Microbiome and plant cell transformation trigger insect gall induction in cassava.

Several specialised insects can manipulate normal plant development to induce a highly organised structure known as a gall, which represents one of the most complex interactions between insects and plants. Thus far, the mechanism for insect-induced plant galls has remained elusive. To study the induction mechanism of insect galls, we selected the gall induced by Iatrophobia brasiliensis (Diptera: Cecidomyiidae) in cassava (Euphorbiaceae: Manihot esculenta Crantz) as our model. PCR-based molecular markers and deep metagenomic sequencing data were employed to analyse the gall microbiome and to test the hypothesis that gall cells are genetically transformed by insect vectored bacteria. A shotgun sequencing discrimination approach was implemented to selectively discriminate between foreign DNA and the reference host plant genome. Several known candidate insertion sequences were identified, the most significant being DNA sequences found in bacterial genes related to the transcription regulatory factor CadR, cadmium-transporting ATPase encoded by the cadA gene, nitrate transport permease protein (nrtB gene), and arsenical pump ATPase (arsA gene). In addition, a DNA fragment associated with ubiquitin-like gene E2 was identified as a potential accessory genetic element involved in gall induction mechanism. Furthermore, our results suggest that the increased quality and rapid development of gall tissue are mostly driven by microbiome enrichment and the acquisition of critical endophytes. An initial gall-like structure was experimentally obtained in M. esculenta cultured tissues through inoculation assays using a Rhodococcus bacterial strain that originated from the inducing insect, which we related to the gall induction process. We provide evidence that the modification of the endophytic microbiome and the genetic transformation of plant cells in M. esculenta are two essential requirements for insect-induced gall formation. Based on these findings and having observed the same potential DNA marker in galls from other plant species (ubiquitin-like gene E2), we speculate that bacterially mediated genetic transformation of plant cells may represent a more widespread gall induction mechanism found in nature.

Butterfly wing color made of pigmented liquid.

A previously undescribed mechanism underlying butterfly wing coloration patterns was discovered in two distantly related butterfly species, Siproeta stelenes and Philaethria diatonica. These butterflies have bright green wings, but the color pattern is not derived from solid pigments or nanostructures of the scales or from the color of the cuticular membrane but rather from a liquid retained in the wing membrane. Wing structure differs between the green and non-green areas. In the non-green region, the upper and lower cuticular membranes are attached to each other, whereas in the green region, we observed a space of 5-10 µm where green liquid is held and living cells are present. A pigment analysis and tracer experiment revealed that the color of the liquid is derived from hemolymph components, bilin and carotenoid pigments. This discovery broadens our understanding of the diverse ways in which butterfly wings obtain their coloration and patterns.

Virulence of native isolates of entomopathogenic fungi (Hypocreales) against the "sweetpotato whitefly" Bemisia tabaci (Hemiptera: Aleyrodidae), including the effects of temperature and fungicides.

Hypocrella, Moelleriella and related species in the Hypocreales (Ascomycota, Sordariomycetes) cause epizootics of whiteflies and scale insects in nature. However, studies on their host specificity, virulence, infection cycles, optimal development under laboratory conditions, and compatibility with other control methods, are unexplored for most species. Under laboratory conditions, the virulence of several isolates of field-collected hypocrealean fungi (Hypocrella, Moelleriella, Regiocrella, and Verticillium) was determined on Bemisia tabaci eggs and 4th instar nymphs. In addition to virulence, the effect of temperature and two commercial fungicides on growth rates and germination of the isolates was evaluated. None of the isolates infected the eggs, while M. libera, M. ochracea, and M. turbinata caused high nymphal mortality. Moelleriella libera was the most virulent isolate. At all temperatures, M. libera, Regiocrella sp. (P17H20), and Verticillium cf. pseudohemipterigenum had the highest germination and growth rates. The optimal growth temperature depended on the isolate, but at 23 °C and 25 °C, the probability of spore germination was higher for most isolates. Finally, the fungicides azoxystrobin and chlorothalonil inhibited growth rates and conidial germination at 24 and 48 h of exposure. This research produces vital knowledge on the virulence and infection cycles of poorly studied native species of entomopathogenic fungi. In addition, the results provide information on the optimal temperature for development in laboratory conditions and susceptibility to fungicides, which could contribute to future biological control strategies.

Ultraconserved elements-based systematics reveals evolutionary patterns of host-plant family shifts and phytophagy within the predominantly parasitoid braconid wasp subfamily Doryctinae.

Phytophagy has promoted species diversification in many insect groups, including Hymenoptera, one of the most diverse animal orders on Earth. In the predominantly parasitoid family Braconidae, an association with insect-induced, plant galls in angiosperms have been reported in three subfamilies, but in particular in the Doryctinae, where it has been recorded to occur in species of ten genera. Allorhogas Gahan is the most species-rich of these genera, with its species having different phytophagous strategies. Here we conducted a comprehensive phylogenomic study for the doryctine gall-associated genera, with an emphasis on Allorhogas, using ultraconserved elements (UCEs). Based on this estimate of phylogeny we: (1) evaluated their taxonomic composition, (2) estimated the timing of origin of the gall-associated clade and divergence of its main subclades, and (3) performed ancestral state reconstruction analyses for life history traits related to their host-plant association. Our phylogenetic hypothesis confirmed Allorhogas as polyphyletic, with most of its members being nested in a main clade composed of various subclades, each comprising species with a particular host-plant family and herbivorous feeding habit. The origin of gall-association was estimated to have occurred during the late Oligocene to early Miocene, with a subsequent diversification of subclades during the middle to late Miocene and Pliocene. Overlap in divergence timing appears to occur between some taxa and their host-associated plant lineages. Evolution of the feeding strategies in the group shows "inquilinism-feeding" as the likely ancestral state, with gall-formation in different plant organs and seed predation having independently evolved on multiple occasions.

First evidence for an aposematic function of a very common color pattern in small insects.

Many small parasitoid wasps have a black head, an orange mesosoma and a black metasoma (BOB color pattern), which is usually present in both sexes. A likely function of this widespread pattern is aposematic (warning) coloration, but this has never been investigated. To test this hypothesis, we presented spider predators (Lyssomanes jemineus), both field-captured and bred in captivity from eggs, to four wasp genera (Baryconus, Chromoteleia, Macroteleia and Scelio), each genus being represented by a BOB morphospecies and black morphospecies. We also used false prey, consisting of lures made of painted rice grains. Behavioral responses were analyzed with respect to presence or absence of the BOB pattern. In order to better understand the results obtained, two additional studies were performed. First, the reflection spectrum of the cuticle of the wasp and a theoretical visual sensibility of the spider were used to calculate a parameter we called "absorption contrast" that allows comparing the perception contrast between black and orange in each wasp genus as viewed by the spider. Second, acute toxicity trials with the water flea, Daphnia magna, were performed to determine toxicity differences between BOB and non-BOB wasps. At least some of the results suggest that the BOB color pattern may possibly play an aposematic role.

Plant galls recorded from Guanacaste Conservation Area-Costa Rica as an integrated concept of a biological database / Registro de galhas em plantas da Área de Conservação Guanacaste, Costa Rica, como conceito integrado de um banco de dados biológico

Abstract: Galling insects are specialist herbivorous that have the ability of manipulating plant tissue to form complex biological structures called galls. Even though different organisms have the ability to induce galls in plants, insect galls have the highest degree of structural complexity. The main goal of this study was to obtain a preliminary systematic record of plant gall morphotypes from the Guanacaste Conservation Area in Costa Rica and integrate the information into a biological database. Plant gall morphotypes were recorded, characterized and deposited into a specialized herbarium established as a reference for the inventory. Moreover, organisms associated with gall morphotypes were included in the inventory when it was possible to obtain and identify them. Galls were collected in the rainy season over a period of three years. In total, we recorded forty-four families, seventy genera, and eighty-seven host plant species. One hundred thirty-one morphotypes of plant galls were identified in the Guanacaste Conservation Area. The family with the highest number of gall morphotypes was Fabaceae (8.4%). Leaves were the organ with the largest number of galls (71%), followed by stems (17.6%), and apical buds (6.9%). The predominant gall shape was globular (25.2%), followed by discoid (18.3%). Fifty-nine percent of the galls had a glabrous texture, which was most common on leaves, with 77%. One hundred twenty of our field records (91.6%) of plant galls were new morphotypes not only for Costa Rica but also the world. As a consequence of this research and considering the prospect of future increases in new gall records (and associated organisms), we proposed having the biological entities resulting from the inventory placed in a cecidiarium. This repository represents a standardized and comprehensive way to manage the data and biological materials associated with the plant galls. We also suggest a nomenclature for standardizing gall morphotype registries and identifications. This work is the first and most detailed inventory of plant galls carried out thus far in the Guanacaste Conservation Area.

Resumo: Os insetos galhadores são herbívoros especializados, que têm a habilidade de manipular os tecidos vegetais, formando uma complexa estrutura biológica. Diferentes organismos têm a capacidade de induzir galhas, porém as de insetos têm maior grau de complexidade estrutural. O principal objetivo desse estudo foi realizar um levantamento sistemático preliminar das galhas de insetos na Área de Conservação Guanacaste, na Costa Rica, e inserir as informações em uma base de dados biológicos. Os morfotipos de galhas foram registrados, caraterizados e depositados em um herbário estabelecido como base de referência deste inventário. Além disso, quando foi possível obter e identificar os organismos associados a cada morfotipo de galha, eles foram incluídos no inventário. As amostras de galhas foram coletadas na estação chuvosa, durante três anos. No total, foram registradas 44 famílias, 70 gêneros e 87 espécies de plantas hospedeiras. Cento e trinta e um morfotipos de galhas foram identificados na Área de Conservação de Guanacaste. A família com o maior número de morfotipos de galhas foi Fabaceae (8.4%). Os órgãos vegetais com o maior número de galhas foram as folhas (71,0%), seguidas dos caules (17,6%), e das gemas apicais com 6,9%. A forma predominante das galhas foi globoide (25,2%), seguida da lenticular (18,3%) e 59% das galhas apresentaram textura glabra, observada mais frequentemente folhas (77%). Cento e vinte morfotipos de galhas (91.6%) representaram novos registros não só na Costa Rica como também no mundo. Como consequência desta pesquisa e considerando as perspectivas de aumentos futuros de novos registros de galhas (e organismos associados), nós propomos que as entidades biológicas resultantes deste inventário sejam depositadas em um cecidiário. Este repositório representa uma maneira padronizada e abrangente de gerenciar e integrar os dados e os materiais biológicos associados às galhas das plantas. Também sugerimos uma terminologia para padronizar os registros e identificações dos morfotipos de galhas. Este inventário de galhas em plantas é o primeiro e o mais detalhado já realizado na Área de Conservação Guanacaste.

Plants and gall hosts of the Tirimbina Biological Reserve, Sarapiqui, Costa Rica: Combining field sampling with herbarium records

There has been an increasing number of inventories of gall-inducing arthropods in the Neotropics. Nonetheless, very few inventories have been carried out in areas where the flora is well documented, and records of galls from herbaria and sites outside the study area have seldom been utilized. In this study we provide a checklist of the native vascular plants of a 345 ha forest reserve in the Caribbean lowlands of Costa Rica and document which of these plants were found to harbor galls. The gall surveys were carried out between November 2013 and December 2016. We also cross-checked our plant list with the previous gall records from elsewhere in the country and searched for galls on herbarium specimens of dicots reported from the reserve. In total, we recorded 143 families and 1174 plant species, of which 401 were hosts of galls. Plant hosts of galls were found in the following non-mutually exclusive categories: 209 in our field sampling, 257 from previous records, and 158 in herbarium specimens. Of our field records of galls, 77 were new for the country, 77 were also found in the herbarium and 110 had been previously recorded in the country. Herbarium specimens and previous records are good indicators of potential gall hosts but around one quarter (40/158) of the gall records in the herbarium were ambiguous as to whether the tissue alteration was a gall or not. Plant family size was related to number of gall hosts, but most dicot families have similar proportions of gall host species. We concluded that more than a third of the native plants in the reserve are gall hosts, but this number increased to 44.93 % when only dicots were considered. Our results demonstrate the utility of providing complete plant inventories when conducting gall inventories, and of using herbarium specimens and previous galls studies to infer gall diversity in a specific area.

Los estudios relacionados con la diversidad de insectos inductores de agallas han venido aumentando en el Neotrópico. Sin embargo, pocos inventarios se han realizado en sitios donde la flora está bien documentada y los registros previos de agallas, así como información disponible de herbario, han sido subutilizados. En este estudio brindamos un inventario de las plantas nativas en una reserva de 345 ha ubicada en las tierras bajas del Caribe costarricense y documentamos cuales de estas son hospederas de agallas. El muestreo de agallas se llevó a cabo entre noviembre del 2013 y diciembre del 2016. Así mismo, comparamos nuestra lista de plantas con los registros previos de agallas encontradas en el resto del país y examinamos en herbario todas las dicotiledóneas registradas en la reserva en busca de agallas. En total, encontramos 143 familias y 1 174 especies de plantas de las cuales 401 especies fueron hospederas de agallas. Estas fueron encontradas de las siguientes categorías no mutuamente exclusivas: 209 mediante colectas de campo, 257 a partir de registros previos y 158 en material de herbario. De las plantas con agallas encontradas en el campo 77 corresponden a registros nuevos para el país, 77 fueron halladas también en herbario y 110 habían sido reportadas anteriormente en el país. El material de herbario y los registros previos demostraron ser buenos indicadores de las potenciales especies de plantas hospederas de agallas, sin embargo, alrededor de una cuarta parte (40/158) de los registros de agallas en herbario son ambiguos (difíciles de determinar si son agallas o no). El tamaño de la familia de las plantas está relacionado con la diversidad de especies de plantas hospederas de agallas, no obstante, la mayoría de las familias de dicotiledóneas tiene una proporción similar de especies hospederas de agallas. Concluimos que al menos un tercio de las especies de plantas nativas de la reserva son hospederas de agallas, pero este número incrementa a 44.93 % cuando solo se consideran las dicotiledóneas. Nuestros resultados demuestran la importancia de proveer inventarios completos de plantas cuando se realizan inventarios de agallas, así como la utilidad de examinar material de herbario y contrastar registros previos para inferir la diversidad de agallas en un sitio.

Overview of the lady beetle tribe Diomini (Coleoptera: Coccinellidae) and description of a new phytophagous, silk-spinning genus from Costa Rica that induces food bodies on leaves of Piper (Piperaceae).

A new genus of lady beetle, Moiradiomus gen. nov. (Coleoptera: Coccinellidae Latreille, 1807: Diomini Gordon, 1999 ), and four new species are described from Costa Rica, representing the first known occurrences of obligate phytophagous lady beetle species outside of the tribe Epilachnini Mulsant, 1846 (sens. Slipinski 2007). The new species are described, illustrated and keyed, and their life histories discussed. Each species of Moiradiomus occurs on a separate species of Piper L., 1753 (Piperaceae Giseke, 1792), where the larva constructs a small silken tent between leaf veins and inside this shelter induces the production of food bodies, which are its exclusive source of food. Background information is provided on lady beetle trophic relations and other insect/Piper symbioses. The taxonomic history of Diomus Mulsant, 1850 and related species in the tribe Diomini is reviewed and existing errors in observation, interpretation, identification, and classification are corrected in order to provide a more meaningful context for understanding the new genus. The tribe Diomini is rediagnosed and recircumscribed to include Diomus, Decadiomus Chapin, 1933, Heterodiomus Brèthes, 1925, Dichaina Weise, 1923, Andrzej Slipinski, 2007, and Moiradiomus. Magnodiomus Gordon, 1999 and Erratodiomus Gordon, 1999 are removed from Diomini and transferred to Hyperaspidini Costa, 1849, subtribe Selvadiina Gordon, 1985 stat. nov. Mimoscymnus Gordon, 1994 and Planorbata Gordon, 1994, originally described in Coccidulini Mulsant, 1846 are also transfered to Hyperaspidini and placed in Mimoscymnina subtribe nov. (type genus Mimoscymnus). The main morphological characters distinguishing Diomini and Hyperaspidini are described and illustrated. A key to genera of Diomini sensu novo is provided. The identification of the Australian Diomus species illustrated in Gordon's publication on North American lady beetles is corrected from D. pumilio Weise, 1885 to D. tenebricosus (Boheman, 1859), however specimens recently collected in California do not match these genitalic illustrations and are identified as true D. pumilio. The following species of Diomus are transferred to Decadiomus as new combinations: D. balteatus (LeConte, 1878), D. floridanus (Mulsant, 1850), D. amabilis (LeConte, 1852), D. liebecki (Horn, 1895), D. myrmidon (Mulsant, 1850), D. humilis (Gordon, 1976), D. pseudotaedatus (Gordon, 1976), D. taedatus (Fall, 1901), D. bigemmeus (Horn, 1895), and D. austrinus (Gordon, 1976). Decadiomus seini Segarra, 2014 is placed as a junior synonym of D. austrinus. The following new species of Moiradiomus are described: M. clotho sp. nov., M. lachesis sp. nov., M. atopos sp. nov., M. nanita sp. nov.

An extreme case of plant-insect codiversification: figs and fig-pollinating wasps.

It is thought that speciation in phytophagous insects is often due to colonization of novel host plants, because radiations of plant and insect lineages are typically asynchronous. Recent phylogenetic comparisons have supported this model of diversification for both insect herbivores and specialized pollinators. An exceptional case where contemporaneous plant-insect diversification might be expected is the obligate mutualism between fig trees (Ficus species, Moraceae) and their pollinating wasps (Agaonidae, Hymenoptera). The ubiquity and ecological significance of this mutualism in tropical and subtropical ecosystems has long intrigued biologists, but the systematic challenge posed by >750 interacting species pairs has hindered progress toward understanding its evolutionary history. In particular, taxon sampling and analytical tools have been insufficient for large-scale cophylogenetic analyses. Here, we sampled nearly 200 interacting pairs of fig and wasp species from across the globe. Two supermatrices were assembled: on an average, wasps had sequences from 77% of 6 genes (5.6 kb), figs had sequences from 60% of 5 genes (5.5 kb), and overall 850 new DNA sequences were generated for this study. We also developed a new analytical tool, Jane 2, for event-based phylogenetic reconciliation analysis of very large data sets. Separate Bayesian phylogenetic analyses for figs and fig wasps under relaxed molecular clock assumptions indicate Cretaceous diversification of crown groups and contemporaneous divergence for nearly half of all fig and pollinator lineages. Event-based cophylogenetic analyses further support the codiversification hypothesis. Biogeographic analyses indicate that the present-day distribution of fig and pollinator lineages is consistent with a Eurasian origin and subsequent dispersal, rather than with Gondwanan vicariance. Overall, our findings indicate that the fig-pollinator mutualism represents an extreme case among plant-insect interactions of coordinated dispersal and long-term codiversification. [Biogeography; coevolution; cospeciation; host switching; long-branch attraction; phylogeny.].

Phylogeny and evolution of life-history strategies in the Sycophaginae non-pollinating fig wasps (Hymenoptera, Chalcidoidea).

BACKGROUND: Non-pollinating Sycophaginae (Hymenoptera, Chalcidoidea) form small communities within Urostigma and Sycomorus fig trees. The species show differences in galling habits and exhibit apterous, winged or dimorphic males. The large gall inducers oviposit early in syconium development and lay few eggs; the small gall inducers lay more eggs soon after pollination; the ostiolar gall-inducers enter the syconium to oviposit and the cleptoparasites oviposit in galls induced by other fig wasps. The systematics of the group remains unclear and only one phylogeny based on limited sampling has been published to date. Here we present an expanded phylogeny for sycophagine fig wasps including about 1.5 times the number of described species. We sequenced mitochondrial and nuclear markers (4.2 kb) on 73 species and 145 individuals and conducted maximum likelihood and Bayesian phylogenetic analyses. We then used this phylogeny to reconstruct the evolution of Sycophaginae life-history strategies and test if the presence of winged males and small brood size may be correlated. RESULTS: The resulting trees are well resolved and strongly supported. With the exception of Apocrytophagus, which is paraphyletic with respect to Sycophaga, all genera are monophyletic. The Sycophaginae are divided into three clades: (i) Eukoebelea; (ii) Pseudidarnes, Anidarnes and Conidarnes and (iii) Apocryptophagus, Sycophaga and Idarnes. The ancestral states for galling habits and male morphology remain ambiguous and our reconstructions show that the two traits are evolutionary labile. CONCLUSIONS: The three main clades could be considered as tribes and we list some morphological characters that define them. The same biologies re-evolved several times independently, which make Sycophaginae an interesting model to test predictions on what factors will canalize the evolution of a particular biology. The ostiolar gall-inducers are the only monophyletic group. In 15 Myr, they evolved several morphological adaptations to enter the syconia that make them strongly divergent from their sister taxa. Sycophaginae appears to be another example where sexual selection on male mating opportunities favored winged males in species with small broods and wingless males in species with large broods. However, some species are exceptional in that they lay few eggs but exhibit apterous males, which we hypothesize could be due to other selective pressures selecting against the re-appearance of winged morphs.