Italo Delalibera

Bacteria Associated with the Guts of Two Wood-Boring Beetles: Anoplophora glabripennis and Saperda vestita (Cerambycidae)

Abstract Commensal microorganisms have significant impacts on the health of many insect hosts. Little is known, however, about the structure of commensal bacterial communities associated with the Cerambycidae, despite the important roles this large family of herbivorous endophytic insects plays in ecosystem processes, economic losses to ornamental and forest trees, and biological invasions. We analyzed the bacterial commensal communities of the exotic Asian longhorned beetle, Anoplophora glabripennis, and the native linden borer, Saperda vestita, by randomly sequencing 16S rRNA gene fragments from bacterial DNA extracted directly from the gut of larvae. The 16S rRNA gene sequences sampled from S. vestita were derived entirely from the γ-Proteobacteria phylum of Bacteria. In contrast, the gut of A. glabripennis larvae contained members of the α-Proteobacteria, β-Proteobacteria, γ-Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. A better understanding of insect-microbe interactions may lead to new strategies to reduce the effects of these pest species.

Contrasts in cellulolytic activities of gut microorganisms between the wood borer, Saperda vestita (Coleoptera: Cerambycidae), and the bark beetles, Ips pini and Dendroctonus frontalis (Coleoptera: Curculionidae)

Abstract The goal of this study was to characterize the cellulose-degrading microorganisms in the guts of wood-inhabiting beetles. We enriched for cellulose-degrading microorganisms by inoculating filter paper in liquid growth medium with macerated guts from larvae and adults of the wood-boring longhorned beetle, Saperda vestita, and the phloeophagous bark beetles, Ips pini and Dendroctonus frontalis. After incubation, microorganisms were isolated in pure culture and tested for their ability to digest carboxymethylcellulose (CMC). Isolates were identified based on their ribosomal RNA gene or intergenic region sequences. Bacteria and fungi from the gut of S. vestita degraded filter paper, whereas the microorganisms from the phloeophagous insects did not. The only bacterium detected that was able to degrade CMC is closely related to Sphingobium yanoikuyae, a member of the α-Proteobacteria class. We found this species in all field-collected S. vestita larvae in 2002 and 2003. Population densities of cellulolytic bacteria in S. vestita ranged from 2.4 × 105 to 3.6 × 106 CFU/gut. Bacteria isolated from the phloeophagous beetles I. pini and D. frontalis did not degrade CMC. Two fungi isolated from the gut of S. vestita adults had strong degradative activity. Sequences of the ITS1, 5.8S, and ITS2 rRNA regions indicated that these fungi are highly similar to Fusarium culmorum and Penicillium crustosum, respectively. This study provides the first description of the gut microbial community of S. vestita and the first documentation of association between cellulolytic microorganisms and a wood-boring beetle.

Biological control of insects in Brazil and China: history, current programs and reasons for their successes using entomopathogenic fungi

Abstract Brazil and China have been successful in the use of microbial control methods to manage several agricultural and forest insects. In both countries, entomopathogenic fungi (EF) have been used for pest management since the 1970s. However, EF production and commercialization have not been constant in either country. Several companies and cooperatives suspended their activities or shut down from the 1970s to the 1990s. This was due to loss of confidence in available mycoinsecticides by Brazilian farmers or due to reduced involvement and government subsidies for biological control in China; and, consequently, mycoinsecticides were largely replaced by inexpensive chemical insecticides. Starting in the 1990s and continuing until today, however, new Brazilian and Chinese private companies have arisen. In Brazil, the area treated with M. anisopliae for spittlebug control alone is estimated to be approximately one million hectares in 2008, 75% of which was for control of spittlebugs in sugarcane plantations and the remainder for spittlebugs in pasture grass (primarily Brachiaria spp.) and other smaller programs. In China, the fungus Beauveria bassiana was used annually in 0.8–1.3 million ha until the 1980s. Several factors were important for the success of these programs, such as: governmental support (at least during the initial steps of biocontrol programs); availability of indigenous virulent fungal isolates; low-cost substrates for mass production; retail prices of mycoinsecticides lower than their chemical counterparts; and sale by contract which allows the products to be immediately available for use, rather than stored. In this report, we discuss the current biocontrol programs using insect fungi in these two developing countries, as well as the future and main challenges they must face to further encourage the adoption of mycoinsecticides.

Characterization of Gut-Associated Bacteria in Larvae and Adults of the Southern Pine Beetle, Dendroctonus frontalis Zimmermann

Abstract We report the first study of gut-associated bacteria of bark beetles using both culture-dependent and culture-independent methods. These insects are major pests of pine trees but also contribute to important ecological functions such as nutrient cycling. We found members of the α- and γ-Proteobacteria and Firmicutes in larvae of the southern pine beetle, Dendroctonus frontalis Zimmermann. Sequences from three larval guts were grouped into one to three operational taxonomic units (OTUs) at 3% difference among sequences. Communities in adult southern pine beetle guts consisted solely of members of the γ-Proteobacteria. These could be grouped into three to five OTUs at 3% difference between sequences. These gut communities have relatively low species richness, which may reflect the specialization needed to exploit a nutrient-poor food source, colonize a chemically complex habitat, and maintain consistent associations with mutualistic fungi. However, there is considerable variation in gut microbiota composition among individual insects, suggesting the need for additional studies on sources of variation and potential substitutability among species performing similar functions.

Role of entomopathogenic fungi in the control of Tetranychus evansi and Tetranychus urticae (Acari: Tetranychidae), pests of horticultural crops

The spider mites Tetranychus urticae Koch and Tetranychus evansi Baker and Pritchard are important pests of horticultural crops. They are infected by entomopathogenic fungi naturally or experimentally. Fungal pathogens known to cause high infection in spider mite populations belong to the order Entomophthorales and include Neozygites spp. Studies are being carried out to develop some of these fungi as mycoacaricides, as stand-alone control measures in an inundative strategy to replace the synthetic acaricides currently in use or as a component of integrated mite management. Although emphasis has been put on inundative releases, entomopathogenic fungi can also be used in classical, conservation and augmentative biological control. Permanent establishment of an exotic agent in a new area of introduction may be possible in the case of spider mites. Conservation biological control can be achieved by identifying strategies to promote any natural enemies already present within crop ecosystems, based on a thorough understanding of their biology, ecology and behaviour. Further research should focus on development of efficient mass production systems, formulation, and delivery systems of fungal pathogens.

Neozygites tanajoae sp. nov., a pathogen of the cassava green mite

The fungal pathogen Neozygites tanajoae Delalibera Jr., Humber & Hajek sp. nov. (Zygomycetes: Entomophthorales) is being used in Africa as a biological control agent against the introduced cassava green mite (CGM), Mononychellus tanajoa (Bondar) (Acari: Tetranychidae). This fungus is specific to CGM and has been referred to as N. floridana (Weiser & Muma) Remaud. & Keller, a common pathogen of many tetranychid mites. In the present study N. tanajoae is investigated at the morphological and molecular levels and physiological attributes of N. tanajoae and N. floridana are compared. Morphological observations of N. tanajoae isolates generally correspond to N. floridana and to other mite pathogenic species of Neozygites. However, this fungus readily can be distinguished from N. floridana based on 18S rDNA sequences, host ranges, nutritional requirements for growth in vitro, tolerances to cold (4 C) and abilities to withstand specific cryopreservation techniques. N. tanajoae isolates from Brazil and Africa have identical 18S rDNA sequences but they presented 5.7 and 9.94% pairwise distance from N. floridana isolates. N. tanajoae proved to differ sufficiently from other mite-pathogenic fungi referred to as N. floridana to justify the description of a new species.

Specificity of a strain ofNeozygites sp. (Zygomycetes: Entomophthorales) toMononychellus tanajoa (Acari: Tetranychidae)

Neozygites sp. is commonly found infecting the cassava green mite,Mononychellus tanajoa (Bondar), in parts of northeast Brazil. The introduction of this fungus into other regions requires the knowledge of its specificity, especially in relation to natural enemies of different cassava pests. Laboratory tests indicated the development of germination tubes ofNeozygites in some females ofTetranychus bastosi Tuttle et al. andT. urticae Koch, with subsequent formation of a reduced number of hyphal bodies in someT. bastosi. No females of the phytoseiid predatorsAmblyseius idaeus (Denmark & Muma) andAmblyseius limonicus Garman & McGregor s.l. were infected byNeozygites sp.

Alternative plant habitats for common phytoseiid predators of the cassava green mite (Acari: Phytoseiidae, Tetranychidae) in northeast Brazil

A survey was conducted to identify possible alternative plant habitats of the most common phytoseiid predators associated with the cassava green mite, Mononychellus tanajoa (Bondar), in their native environment in northeast Brazil. Thirty-two phytoseiid species were collected, including Amblyseius aripo (DeLeon), Amblyseius idaeus (Denmark and Muma) and Amblyseius limonicus Garman and McGregor s.l., the three predators previously shown as the most common on cassava. In increasing order, A. idaeus, Phytoseius guianensis DeLeon and A. aripo were the most common phytoseiids collected on the plant habitats examined. A. limonicus s.l. was one of the least common phytoseiids on plants other than cassava. Altermative plant habitats seem important in harboring A. aripo and A. idaeus, but not A. limonicus s.l..

Potential of the mite-pathogenic fungus Neozygites floridana (Entomophthorales: Neozygitaceae) for control of the cassava green mite Mononychellus tanajoa (Acari: Tetranychidae)

The cassava green mite, Mononychellus tanajoa (Bondar), is an exotic pest in Africa and is the target of a classical biological control programme. Field data from the Neotropics, where it is indigenous, are presented for the first time, charting the variation in abundance of M. tanajoa over several seasons. This was highly variable, with a characteristic trough mid-year and a peak at the turn of the year. This pattern corresponded positively with rainfall levels, appearing to fit a phenology also characteristic of African studies, where rainfall at the start of the wet season promotes a leaf flush and so growth in M. tanajoa populations. Analyses implied some impact of leaf-inhabiting predatory mites (predominantly Neoseiulus idaeus Denmark & Muma) and a considerable impact of the fungal pathogen Neozygites floridana Fisher on M. tanajoa populations. This pathogen was not observed in the host population for several (generally dry) periods implying survival outside the host, perhaps as resting spores. This is a particularly desirable characteristic of a biological control agent. It is therefore proposed that N. floridana might be of particular use in drier cassava-growing areas where rainfall at the outset of the wet season is not sufficiently intense to cause heavy M. tanajoa mortality but may be sufficient to stimulate epizootics of the fungal pathogen, protecting the flush of new cassava growth.

Toxicological evaluation of genetically modified cotton (Bollgard®) and Dipel® WP on the non-target soil mite Scheloribates praeincisus (Acari: Oribatida)

Insecticides derived from the bacterium Bacillus thuringiensis (Bt) and plants genetically modified (GM) to express B.thuringiensis toxins are important alternatives for insect pest control worldwide. Risk assessment of B.thuringiensis toxins to non-target organisms has been extensively studied but few toxicological tests have considered soil invertebrates. Oribatid mites are one of the most diverse and abundant arthropod groups in the upper layers of soil and litter in natural and agricultural systems. These mites are exposed to the toxic compounds of GM crops or pesticides mainly when they feed on vegetal products incorporated in the soil. Although some effects of B. thuringiensis products on Acari have been reported, effects on oribatid mites are still unknown. This study investigated the effects of the ingestion of Bt cotton Bollgard® and of the B. thuringiensis commercial product Dipel® WP on the pantropical species Scheloribates praeincisus (Scheloribatidae). Ingestion of Bollgard and Dipel did not affect adult and immature survivorship and food consumption (estimated by number of fecal pellets produced daily) or developmental time of immature stages of S. praeincisus. These results indicate the safety of Bollgard and Dipel to S. praeincisus under field conditions where exposition is lower and other food sources besides leaves of Bt plants are available. The method for toxicological tests described here can be adapted to other species of Oribatida, consisting on a new option to risk assessment studies.