Taxonomy and phylogeny of European Monochamus species: first molecular and karyological data

The worldwide distributed genus Monochamus Megerle, 1821 (Coleoptera Cerambicydae) comprises beetles that may become pests of economic importance in conifer stands in the Nearctic and Palearctic Regions. Besides direct damage due to the larval tunnelling habits, they have also been recognized as main vectors of the phytoparasitic nematode Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) (Nematoda Aphelenchoididae). We analysed the complete mitochondrial cytochrome oxidase I gene and a fragment of the small subunit RNA gene sequences (1536 base pairs) in the five European species. These are: Monochamus galloprovincialis (Olivier, 1795), morphologically distinguished in two subspecies M. galloprovincialis galloprovincialis (Olivier, 1795) and M. galloprovincialis pistor (Germar, 1818); Monochamus sutor (Linneus 1758); Monochamus saltuarius (Gebler 1830); Monochamus sartor (Fabricius, 1787) and Monochamus urussovi (Fischer, 1806). For appropriate comparisons, also the Asiatic Monochamus alternatus Hope, 1842 and a Japanese M. saltuarius sample have been analysed. Both genes show an absolute identity between the two subspecies of M. galloprovincialis and a strong affinity between M. sartor and M. urussovi: the morphological subdivisions of the former taxon in two subspecies and of the latter in two entities of specific level are therefore not supported genetically. On the other hand, the Italian and the Japanese samples of M. saltuarius always cluster together in all trees, and for the remaining taxa, no doubt about their rank of specific differentiation emerges from present analyses. From a phyletic point of view, tree topology indicates the Japanese M. alternatus as the most differentiated taxon and the Euroasiatic M. saltuarius as basal to all other strictly European entities. Chromosome analyses show that the diploid autosomal complement ranges from 18 in M. saltuarius to 20 in M. galloprovincialis, and 22 in M. sartor, but a XX–Xyp sex determining system is shared by all analysed taxa. The M. saltuarius karyotype appears as the most primitive from which the others may be derived through Robertsonian fissions. Karyological data therefore agree with molecular analyses in indicating a basal position of Euroasiatic M. saltuarius with respect to the group of European Monochamus taxa; among these, M. galloprovincialis and M. sartor represent two clearly diverging evolutionary units. Furthermore, karyotype analyses substantiate molecular conclusions about the identity between M. galloprovincialis galloprovincialis and M. galloprovincialis pistor. Zusammenfassung Die weltweit verbreitete Gattung Monomachus Megerle, 1821 (Coleoptera, Cerambycidae) enthalt Kaferarten, die in den Nadelwaldern der nearktischen und palaarktischen Regionen zu wirtschaftlich bedeutenden Schadlingen werden konnen. Neben dem direkten Schaden durch die minierenden Larven sind sie auch als Hauptvektoren des Nematoden Bursaphelenchus xylophilus bekannt geworden. Wir haben das gesamte Gen der mitochondrialen Cytochromoxidase I und ein Stuck des ssRNA-Gens (1536 bp) der funf europaischen Arten (M. galloprovincialis, mit den zwei Unterarten M. g. galloprovincials und M. g. pistor, M. sutor, M. saltuarius, M. sartor und M. urossovi, sowie fur einen Vergleich auch von der asiatischen Art M. alternatus und aus dem japanischen Verbreitungsgebietes der Art M. saltuarius analysiert. Beide Gene erweisen sich fur die beiden Unterarten M. g. galloprovincials und M. g. pistor als vollkommen identisch und weisen auf eine nahe Verwandtschaft zwischen M. sartor und M. urossovi hin; die morphologische Unterteilung in die beiden Unterarten und die Auftrennung dieser beiden Spezies auf Artniveau wird also genetisch nicht unterstutzt. Auf der anderen Seite clustern die italienischen und die japanischen Stichproben von M. saltuarius in allen Dendrogrammen sehr eng, so das ihr Status als Art gegenuber den anderen Taxons nicht bezweifelt werden kann. Vom phylogenetischen Standpunkt beurteilt weist die Topologie der Baume darauf hin, das die japanische Art M. alternatus die am meisten abweichende Spezies und die eurasische Art M. saltuarius die ursprunglichste unter den streng europaischen Monomachus-Arten ist. Die Chromosomenanalyse ergibt, das die diploide Chromosomenzahl sich von 18 bei M. saltuarius auf 20 bei M. galloprovincialis und auf 22 bei M. sartor erhoht, das geschlechtsdeterminierende System aber bei allen Taxa gemeinsam dem Typ XX-Xyp entspricht. Der Karyotyp von M. saltuarius scheint der ursprunglichste zu sein, von dem die anderen durch Robertsonsche Fissionen abgeleitet werden konnen. Die karyologischen Daten stimmen auch mit den molekularen uberein, indem sie der eurasischen Art M. saltuarius eine, zu den europaischen Arten basale Position zuweisen. Unter diesen stellen die Arten M. galloprovincialis und M. sartor zwei deutlich divergierende evolutionare Einheiten dar. Auserdem bestatigen die karyotypischen Analysen die molekulare Schlusfolgerung, das die zwei Unterarten M. g. galloprovincials und M. g. pistor identisch sind

An overview on the natural enemies of Rhynchophorus palm weevils, with focus on R. ferrugineus.

Rhynchophorus palm weevils are large insects belonging to the family Dryophthoridae. All Rhynchophorus species are polyphagous and have a similar life history but some are major pests because of the serious economic damage they cause, in particular to several species of the family Arecaceae. Here we review the natural enemies of Rhynchophorus species in both their native and introduced regions of the world, to assess the possibility of biological control of this taxon. Moreover, particular attention is paid to the well-studied and harmful species Rhynchophorus ferrugineus, about which more information is available, and to its natural enemies in the Mediterranean region, because the impact of this pest in this recently colonized area is particularly remarkable and also the recent trend in species management is looking for indigenous natural enemies. More than 50 natural enemies have been reported to attack Rhynchophorus species, even if most of them are associated to R. ferrugineus (Olivier), highlighting the lack of information on the other species of the genus. Pros and cons of all the biological control agents are then discussed: among the considered organisms, fungi are noteworthy to be considered for inclusion in integrated pest management programs. Overall, our overview underlines the need to increase knowledge on natural enemies of all the species of the genus Rhynchophorus, to isolate more virulent strains and to determine the optimum conditions for the actions of the biocontrol agents

TaqMan probe assays on different biological samples for the identification of three ambrosia beetle species, Xylosandrus compactus (Eichoff), X. crassiusculus (Motschulsky) and X. germanus (Blandford) (Coleoptera Curculionidae Scolytinae)

Molecular assays based on qPCR TaqMan Probes were developed to identify three species of the genus Xylosandrus, X. compactus, X. crassiusculus and X. germanus (Coleoptera Curculionidae Scolytinae). These ambrosia beetles are xylophagous species alien to Europe, causing damages to many ornamental and fruiting trees as well as shrubs. DNA extraction was carried out from adults, larvae and biological samples derived from insect damages on infested plants. For X. compactus, segments of galleries in thin infested twigs were cut and processed; in the case of X. crassiusculus, raw frass extruded from exit holes was used, while DNA of X. germanus was extracted from small wood chips removed around insect exit holes. The assays were inclusive for the target species and exclusive for all the non-target species tested. The LoD was 3.2 pg/μL for the frass of X. crassiusculus and 0.016 ng/μL for the woody matrices of the other two species. Both repeatability and reproducibility were estimated on adults and woody samples, showing very low values ranging between 0.00 and 4.11. Thus, the proposed diagnostic assays resulted to be very efficient also on the woody matrices used for DNA extraction, demonstrating the applicability of the protocol in the absence of dead specimens or living stages

Antimicrobial activity of the red palm weevil Rhynchophorus ferrugineus

The red palm weevil, Rhynchophorus ferrugineus (Olivier) (Coleoptera Dryophthoridae), is an important pest of palms. Knowl- edge of both its natural enemies and its defensive mechanisms against predators and microorganisms is important to develop methods for an integrated pest control. Antimicrobial activity of the cuticular surface of adults and larvae, as well as of eggs, of this invasive species was investigated. This activity was tested against the Gram-positive bacteria Bacillus subtilis (Ehrenberg) Cohn and Bacillus thuringiensis Berliner, the Gram-negative bacterium Escherichia coli Escherich, and the entomopathogenic fungi Beauveria bassiana (Balsamo) Vuillemin and Metarhizium anisopliae (Metchnikoff) Sorokin. A similar analysis was con- ducted with the hemolymph of R. ferrugineus\u2019 larvae infected by Pseudomonas aeruginosa (Schroter) Migula, E. coli and Staphy- lococcus aureus Rosenbach. Polar surface fraction of extracts from adults and large larvae inhibits Gram-positive bacteria and the B. bassiana\u2019s growth, but not the growth of E. coli and M. anisopliae. Similarly, the hemolymph of larvae and the surface extracts of both small larvae and eggs seemed not to show any inhibition. Chemical analyses of the fraction exhibiting antimicrobial activ- ity show the presence of some polar compounds ranging between 1000 and 1500 Dalton. This study improves our knowledge on the biology of R. ferrugineus and helps to suggest strategies for the biocontrol of this pest

Taxonomy and phylogeny of European Monochamus species: First molecular and karyological data

The worldwide distributed genus Monochamus Megerle, 1821 (Coleoptera Cerambicydae) comprises beetles that may become pests of economic importance in conifer stands in the Nearctic and Palearctic Regions. Besides direct damage due to the larval tunnelling habits, they have also been recognized as main vectors of the phytoparasitic nematode Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) (Nematoda Aphelenchoididae). We analysed the complete mitochondrial cytochrome oxidase I gene and a fragment of the small subunit RNA gene sequences (1536 base pairs) in the five European species. These are: Monochamus galloprovincialis (Olivier, 1795), morphologically distinguished in two subspecies M. galloprovincialis galloprovincialis (Olivier, 1795) and M. galloprovincialis pistor (Germar, 1818): Monochamus sutor (Linneus 1758); Monochamus saltuarius (Gebler 1830); Monochamus sartor (Fabricius, 1787) and Monochamus urussovi (Fischer, 1806). For appropriate comparisons, also the Asiatic Monochamus alternatus Hope, 1842 and a Japanese M. saltuarius sample have been analysed. Both genes show an absolute identity between the two subspecies of M. galloprovincialis and a strong affinity between M. sartor and M. urussovi: the morphological subdivisions of the former taxon in two subspecies and of the latter in two entities of specific level are therefore not supported genetically. On the other hand, the Italian and the Japanese samples of M. saltuarius always cluster together in all trees, and for the remaining taxa, no doubt about their rank of specific differentiation emerges from present analyses. From a phyletic point of view, tree topology indicates the Japanese M. alternatus as the most differentiated taxon and the Euroasiatic M. saltuarius as basal to all other strictly European entities. Chromosome analyses show that the diploid autosomal complement ranges from 18 in M. saltuarius to 20 in M. galloprovincialis, and 22 in M. sartor, but a XX-Xyp sex determining system is shared by all analysed taxa. The M. saltuarius karyotype appears as the most primitive from which the others may be derived through Robertsonian fissions. Karyological data therefore agree with molecular analyses in indicating a basal position of Euroasiatic M. saltuarius with respect to the group of European Monochamus taxa; among these, M. galloprovincialis and M. sartor represent two clearly diverging evolutionary units. Furthermore, karyotype analyses substantiate molecular conclusions about the identity between M. galloprovincialis galloprovincialis and M. galloprovincialis pistor

Insects as source of angiotensin converting enzyme inhibitory peptides

Hypertension is well known as one of the major risk for cardiovascular diseases which annually affect millions of people. The angiotensin converting enzyme (ACE) plays a key role in blood pressure regulation process. Indeed, hypertension treatment by synthetic ACE inhibitors (e.g. captopril, lisinopril and ramipril) is effective; however, their use can cause serious side effects, such as hypotension, cough, reduced renal function and angioedema. Thus, research was focused on natural ACE inhibitory peptides sources such as foodstuffs and also, more recently, edible insects In the last decades, ACE inhibitory activity has been detected in protein hydrolysates from insect species belonging to the orders of Coleoptera, Diptera, Hymenoptera, Lepidoptera and also Orthoptera. Further investigations led to identify specific ACE inhibitory peptides from the silkworm Bombyx mori (Lepidoptera: Bombycidae), the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae), the cotton leafworm Spodoptera littoralis (Lepidoptera: Noctuidae) and also from the weaver ant Oecophylla smaragdina (Hymenoptera: Formicidae). Even if ACE inhibitory activity of these bioactive peptides has been in vitro assayed and is comparable to those of some bioactive peptides derived from other animal protein sources, the in vivo effectiveness of most of these bioactive peptides still needs to be confirmed. The aim of this review is to present an outline of the currently available data on the potential use of insects for hypertension treatment with a focus on the ACE inhibitory peptides identified in these invertebrates to date

Taxonomy and phylogeny of European Monochamus species: first molecular and karyological data.

The worldwide distributed genus Monochamus Megerle, 1821 (Coleoptera Cerambicydae) comprises beetles that may become pests of economic importance in conifer stands in the Nearctic and Palearctic Regions. Besides direct damage due to the larval tunnelling habits, they have also been recognized as main vectors of the phytoparasitic nematode Bursaphelenchus xylophilus (Steiner & Buhrer, 1934) (Nematoda Aphelenchoididae). We analysed the complete mitochondrial cytochrome oxidase I gene and a fragment of the small subunit RNA gene sequences (1536 base pairs) in the five European species. These are: Monochamus galloprovincialis (Olivier, 1795), morphologically distinguished in two subspecies M. galloprovincialis galloprovincialis (Olivier, 1795) and M. galloprovincialis pistor (Germar, 1818); Monochamus sutor (Linneus 1758); Monochamus saltuarius (Gebler 1830); Monochamus sartor (Fabricius, 1787) and Monochamus urussovi (Fischer, 1806). For appropriate comparisons, also the Asiatic Monochamus alternatus Hope, 1842 and a Japanese M. saltuarius sample have been analysed. Both genes show an absolute identity between the two subspecies of M. galloprovincialis and a strong affinity between M. sartor and M. urussovi: the morphological subdivisions of the former taxon in two subspecies and of the latter in two entities of specific level are therefore not supported genetically. On the other hand, the Italian and the Japanese samples of M. saltuarius always cluster together in all trees, and for the remaining taxa, no doubt about their rank of specific differentiation emerges from present analyses. From a phyletic point of view, tree topology indicates the Japanese M. alternatus as the most differentiated taxon and the Euroasiatic M. saltuarius as basal to all other strictly European entities. Chromosome analyses show that the diploid autosomal complement ranges from 18 in M. saltuarius to 20 in M. galloprovincialis, and 22 in M. sartor, but a XX-Xy(p) sex determining system is shared by all analysed taxa. The M. saltuarius karyotype appears as the most primitive from which the others may be derived through Robertsonian fissions. Karyological data therefore agree with molecular analyses in indicating a basal position of Euroasiatic M. saltuarius with respect to the group of European Monochamus taxa; among these, M. galloprovincialis and M. sartor represent two clearly diverging evolutionary units. Furthermore, karyotype analyses substantiate molecular conclusions about the identity between M. galloprovincialis galloprovincialis and M. galloprovincialis pistor

Galleria mellonella (Lepidoptera Pyralidae): an edible insect of nutraceutical interest

The nutritive values of G. mellonella were until now investigated as feed for insectivore animals and notwithstanding the interesting nutritive content of the larvae and the possibility to affect n-3 fatty acid content by diets, this species is not considered of some interest for human diet. The focus of the present study is to value the effects of two diets with (D1+) and without (D1) the addition of leenseeds, on reaching n-3 fatty acid content and n-6/n-3 fatty acids ratio of interest for human health, in particular in the prevention of cardiovascular diseases. Results highlighted higher n-3 fatty acid values in G. mellonella larvae fed on D1 (2.0g\100g) and on D1+ (14.82g/100g) diets than reported in literature and a lower n-6/n-3 ratio in larvae from D1+ (0.859) than in those recorded from D1 (4.68). Anyway, larvae fed on both the diets showed n-6/n-3 ratio optimal for human health. The study showed the nutraceutical potential of G. mellonella which deserves to be more investigated in the future

THE POTENTIAL BENEFICIAL EFFECTS OF TENEBRIO MOLITOR (COLEOPTERA TENEBRIONIDAE) AND GALLERIA MELLONELLA (LEPIDOPTERA PYRALIDAE) ON HUMAN HEALTH

The nutritional composition of the edible insect species Tenebrio molitor (Coleoptera: Tenebrionidae) and Galleria mellonella (Lepidoptera: Pyralidae) was evaluated in view of their feasible consumption in human diet. Larvae and pupae of both the edible insect species resulted to be a good energy source because of their high total fat content (37.42-48.97% of dry weight). Fatty acid composition was similar in T. molitor larvae and pupae. Conversely, G. mellonella larvae contained a higher percentage of medium and long-chain saturated fatty acids (SFA) than pupae (p= 0.015) while G. mellonella pupae showed a higher content of polyunsaturated fatty acids (PUFA) and a lower n-6/n-3 ratio than larvae (p= 0.003 and p= 0.006, respectively). Both the species contained low cholesterol values (13.56-29.08 mg/100 g of dry weight). The ability of the protein extracts from these insects to reduce blood pressure by inhibiting angiotensin converting enzyme (ACE) has been also investigated. Significantly lower IC50 values were detected either in T. molitor (0.077-0.097 mg/mL) or G. mellonella (0.046-0.605 mg/mL) protein fractions hydrolyzed by gastrointestinal proteases than the corresponding crude extracts. Even if the mealworm T. molitor and the waxmoth G. mellonella show satisfactory values of UFA, low cholesterol contents and the ability to inhibit ACE in vitro, T. molitor larvae provide the SFA/UFA ratio (0.41) that better meets diet requirements for human health, as well as a high environmental efficiency of production. These results suggest the potential use of T. molitor larvae in food industry as ingredient in fortified foods and/or diet supplements for human consumption