult Morphs of Nonhost-Alternating, Holocyclic Sitobion avenae (Hemiptera: Aphididae).

Aphids, mainly distributed in temperate zones, exhibit seasonal generation-alternating phenomena. Across the life cycle, different morphs are produced. Sitobion avenae (Fabricius 1775) is a major pest of wheat worldwide. To elucidate olfactory perception of morph-specific behavior across their life cycle, we investigated antennal sensilla among seven morphs using scanning electron microscopy. Trichoid, placoid, coeloconic, and campaniform sensilla were identified. Trichoid sensilla, big multiporous placoid sensilla (primary rhinarium), a group of sensilla (primary rhinaria), and campaniform sensilla showed similar distribution and resemblance among morphs, whereas small multiporous placoid sensilla (secondary rhinaria) exhibited obvious differences. Compared to apterous morphs, alate morphs possessed a greater abundance of secondary rhinaria, with the greatest found in males on antennal segments III-V. Alate virginoparae and alate sexuparae ranged from six to fourteen rhinaria on antennal segment III. Fundatrices, apterous virginoparae and apterous sexuparae only had one or two secondary rhinaria on antennal segment III while they disappeared in oviparae. Secondary rhinaria, lying in a cuticle cavity, are convex or concave in their central part. In males, both forms were present, with a greater proportion of convex form than that of the concave form. Fundatrices and virginoparae had the convex form while sexuparae had the concave form. Polyphenism of secondary rhinaria might suggest their association with the olfactory functions of morph-specific behavior. These results have improved our understanding of the adaptive evolution of the antennal sensilla in nonhost-alternating, holocyclic aphids.

Wing plasticity and associated gene expression varies across the pea aphid biotype complex.

Developmental phenotypic plasticity is a widespread phenomenon that allows organisms to produce different adult phenotypes in response to different environments. Investigating the molecular mechanisms underlying plasticity has the potential to reveal the precise changes that lead to the evolution of plasticity as a phenotype. Here, we study wing plasticity in multiple host-plant adapted populations of pea aphids as a model for understanding adaptation to different environments within a single species. We describe the wing plasticity response of different "biotypes" to a crowded environment and find differences within as well as among biotypes. We then use transcriptome profiling to compare a highly plastic pea aphid genotype to one that shows no plasticity and find that the latter exhibits no gene expression differences between environments. We conclude that the loss of plasticity has been accompanied by a loss of differential gene expression and therefore that genetic assimilation has occurred. Our gene expression results generalize previous studies that have shown a correlation between plasticity in morphology and gene expression.

Trophobiotic organ and perianal structures in aphid genus Stomaphis WALKER (Aphididae, Lachninae).

Stomaphis Walker is an obligatorily myrmecophilous, tree-trunk feeding aphid genus, having many peculiar features in its morphology. One poorly studied trait is its perianal anatomy, with suspected existence of trophobiotic organ and intriguing tubercles near abdominal segment VIII. Tubercles on rear end of abdomen in Stomaphis were considered to be marginal tubercles, structure typical of aphids. Only after their investigation under scanning electron microscope they revealed to possess a very rough surface, consisting of many protuberant, sclerotic scales with finger-like processes. Analysis and comparison with other aphids suggests, that these structures - perianal tubercles - may be considered remnants of abdominal sternite IX, atrophied in aphids. Due to their similarity to the surface of cauda of non myrmecophilous aphids, they are suspected to serve as sort of auxiliary caudas, protecting from sticky secretions of aphids e. g. honeydew or mucus while giving birth or ovipositing in confined space. A very well developed trophobiotic organ in this genus has been shown to exist for the first time.

Wax glands of the horned gall aphid, Schlechtendalia chinensis, at different stages.

The horned gall aphid, Schlechtendalia chinensis, inhabits the productive species of Chinese gallnuts, which have economic value. Aphid wax glands are crucial for the survival of the insects, since the secreted waterproofing wax is important to protect the aphids from predators, pathogens and honeydew contamination. In this study, we investigated the structure of wax glands and their role in different aphid stages using light and electron microscopy. Our results show that aphids of all stages except the newly hatched fundatrix possess six parallel dorsal lines and have a total of 56 wax gland plates. Although no wax glands occur on the dorsum of the newly hatched fundatrix (first instar), the glands do appear once a fundatrix enters the second instar. The wax gland plate is composed of 2-22 polygonal depressions, each of which corresponds to a secretory cell covered by cuticle. The wax glands of this aphid belong to the class 1 glands, which are formed by epidermal secretory cells. The structure of the wax glands varies in the different stages and these changes may be adaptive to the changeable microenvironments in which the aphids live.

A large genomic insertion containing a duplicated follistatin gene is linked to the pea aphid male wing dimorphism.

Wing dimorphisms have long served as models for examining the ecological and evolutionary tradeoffs associated with alternative phenotypes. Here, we investigated the genetic cause of the pea aphid (Acyrthosiphon pisum) male wing dimorphism, wherein males exhibit one of two morphologies that differ in correlated traits that include the presence or absence of wings. We mapped this trait difference to a single genomic region and, using third generation, long-read sequencing, we identified a 120 kb insertion in the wingless allele. This insertion includes a duplicated follistatin gene, which is a strong candidate gene in the minimal mapped interval to cause the dimorphism. We found that both alleles were present prior to pea aphid biotype lineage diversification, we estimated that the insertion occurred millions of years ago, and we propose that both alleles have been maintained in the species, likely due to balancing selection.

Neuroanatomical correlates of mobility: Sensory brain centres are bigger in winged than in wingless parthenogenetic pea aphid females.

Many aphid species reproduce parthenogenetically throughout most of the year, with individuals having identical genomes. Nevertheless, aphid clones display a marked polyphenism with associated behavioural differences. Pea aphids (Acyrthosiphon pisum), when crowded, produce winged individuals, which have a larger dispersal range than wingless individuals. We examined here if brain structures linked to primary sensory processing and high-order motor control change in size as a function of wing polyphenism. Using micro-computing tomography (micro-CT) scans and immunocytochemical staining with anti-synapsin antibody, we reconstructed primary visual (optic lobes) and olfactory (antennal lobes) neuropils, together with the central body of winged and wingless parthenogenetic females of A. pisum for volume measurements. Absolute neuropil volumes were generally bigger in anti-synapsin labelled brains compared to micro-CT scans. This is potentially due to differences in rearing conditions of the used aphids. Independent of the method used, however, winged females consistently had larger antennal lobes and optic lobes than wingless females in spite of a larger overall body size of wingless compared to winged females. The volume of the central body, on the other hand was not significantly different between the two morphs. The larger primary sensory centres in winged aphids might thus provide the neuronal substrate for processing different environmental information due to the increased mobility during flight.

Laser surgery reveals the biomechanical and chemical signaling functions of aphid siphunculi (cornicles).

Aphids are an attractive food source to many predators and parasitoids because of their small size, soft bodies and slow movement. To combat predation, aphids evolved both behavioral and chemical defensive mechanisms that are operated via siphunculi (cornicles), differently developed structures that more or less extend from their abdomen. Although both direct and indirect linkages between siphunculi and their defensive mechanisms have been explored, their ultimate effects on aphid fitness are still broadly debated. To explicitly test the influence of siphunculi on brown citrus aphid, Aphis (Toxoptera) citricida (Kirkaldy), fitness, we razor-cut and laser-sealed the siphunculi. Siphunculi removal resulted in two distinct behavior modifications, (false aggregation and increased drop-off rates) that led to decreased survival and the loss of the ability to right themselves from an inverted position. These results together indicate that siphunculi play an important role in survival, and removal of these organs will have negative effect on aphid fitness. Furthermore, results suggested that released alarm pheromone may play an important role in communication among aphid clone-mate, and omitting it results in miscommunication and competition among clonemates. These findings will help in better understanding the aphid biology.

DNA barcoding and a precise morphological comparison revealed a cryptic species in the Nippolachnus piri complex (Hemiptera: Aphididae: Lachninae).

Nippolachnus is a small Palaearctic-Oriental genus of very characteristic aphids that live on the leaves of woody Rosaceae. One species, N. piri, has hitherto been regarded to be widely distributed and relatively polyphagous. Members of this genus are considered to be easy to recognize due to the absence of the ocular tubercle and triommatidia on the head. We conducted research on the morphology and generic characters of Nippolachnus piri complex using scanning electron microscopy (for the first time) and DNA barcoding. We analyzed N. piri populations on Pyrus and other plants (Eriobotrya, Rhaphiolepis and Sorbus) in Japan and the Republic of Korea. Specifically, a high genetic divergence value was found between the N. piri populations associated with different host plants. SEM investigation of the head capsule revealed that a triommatidium is present under the compound eye, despite their lack of an ocular tubercle. We propose Nippolachnus micromeli Shinji, 1924 stat. nov. as a cryptic species in the N. piri complex based on a morphological comparison, DNA barcoding and different host-plant associations. Illustrations and descriptions of studied species are given. Morphological keys to the apterae and alatae of all known species of the genus Nippolachnus are also provided.

Extensive Differential Splicing Underlies Phenotypically Plastic Aphid Morphs.

Phenotypic plasticity results in a diversity of phenotypes from a single genotype in response to environmental cues. To understand the molecular basis of phenotypic plasticity, studies have focused on differential gene expression levels between environmentally determined phenotypes. The extent of alternative splicing differences among environmentally determined phenotypes has largely been understudied. Here, we study alternative splicing differences among plastically produced morphs of the pea aphid using RNA-sequence data. Pea aphids express two separate polyphenisms (plasticity with discrete phenotypes): a wing polyphenism consisting of winged and wingless females and a reproduction polyphenism consisting of asexual and sexual females. We find that pea aphids alternatively splice 34% of their genes, a high percentage for invertebrates. We also find that there is extensive use of differential spliced events between genetically identical, polyphenic females. These differentially spliced events are enriched for exon skipping and mutually exclusive exon events that maintain the open reading frame, suggesting that polyphenic morphs use alternative splicing to produce phenotype-biased proteins. Many genes that are differentially spliced between polyphenic morphs have putative functions associated with their respective phenotypes. We find that the majority of differentially spliced genes is not differentially expressed genes. Our results provide a rich candidate gene list for future functional studies that would not have been previously considered based solely on gene expression studies, such as ensconsin in the reproductive polyphenism, and CAKI in the wing polyphenism. Overall, this study suggests an important role for alternative splicing in the expression of environmentally determined phenotypes.

A comparative morphological revision of the aphid genus Myzaphis van der Goot, 1913 (Insecta: Hemiptera: Aphididae) revealed a new genus and three new species.

The aphid genus Myzaphis van der Goot, 1913 from the tribe Macrosiphini is revised to include eight species. Apterous and alate viviparous females, known fundatrices and known sexual morphs (oviparous females and males) of Myzaphis bucktoni, M. juchnevitschae, M. rosarum, M. tianshanica and M. turanica are re-described and illustrated. Lectotype and paralectotypes of Myzaphis bucktoni and M. turanica are designated. The status of M. komatsubarae nomen dubium is discussed. Myzaphis avariolosa is regarded as a species belonging to the genus Ericaphis. Three new species: M. oezdemirae Kanturski & Barjadze sp. nov., M. tuatayae Kanturski & Barjadze sp. nov. from Turkey and M. rezwanii Kanturski & Barjadze sp. nov. from Iran are described and illustrated. Myzaphis bucktoni is recorded from Portugal for the first time. Diagnosis of the genus Myzaphis van der Goot, 1913 is redefined and a new genus Richardsaphis Kanturski & Barjadze gen. nov. is erected with the type species R. canadensis (Richards) comb. nov. Richardsaphis is for the first time recorded from the USA and hitherto unknown oviparous female and alate male are described and illustrated. Original keys to species of the genus Myzaphis and aphid genera of the tribe Macrosiphini with 2-2-2 first tarsal chaetotaxy are also provided.

New genus and species of the extinct aphid family Szelegiewicziidae and their implications for aphid evolution.

Recently, we are witnessing an increased appreciation for the importance of the fossil record in phylogenetics and testing various evolutionary hypotheses. However, this approach brings many challenges, especially for such a complex group as aphids and requires a thorough morphological analysis of the extinct groups. The extinct aphid family Szelegiewicziidae is supposed to be one of the oviparous lineages in aphid evolution. New material from the rock fossil deposits of Shar Teg (Upper Jurassic of Mongolia), Baissa (Lower Cretaceous of Siberia-Russia), and Burmese amber (Upper Cretaceous of Myanmar) allowed us to undertake a more detailed examination of the morphological features and carry out an analysis of the taxonomical composition and evolution of the family. This led us to the conclusion that evolution of the body plan and wing structure was similar in different, often not closely related groups, probably as a result of convergence. Additionally, we present a description of a new genus and two species (Tinaphis mongolica Zyla &Wegierek, sp. nov., and Feroorbis burmensis Wegierek & Huang, gen. et sp. nov.) that belong to this family.

Geographical variation in morphology of Chaetosiphella stipae stipae Hille Ris Lambers, 1947 (Hemiptera: Aphididae: Chaitophorinae).

Chaetosiphella stipae stipae is a xerothermophilous aphid, associated with Palaearctic temperate steppe zones or dry mountain valleys, where there are grasses from the genus Stipa. Its geographical distribution shows several populations that are spread from Spain, across Europe and Asia Minor, to Mongolia and China. Geographical variation in chaetotaxy and other morphological features were the basis to consider whether individuals from different populations are still the same species. Moreover, using Ch. stipae stipae and Stipa species occurrences, as well as climatic variables, we predict potential geographical distributions of the aphid and its steppe habitat. Additionally, for Stipa species we projected current climatic conditions under four climate change scenarios for 2050 and 2070. While highly variable, our results of morphometric analysis demonstrates that all Ch. stipae stipae populations are one very variable subspecies. And in view of predicted climate change, we expect reduction of Stipa grasslands. The disappearance of these ecosystems could result in stronger separation of the East-European and Asian steppes as well as European 'warm-stage' refuges. Therefore, the geographic morphological variability that we see today in the aphid subspecies Ch. stipae stipae may in the future lead to speciation and creation of separate subspecies or species.

Aphis species (Hemiptera, Aphididae) living on Mulinum (Apiaceae) in South America, with a description of a new species.

Aphis species living on the South American native genus Mulinum are studied. Aphis vurilocensis Nieto Nafría, Brown and López Ciruelos, sp. n. is described from apterous viviparous females. Alate viviparous females, oviparous females and winged males of Aphis roberti are described. Knowledge of intraspecific variability of apterous viviparous females of A. martinezi, A. paravanoi and A. roberti is developed. An identification key of apterous viviparous females of Aphis species living on Mulinum is presented.

Description of the sexuales of Myzodium modestum (Hottes) (Hemiptera: Aphididae) discovered in the Swiss Alps.

The sexuales (apterous oviparous female and alate male) of Myzodium modestum (Hottes) are described for the first time from specimens captured in the Swiss Alps. This is also the first record of this species in Switzerland. This is the first evidence that an aphid may be able to complete its life cycle on mosses.

Hybothoracaphis, a new genus of Nipponaphidini (Hemiptera, Aphididae, Hormaphidinae) from China and its phylogenetic placement based on multiple genes.

Hybothoracaphis gen. nov. with one new species Hybothoracaphis laevigata sp. nov. on Quercus aquifolioides from Tibet, China is described and illustrated in the aphid tribe Nipponaphidini. The phylogenetic position of the new genus within Nipponaphidini is investigated using multiple genes, and a key to Hybothoracaphis and allied genera is provided.

Differential expression of genes in the alate and apterous morphs of the brown citrus aphid, Toxoptera citricida.

Winged and wingless morphs in insects represent a trade-off between dispersal ability and reproduction. We studied key genes associated with apterous and alate morphs in Toxoptera citricida (Kirkaldy) using RNAseq, digital gene expression (DGE) profiling, and RNA interference. The de novo assembly of the transcriptome was obtained through Illumina short-read sequencing technology. A total of 44,199 unigenes were generated and 27,640 were annotated. The transcriptomic differences between alate and apterous adults indicated that 279 unigenes were highly expressed in alate adults, whereas 5,470 were expressed at low levels. Expression patterns of the top 10 highly expressed genes in alate adults agreed with wing bud development trends. Silencing of the lipid synthesis and degradation gene (3-ketoacyl-CoA thiolase, mitochondrial-like) and glycogen genes (Phosphoenolpyruvate carboxykinase [GTP]-like and Glycogen phosphorylase-like isoform 2) resulted in underdeveloped wings. This suggests that both lipid and glycogen metabolism provide energy for aphid wing development. The large number of sequences and expression data produced from the transcriptome and DGE sequencing, respectively, increases our understanding of wing development mechanisms.

Minor Coat and Heat Shock Proteins Are Involved in the Binding of Citrus Tristeza Virus to the Foregut of Its Aphid Vector, Toxoptera citricida.

Vector transmission is a critical stage in the viral life cycle, yet for most plant viruses how they interact with their vector is unknown or is explained by analogy with previously described relatives. Here we examined the mechanism underlying the transmission of citrus tristeza virus (CTV) by its aphid vector, Toxoptera citricida, with the objective of identifying what virus-encoded proteins it uses to interact with the vector. Using fluorescently labeled virions, we demonstrated that CTV binds specifically to the lining of the cibarium of the aphid. Through in vitro competitive binding assays between fluorescent virions and free viral proteins, we determined that the minor coat protein is involved in vector interaction. We also found that the presence of two heat shock-like proteins, p61 and p65, reduces virion binding in vitro Additionally, treating the dissected mouthparts with proteases did not affect the binding of CTV virions. In contrast, chitinase treatment reduced CTV binding to the foregut. Finally, competition with glucose, N-acetyl-ß-d-glucosamine, chitobiose, and chitotriose reduced the binding. These findings together suggest that CTV binds to the sugar moieties of the cuticular surface of the aphid cibarium, and the binding involves the concerted activity of three virus-encoded proteins. IMPORTANCE: Limited information is known about the specific interactions between citrus tristeza virus and its aphid vectors. These interactions are important for the process of successful transmission. In this study, we localized the CTV retention site as the cibarium of the aphid foregut. Moreover, we demonstrated that the nature of these interactions is protein-carbohydrate binding. The viral proteins, including the minor coat protein and two heat shock proteins, bind to sugar moieties on the surface of the foregut. These findings will help in understanding the transmission mechanism of CTV by the aphid vector and may help in developing control strategies which interfere with the CTV binding to its insect vector to block the transmission.