Conidial development in selected ambrosial species of the genus <Emphasis Type="Italic">Raffaelea</Emphasis>

Conidiogenesis of the type species and two additional species of the genus Raffaelea Arx & Hennebert are described. In contrast to previous conclusions based on light microscopy that indicate sympodial production of the conidia, we found that conidium development was by annellidic percurrent proliferation in the type species Raffaelea ambrosiae, as well as in Raffaelea arxii and Raffaelea albimanens. Consequently, this mode of conidium development is similar to the conidiogenesis of anamorphs within the Ophiostomatales, but distinguished by sporodochia formed within the genus Raffaelea. These findings reduce the differences between Raffaelea and other anamorphs with annellidic percurrent proliferation of the conidiogenous cells within the Ophiostomatales to different conidiomata and conidiophores, and they support previous reports of the close phylogenetic relationship between Raffaelea and Ophiostoma.     

Transmission of <Emphasis Type="Italic">Metarhizium brunneum</Emphasis> conidia between male and female <Emphasis Type="Italic">Anoplophora glabripennis</Emphasis> adults

Transmission of conidia between mates of the Asian longhorned beetle, Anoplophora glabripennis (Motschulsky) (Coleoptera: Cerambycidae), was studied using two isolates of the entomopathogenic fungus Metarhizium brunneum Petch (Hypocreales: Clavicipitaceae) (formerly M. anisopliae). After one beetle was inoculated and caged with a mate for 6 h, conidia were rinsed off each beetle using Tween-80 and pentane to count conidia transferred. Treated males transmitted more conidia to females than treated females transferred to males. Untreated partners did not die as quickly as their inoculated mates, but died significantly faster than controls. For beetles inoculated with ARSEF 7711, the difference in time to death between inoculated beetles and their untreated mates was shorter when males were inoculated than when females were inoculated. We hypothesize that greater conidial transmission from males to females was due to their relative positions during copulation and the prolonged post-copulatory mate-guarding characteristic of males.     

Nutritional symbionts of a putative vector, <Emphasis Type="Italic">Xyleborus bispinatus</Emphasis>, of the laurel wilt pathogen of avocado, <Emphasis Type="Italic">Raffaelea lauricola</Emphasis>

Ambrosia beetles subsist on fungal symbionts that they carry to, and cultivate in, their natal galleries. These symbionts are usually saprobes, but some are phytopathogens. Very few ambrosial symbioses have been studied closely, and little is known about roles that phytopathogenic symbionts play in the life cycles of these beetles. One of the latter symbionts, Raffaelea lauricola, causes laurel wilt of avocado, Persea americana, but its original ambrosia beetle partner, Xyleborus glabratus, plays an uncertain role in this pathosystem. We examined the response of a putative, alternative vector of R. lauricola, Xyleborus bispinatus, to artificial diets of R. lauricola and other ambrosia fungi. Newly eclosed, unfertilized females of X. bispinatus were reared in no-choice assays on one of five different symbionts or no symbiont. Xyleborus bispinatus developed successfully on R. lauricola, R. arxii, R. subalba and R. subfusca, all of which had been previously recovered from field-collected females of X. bispinatus. However, no development was observed in the absence of a symbiont or on another symbiont, Ambrosiella roeperi, recovered from another ambrosia beetle, Xylosandrus crassiusculus. In the no-choice assays, mycangia of foundress females of X. bispinatus harbored significant colony-forming units of, and natal galleries that they produced were colonized with, the respective Raffaelea symbionts; with each of these fungi, reproduction, fecundity and survival of the beetle were positively impacted. However, no fungus was recovered from, and reproduction did not occur on, the A. roeperi and no symbiont diets. These results highlight the flexible nature of the ambrosial symbiosis, which for X. bispinatus includes a fungus with which it has no evolutionary history. Although the “primary” symbiont of the neotropical X. bispinatus is unclear, it is not the Asian R. lauricola.     

Presence and Diversity of <Emphasis Type="Italic">Streptomyces</Emphasis> in <Emphasis Type="Italic">Dendroctonus</Emphasis> and Sympatric Bark Beetle Galleries Across North America

Recent studies have revealed several examples of intimate associations between insects and Actinobacteria, including the Southern Pine Beetle Dendroctonus frontalis and the Spruce Beetle Dendroctonus rufipennis. Here, we surveyed Streptomyces Actinobacteria co-occurring with 10 species of Dendroctonus bark beetles across the United States, using both phylogenetic and community ecology approaches. From these 10 species, and 19 other scolytine beetles that occur in the same trees, we obtained 154 Streptomyces-like isolates and generated 16S sequences from 134 of those. Confirmed 16S sequences of Streptomyces were binned into 36 distinct strains using a threshold of 0.2% sequence divergence. The 16S rDNA phylogeny of all isolates does not correlate with the distribution of strains among beetle species, localities, or parts of the beetles or their galleries. However, we identified three Streptomyces strains occurring repeatedly on Dendroctonus beetles and in their galleries. Identity of these isolates was corroborated using a house-keeping gene sequence (efTu). These strains are not confined to a certain species of beetle, locality, or part of the beetle or their galleries. However, their role as residents in the woodboring insect niche is supported by the repeated association of their 16S and efTu from across the continent, and also having been reported in studies of other subcortical insects.     

Fungus Symbionts Colonizing the Galleries of the Ambrosia Beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis>

Isolations were made to determine the fungal symbionts colonizing Platypus quercivorus beetle galleries of dead or dying Quercus laurifolia, Castanopsis cuspidata, Quercus serrata, Quercus crispula, and Quercus robur. For these studies, logs from oak wilt-killed trees were collected from Kyoto Prefecture, Japan. Fungi were isolated from the: (1) entrances of beetle galleries, (2) vertical galleries, (3) lateral galleries, and (4) the larval cradle of P. quercivorus in each host tree. Among the fungus colonies which appeared on YM agar plates, 1,219 were isolated as the representative isolates for fungus species inhabiting in the galleries based on their cultural characteristics. The validity of the visual classification of the fungus colonies was checked and if necessary properly corrected using microsatellite-primed PCR fingerprints. The nucleotide sequence of the D1/D2 region of the large subunit nuclear rRNA gene detected 38 fungus species (104 strains) of which three species, i.e., Candida sp. 3, Candida kashinagacola (both yeasts), and the filamentous fungus Raffaelea quercivora were isolated from all the tree species. The two yeasts were most prevalent in the interior of galleries, regardless of host tree species, suggesting their close association with the beetle. A culture-independent method, terminal restriction fragment length polymorphism (T-RFLP) analysis was also used to characterize the fungus flora of beetle galleries. T-RFLP patterns showed that yeast species belonging to the genus Ambrosiozyma frequently occurred on the gallery walls along with the two Candida species. Ours is the first report showing the specific fungi inhabiting the galleries of a platypodid ambrosia beetle.     

<Emphasis Type="Italic">Ambrosiella</Emphasis><Emphasis Type="Italic">beaveri,</Emphasis> sp. nov., Associated with an exotic ambrosia beetle, <Emphasis Type="Italic">Xylosandrus mutilatus</Emphasis> (Coleoptera: Curculionidae,Scolytinae), in Mississippi,USA

Xylosandrus mutilatus is an Asian ambrosia beetle that has recently established in Mississippi, Texas, Alabama, and possibly Florida, USA. We investigated the fungi associated with the mycangia (specialized fungus-transporting structures) of X. mutilatus in Mississippi. Mycangia consistently yielded an Ambrosiella sp. which was subsequently found to be closely related to, but distinct from, other Ambrosiella species affiliated with Ceratocystis. This Ambrosiella is described herein as Ambrosiella beaveri sp. nov. Also isolated were Geosmithia lavendula, G. obscura, and a yeast, Candida homelintoma. It is likely Ambrosiella beaveri was introduced along with the beetle into North America.     

Phototactic behavior of the ambrosia beetle <Emphasis Type="Italic">Platypus quercivorus</Emphasis> (Murayama) (Coleoptera: Platypodidae) before and after flight

The ambrosia beetle Platypus quercivorus (Coleoptera: Platypodidae) is a vector of the pathogenic fungi, Raffaelea quercivora (Ophiostomatales: Ophiostomataceae), which causes Japanese oak wilt disease. Previous studies have shown that the beetle displays positive phototactic behavior; however, the adaptive significance of this behavior remains unclear. We postulated that positive phototaxis is necessary to allow the beetle to fly skyward immediately after emergence from a tree, and that this taxis changes following a certain period of flight. The present study aimed to clarify the changes in phototactic behavior of P. quercivorus before and after flight by using individual beetles emerging from the trunk of a Quercus crispula (Fagales: Fagaceae) tree that was attacked in 2014. The response of 60 beetles to light was tested ten times each, before and after flight in a flight mill. A generalized linear mixed model was constructed to predict the probability of positive phototaxis of P. quercivorus before and after flight. A best-fit model showed that the probability of positive phototaxis was lower after flight than before. The results suggest that positive phototaxis of P. quercivorus is decreased after flight.     

Biological control of <Emphasis Type="Italic">Lantana camara</Emphasis> in South Africa: targeting a different niche with a root-feeding agent, <Emphasis Type="Italic">Longitarsus</Emphasis> sp.

The root-feeding flea beetle, Longitarsus sp. (Coleoptera: Chrysomelidae: Alticinae), was studied as a potential biological control agent for Lantana camara L. (Verbenaceae) in South Africa. Host range tests were carried out on 52 plant species in 11 families. Although 11 plant species, all in the family Verbenaceae, supported complete development of Longitarsus sp. during no-choice tests, the beetles showed very strong preferences for L. camara during paired-choice and multi-choice tests. The results confirm that the beetles have a narrow host range, and that under natural conditions they are highly unlikely to utilise plants other than L. camara. In the unlikely event that some of the Lippia spp. are attacked in the field, they are not expected to sustain populations of the flea beetle over time. Attributes that should enhance the biocontrol potential of Longitarsus sp. include: the adults are long-lived and highly mobile; and, the larvae cause extensive direct damage to the roots of L. camara, which could in turn expose the plants to soil-born pathogens. All indications are that Longitarsus sp. could make a substantial contribution to the biological control of L. camara in many countries around the world because the beetles pose no threat to non-target plant species and they damage a part of the plant (i.e. roots) not yet affected by any other agent species.     

Social encapsulation of the small hive beetle (<Emphasis Type="Italic">Aethina tumida</Emphasis> Murray) by European honeybees (<Emphasis Type="Italic">Apis mellifera</Emphasis> L.)

Summary European and African subspecies of honeybees (Apis mellifera L.) utilize social encapsulation to contain the small hive beetle (Aethina tumida Murray), a honeybee colony scavenger. Using social encapsulation, African honeybees successfully limit beetle reproduction that can devastate host colonies. In sharp contrast, European honeybees often fail to contain beetles, possibly because their social encapsulation skills may be less developed than those of African honeybees. In this study, we quantify beetle and European honeybee behaviours associated with social encapsulation, describe colony and time (morning and evening) differences in these behaviours (to identify possible circadian rhythms), and detail intra-colonial, encapsulated beetle distributions. The data help explain the susceptibility of European honeybees to depredation by small hive beetles. There were significant colony differences in a number of social encapsulation behaviours (the number of beetle prisons and beetles per prison, and the proportion of prison guard bees biting at encapsulated beetles) suggesting that successful encapsulation of beetles by European bees varies between colonies. We also found evidence for the existence of circadian rhythms in small hive beetles, as they were more active in the evening rather than morning. In response to increased beetle activity during the evening, there was an increase in the number of prison guard bees during evening. Additionally, the bees successfully kept most (~93%) beetles out of the combs at all times, suggesting that social encapsulation by European honeybees is sufficient to control small populations of beetles (as seen in this study) but may ultimately fail if beetle populations are high.Received 20 January 2003; revised 21 April 2003; accepted 29 April 2003.     

Honey bee, <Emphasis Type="Italic">Apis mellifera</Emphasis> L., confinement behavior toward beetle invaders

We performed two experiments to study the hiding behavior of various beetles introduced into colonies of European honey bees, Apis mellifera L. In the first experiment, we studied the spatial distribution within confinement sites of six beetle species at eight time intervals following their introduction into honey bee observation hives. For each beetle species, we also determined whether the beetle’s level of integration into honey bee colonies correlated with its ability to hide at confinement sites within colonies. In experiment 1, we used five species of nitidulid beetles and one species of tenebrionid beetle, collectively representing three differing levels of integration into honey bee colonies. These species (and their level of integration) included Aethina tumida (highly integrated), Lobiopa insularis, and Epuraea luteola (accidentals), and Carpophilus humeralis, C. hemipterus, and Tribolium castaneum (non-integrated). There were always more A. tumida found in confinement sites than beetles of the other species. This difference became more pronounced over the 24-h observation period. In experiment 2, we determined whether previous A. tumida occupation of confinement sites predisposed those sites to hosting invading A. tumida never before exposed to honey bee colonies. The results from this study indicate that invading A. tumida find hiding sites within honey bee colonies more rapidly if other A. tumida previously occupied the confinement sites. Collectively, these studies suggest that A. tumida is unique among beetle invaders with respect to its ability to seek out and occupy confinement sites inside honey bee colonies.     

Inbreeding influences herbivory in <Emphasis Type="Italic">Cucurbita pepo</Emphasis> ssp. <Emphasis Type="Italic">texana</Emphasis> (Cucurbitaceae)

In a series of field experiments Diabrotica beetle herbivory was found to influence the magnitude of inbreeding depression in Cucurbita pepo ssp. texana, an annual monoecious vine. Beetles damage flowers and fruits and chew dime-sized holes in leaf tissue between major veins. Inbred plants were found to be more likely to be damaged by beetles and to have more leaves damaged per plant than outcrossed plants. A positive linear association was found between the coefficient of inbreeding and the magnitude of leaf damage, whereas a negative association was found between coefficient of inbreeding and several male and female fitness traits. When pesticides were used to control beetle herbivory, the interaction between coefficient of inbreeding and pesticide treatment was significant for fruit production and marginally significant for pollen quantity per anther. Therefore, the magnitude of inbreeding depression in C. pepo ssp . texana varies depending on the severity of beetle herbivory.     

<Emphasis Type="Italic">Geosmithia</Emphasis> Fungi are Highly Diverse and Consistent Bark Beetle Associates: Evidence from their Community Structure in Temperate Europe

Geosmithia spp. (Ascomycota: Hypocreales) are little-studied, dry-spored fungi that occur in galleries built by many phloeophagous bark beetles. This study mapped the distribution and environmental preferences of Geosmithia species occurring in galleries of temperate European bark beetles. One hundred seven host tree samples of 16 tree species infested with 23 subcortical insect species were collected from across Europe during the years 1997–2005. Over 600 Geosmithia isolates from the beetles were sorted into 17 operational taxonomic units (OTUs) based on their phenotype similarity and phylogeny of internal transcribed spacer (ITS) region of rDNA (ITS1-5.8S-ITS2). The OTUs represent six known species and eight undescribed taxa. Ninety-two samples infested with subcortical insects were characterized by the presence/absence of OTUs and the similarity among the samples was evaluated. Geographically distant populations of the same beetle species host relatively uniform Geosmithia communities across large geographic areas (ranging from southern Bulgaria to the Czech Republic). This suggests effective dispersal of Geosmithia spp. by bark beetles. Clustering of similar samples in ordination analysis is correlated predominantly with the isolation source (bark beetles and their respective feeding plant), but not with their geographical origin. The composition of the Geosmithia OTU community of each bark beetle species depends on the degree of isolation of the species’ niches. Thus, Geosmithia communities associated with regularly co-occurring bark beetle species are highly similar. The similarity decreases with decreasing frequency of beetle species’ co-occurrence, a pattern resembling that of entomochoric ophiostomatoid fungi. These findings suggest that: 1) communities of Geosmithia spp. are vector-specific; 2) at least in some cases, the association between Geosmithia OTUs and bark beetles may have been very stable and symbioses are likely to be a fundamental factor in the speciation of Geosmithia fungi; and 3) that even nonsticky spores of Geosmithia are suitable for maintaining an insect–fungus association, contrary to previous hypotheses. An erratum to this article can be found at     

<Emphasis Type="Italic">Ruwenzoria</Emphasis>, a new genus of the Xylariaceae from Central Africa

During a foray to the mountain rainforests of the Democratic Republic of the Congo, a peculiar species of Xylariaceae was found, which could not be accommodated in any of the existing genera. It is recognised as representative of a new genus, named Ruwenzoria, owing to the presence of a new combination of teleomorphic and anamorphic characters that are regarded as significant for generic segregation within the Xylariaceae. Studies on its secondary metabolites in stromata and cultures by high performance liquid chromatography, coupled with diode array and mass spectrometric detection (HPLC-DAD/MS), and on its phylogenetic affinities based on 5.8S/ITS rDNA sequence data, respectively, revealed a close relationship of the new taxon to the genera Daldinia and Entonaema, from which it differs by having neither a hollow, gelatinous nor a conspicuously zonate stromatal interior, and an anamorph featuring enteroblastic rather than holoblastic conidiogenesis. A specimen from the same geographic region, previously identified as Daldinia bakeri by R.W.G. Dennis was found to constitute a mixture of stromata of Ruwenzoria and an additional, undescribed Daldinia species. The latter fungus is not formally described due to the scantiness of the material, but its morphological characteristics are illustrated.     

<Emphasis Type="Italic">Candida kashinagacola</Emphasis> sp. nov., <Emphasis Type="Italic">C. pseudovanderkliftii</Emphasis> sp. nov. and <Emphasis Type="Italic">C. vanderkliftii</Emphasis> sp. nov., three new yeasts from ambrosia beetle-associated sources

Three new yeast species, Candida kashinagacola (JCM 15019(T) = CBS 10903(T)), C. pseudovanderkliftii (JCM 15025(T) = CBS 10904(T)), and C. vanderkliftii (JCM 15029(T) = CBS 10905(T)) are described on the basis of comparison of nucleotide sequences of large subunit ribosomal DNA D1/D2 region (LSU rDNA D1/D2). The nearest assigned species of the three new species was Candida llanquihuensis. Candida kashinagacola and C. pseudovanderkliftii differed from C. llanquihuensis by 3.8% nucleotide substitution of the region, while C. vanderkliftii did by 4.4%. Three new species differed in a number of physiological and growth characteristics from any previously assigned species and from one another. A phylogenetic tree based on the sequences of LSU rDNA D1/D2 showed that these new species together with Candida sp. ST-246, Candida sp. JW01-7-11-1-4-y2, Candida sp. BG02-7-20-001A-2-1 and C. llanquihuensis form a clade near Ambrosiozyma species. The new species did not assimilate methanol as a sole source of carbon, which supported the monophyly of these non methanol-assimilating species which are closely related to the methylotrophic yeasts. Candida kashinagacola was frequently isolated from the beetle galleries of Platypus quercivorus in three different host trees (Quercus serrata, Q. laurifolia and Castanopsis cuspidata) located in the sourthern part of Kyoto, Japan, thus indicating that this species may be a primary ambrosia fungus of P. quercivorus. On the other hand, C. pseudovanderkliftii and C. vanderkliftii were isolated only from beetle galleries in Q. laurifolia. Candida vanderkliftii was isolated from beetle gallery of Platypus lewisi as well as those of P. quercivorus. Candida pseudovanderkliftii and C. vanderkliftii are assumed to be auxiliary ambrosia fungi of P. quercivorus.     

A new species of <Emphasis Type="Italic">Laboulbenia</Emphasis> parasitic on tiger beetles

Laboulbenia phaeoxanthae sp. nov. (Ascomycota: Laboulbeniales) parasitic on the tiger beetle Phaeoxantha aequinoctialis bifasciata (Coleoptera: Carabidae: Cicindelinae) from Brazil is described. The new species seems to be closely related to Laboulbenia pheropsophi, but can be distinguished from the latter by the much more slender and elongate habit, and by the different structure of the appendages. The new species is only the second Laboulbeniales species reported on tiger beetles. The widespread Laboulbenia variabilis was previously recorded on Tetracha fulgida from Ecuador.     

<Emphasis Type="Italic">Batrachochytrium</Emphasis><Emphasis Type="Italic">dendrobatidis</Emphasis> and Chytridiomycosis in Anuran Amphibians of Colombia

In order to investigate the possible presence of the chytrid fungus Batrachochytrium dendrobatidis (Chytridiomycota: Chytridiales) in frogs (Amphibia: Anura) of Colombia, we made a retrospective examination of formalin-fixed specimens preserved in natural history collections. Using the staining technique of hematoxylin and eosin to identify B. dendrobatidis in histological slices, we found evidence of the fungus in 3 of the 53 frog species examined from a total of 672 specimens collected in 17 departments within Colombia between 1968 and 2006. The infected specimens were found dead or dying in recent years in high elevation sites, suggesting that chytridiomycosis (the disease caused by the fungus) may represent a significant threat to Colombian amphibians. We conclude that a more extensive search for B. dendrobatidis in museum specimens and wild-caught frogs should be undertaken as soon as possible, using both histological and molecular genetic techniques, in order to further characterize the geographic and taxonomic extent of infections of B. dendrobatidis.     

<Emphasis Type="Italic">Sporothrix inflata</Emphasis>, a root-inhabiting fungus of <Emphasis Type="Italic">Quercus robur</Emphasis> and <Emphasis Type="Italic">Q. petraea</Emphasis>

The anamorphic fungus Sporothrix inflata, known as a soil-borne fungus with worldwide distribution, was isolated for the first time from the cortex and central cylinder of living and dead roots of healthy and diseased oak trees (Quercus robur and Q. petraea). Isolation frequencies of S. inflata from oak roots varied according to the health status of trees, oak species, study sites, soil depth and root diameter. Colony morphology and growth rate of isolates are influenced by colony age and type of culture medium.