h i g h l i g h t s Rhynchophorus species are polyphagous and some are pests of several palm species. We reviewed their natural enemies in both their native and introduced regions. More than 50 natural enemies have been reported to attack Rhynchophorus species. Fungi are the most promising ones for biological control.

Oscheius onirici sp. n. (Nematoda: Rhabditidae) was isolated from a karst cave soil of Central Italy. Molecular and morphological analyses were performed. Total DNA was extracted from individual nematodes and the mitochondrial COI, the ITS containing region, the D2-D3 expansion domains of the 28S rRNA gene and the 18S rRNA gene were amplified and sequenced. BLAST search at NCBI by using all molecular markers revealed that this taxon is similar to Oscheius species. Phylogenetic trees of ITS, 28S and 18S rDNA revealed that O. onirici sp. n. belongs to Dolichura-group. Oscheius onirici sp. n. is characterized by small body size and stoma rhabditoid type. Female reproductive system is amphidelphic. Males are rare with peloderan bursa, spicules slender and small, nine pairs of papillae of different lengths, arranged in a 1+1+1/3+3 pattern. Entomopathogenicity bioassay revealed that this nematode is capable of infecting larvae of Galleria mellonella and Tenebrio molitor.

In the present study we investigate the mechanism behind temperature controlled cargo uptake using a truncated octahedral DNA cage scaffold functionalized with one, two, three or four hairpin forming DNA strands inserted in one corner of the structure. This investigation was inspired by our previous demonstration of temperature controlled reversible encapsulation of the cargo enzyme, horseradish peroxidase, in the cage with four hairpin forming strands. However, in this previous study the mechanism of cargo uptake was not directly addressed (Juul, et al., Temperature-Controlled Encapsulation and Release of an Active Enzyme in the Cavity of a Self-Assembled DNA Nanocage, ACS Nano, 2013, 7, 9724-9734). In the present study we use a combination of molecular dynamics simulations and in vitro analyses to unravel the mechanism of cargo uptake in hairpin containing DNA cages. We find that two hairpin forming strands are necessary and sufficient to facilitate efficient cargo uptake, which argues against a full opening-closing of one corner of the structure being responsible for encapsulation. Molecular dynamics simulations were carried out to evaluate the atomistic motions responsible for encapsulation and showed that the two hairpin forming strands facilitated extension of at least one of the face surfaces of the cage scaffold, allowing entrance of the cargo protein into the cavity of the structure. Hence, the presented data demonstrate that cargo uptake does not involve a full opening of the structure. Rather, the uptake mechanism represents a feature of increased flexibility integrated in this nanocage structure upon the addition of at least two hairpin-forming strands.

The natural presence of entomopathogenic nematodes (EPNs) has been investigated in the Piedmont region (Northern Italy) in areas infested by the Japanese beetle Popillia japonica. Thirty-nine out of 155 soil samples (25.2%) were positive for EPNs. Most of the samples contained only steinermatids (92.3%), 5.1% contained heterorhabditids, and one sample (2.6%) contained both genera. All the recovered isolates were identified at species level both morphologically and molecularly. Steinernema carpocapsae was the most abundant and it was mainly distributed in open habitats, such as perennial meadows, uncultivated soils, and cropland, characterized by sandy loam soil texture and acidic pH. Steinernema feltiae has been found associated mainly with closed habitats such as coniferous and deciduous woodland, characterized by sandy loam-texture and extremely acidic soil. The three isolates of Heterorhabditis bacteriophora were collected only in open habitats (perennial meadows and uncultivated fields) characterized by strongly acidic soils with sandy loam texture. The virulence of all EPN natural strains was evaluated by laboratory assays against P. japonica third-instar larvae collected during two different periods of the year (spring, autumn). The results showed that larval mortality was higher for pre-wintering larvae than post-wintering ones. The five more promising EPN isolates were tested in the semi-field assay in which H. bacteriophora natural strains have been shown to be more efficient in controlling P. japonica grubs. All of these results are finally discussed considering the use of these natural EPNs as biological control agents against P. japonica, within an eco-friendly perspective of management.

The Japanese beetle Popillia japonica Newman is a US and EU quarantine insect pest that has recently invaded northern Italy. Its ability to rapidly spread in new areas makes this insect a threat to agriculture. In the last decades, several trials on biological control of the Japanese beetle by entomopathogenic nematodes and fungi have been carried out with variable efficacy. However, the necessity of an integrated pest management approach to improve control has arisen. Long-lasting insecticide-treated nets (LLINs) have been used to control other agricultural pests with an attract-and-kill strategy. Here, we present results from laboratory evaluation of 2 LLINs, Storanet 1 (BASF TM ) and ZeroFly 1 (Vestergaard TM ), against P. japonica adults. Both were effective in killing the beetles; however, some differences emerged if different exposure times were compared: ZeroFly 1 always gave 100% mortality in tests from 5-s to 30-min exposure; Storanet 1 showed 100% mortality only with 30-min exposure and going down to 89%-99% mortality for 5-s to 15-min exposure. A description of the paralysis process occurring at 5-s exposure is given. Possible field application of LLINs within programs of integrated pest management is discussed. Integr Environ Assess Manag 2019;15:259-265. C 2018 SETAC

In 2014, the Japanese beetle Popillia japonica (Coleoptera: Scarabaeidae) was first spotted in northern Italy in the Nature Park of the Ticino Valley, its first detection in continental Europe. This polyphagous invasive species has the potential to cause serious losses to horticulture and agriculture. Particularly for its management in a Nature Park, environmentally friendly strategies are necessary. To develop baseline data for a biological control approach to the Italian outbreak of P. japonica, we conducted laboratory and field experiments testing several indigenous and commercial strains of the entomopathogenic nematode (EPN) species Heterorhabditis bacteriophora and Steinernema carpocapsae against P. japonica larvae. In the laboratory, strains of H. bacteriophora caused greater mortality (ranging from 57% to 100%) than those of S. carpocapsae (3% to 77%). In micro-plot field tests carried out at three different times, the most virulent indigenous EPN strain, H. bacteriophora ItH-LU1, showed again the best results ranging from 44% to 93% against young larvae. Finally, in a large-plot field trial, the commercial H. bacteriophora product provided 46% larval mortality. This study shows that H. bacteriophora strains have good potential as biological control agents of larvae of the invasive P. japonica in northern Italy. K E Y W O R D Sbiocontrol, insect pest, invasive species, Japanese beetle, pest management, white grub

Rhynchophorus ferrugineus is considered the worst pest of palm species, and few natural enemies are reported for this parasite in its area of origin. Here, we report the first recovery of the entomopathogenic fungus Metarhizium pingshaense associated with R. ferrugineus from Vietnam. The morphological, biochemical, and toxicological features of this strain were studied and compared with those of another Metarhizium strain associated with this weevil in Sicily (Italy), an area of recent introduction. The potential use of these fungi as biocontrol agents was tested against adult insects in laboratory trials and a similar mortality rate was found. Both strains were able to produce toxins and cuticledegrading proteases, but they showed dissimilar enzymatic and toxicological profiles, suggesting a different virulence activity.

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