Background: SINEs (Short INterspersed Elements) are homoplasy-free and co-dominant genetic markers which are considered to represent useful tools for population genetic studies, and could help clarifying the speciation processes ongoing within the major malaria vector in Africa, Anopheles gambiae s.s. Here, we report the results of the analysis of the insertion polymorphism of a nearly 200 bp-long SINE (SINE200) within genome areas of high differentiation (i.e. "speciation islands") of M and S A. gambiae molecular forms. Methods: A SINE-PCR approach was carried out on thirteen SINE200 insertions in M and S females collected along the whole range of distribution of A. gambiae s.s. in sub-Saharan Africa. Ten specimens each for Anopheles arabiensis, Anopheles melas, Anopheles quadriannulatus A and 15 M/S hybrids from laboratory crosses were also analysed. Results: Eight loci were successfully amplified and were found to be specific for A. gambiae s.s.: 5 on 2L chromosome and one on X chromosome resulted monomorphic, while two loci positioned respectively on 2R (i.e. S200 2R12D) and X (i.e. S200 X6.1) chromosomes were found to be polymorphic. S200 2R12D was homozygote for the insertion in most S-form samples, while intermediate levels of polymorphism were shown in M-form, resulting in an overall high degree of genetic differentiation between molecular forms (Fst = 0.46 p < 0.001) and within M-form (Fst = 0.46 p < 0.001). The insertion of S200 X6.1 was found to be fixed in all M- and absent in all S-specimens. This led to develop a novel easy-to-use PCR approach to straightforwardly identify A. gambiae molecular forms. This novel approach allows to overcome the constraints associated with markers on the rDNA region commonly used for M and S identification. In fact, it is based on a single copy and irreversible SINE200 insertion and, thus, is not subjected to peculiar evolutionary patterns affecting rDNA markers, e.g. incomplete homogenization of the arrays through concerted evolution and/or mixtures of M and S IGS-sequences among the arrays of single chromatids. Conclusion: The approach utilized allowed to develop new easy-to-use co-dominant markers for the analysis of genetic differentiation between M and S-forms and opens new perspectives in the study of the speciation process ongoing within A. gambiae.

Stingless bees (Hymenoptera: Meliponini) are among the most important pollinators of tropical forests. Peru is considered a hotspot of biodiversity of Meliponini, but many areas of this country (e.g., Peruvian Amazon) remain unexplored. We aimed to produce a first inventory of stingless bee species dwelling in humid and seasonally dry forests of northern Peru by combining traditional (morphologically-based) taxonomy and DNA barcoding. Specimens were collected in 2020 at five sites located in San Martin and Piura regions. We identified 12 genera of Meliponini. Among those, Trigona and Plebeia were the most abundant (45.9% and 12.8% respectively), whereas Nannotrigona and Scaura were the least represented ones (2.3%). We assigned a reliable species identification to about 30% of specimens (Trigona amazonensis, T. muzoensis, T. williana, Partamona testacea, Scaura tenuis, Tetragona goettei, and Tetragonisca angustula). Yet, more than a half of the specimens received a provisional identification (e.g., Geotrigona cf. fulvohirta, T. cf. amalthea, T. cf. fuscipennis, T. cf. hypogea, Melipona cf. cramptoni, Partamona cf. epiphytophila, Ptilotrigona cf. perenae, Scaura cf. latitarsis, Tetragona cf. clavipes, Trigonisca cf. atomaria). We also highlighted an extensive polyphyly that affected a number of currently recognized species (e.g., T. fulviventris, T. guianae, Plebeia franki, P. frontalis, M. eburnea, M. illota), whose members were split into various clades. Finally, 16% of individuals failed to be identified at the species level (Trigona sp. 1, T. sp. 2, Nannotrigona sp., Partamona sp., Scaptotrigona sp. 1, S. sp. 2, Trigonisca sp. 1, and Trigonisca sp. 2). We discuss our findings according to the current faunistic and biogeographic knowledge of Meliponini in Peru and the Neotropical region. We also remark on the importance of conducting a taxonomic revision of stingless bees and improving both their morphology-based identification keys and BOLD repository. Finally, we claim that integrative taxonomy shall be strongly implemented to truly assess the biodiversity of Neotropical stingless bees, allowing conserving these important pollinators and the associated traditional meliponiculture in an effective manner.

The organization of the mitochondrial DNA (mtDNA) control region (CR) of the pollen beetle Meligethes thalassophilus is described. This mtDNA CR represents the longest sequenced for beetles so far, since the entire nucleotide sequence ranges from approximately 5000 to approximately 5500 bp. The CR of M. thalassophilus is organized in three distinct domains: a conserved domain near the tRNAIle gene, a variable domain flanking the 12S rRNA gene, and a relatively large central tandem array made up of a variable number of approximately 170 bp repeats that is responsible for the intraspecific length variation observed. Like other CRs found in insects, the M. thalassophilus CR contains two long homopolymeric runs that may be involved in mtDNA replication. Furthermore, conserved stem-and-loop structures in the repetitive domain were identified and their possible role in generating length variation is examined. Intraspecific comparison of the tandem repeat elements of M. thalassophilus suggests mechanisms of concerted evolution leading to homogenization of the repetitive region. The utility of such an array of tandem repeats as a genetic marker for assessing population-level variability and evolutionary relationships among populations is discussed. Finally, the technical difficulties found in isolating the mtDNA CR in beetles are remarked upon.

Polymerase chain reaction (PCR)-based genotyping of mutations in the voltage-sensitive sodium channel (vssc) associated with resistance to pyrethroid insecticides is widely used and represents a potential early warning and monitoring system for insecticide resistance arising in mosquito populations, which are vectors of different human pathogens. In the secondary vector Aedes albopictus—an Asian species that has invaded and colonized the whole world, including temperate regions—sequencing of domain II of the vssc gene is still needed to detect the V1016G mutation associated with pyrethroid resistance. In this study we developed and tested a novel allele-specific PCR (AS-PCR) assay to genotype the V1016G mutation in this species and applied it to the analysis of wild populations from Italy. The results confirm the high accuracy of the novel AS-PCR and highlight frequencies of the V1016G allele as >5% in most sampling sites, with peaks of 20–45% in coastal touristic sites where pyrethroid treatments are extensively implemented, mostly for mosquito nuisance reduction. The high frequency of this mutation observed in Italian Ae. albopictus populations should serve as a warning bell, advocating for increased monitoring and management of a phenomenon which risks neutralizing the only weapon today available to counteract (risks of) arbovirus outbreaks.

The LIFE project “MIPP” - Monitoring of Insects with Public Participation (11 NAT/IT/000252) is focused on selected insect species (five Coleoptera, three Lepidoptera, one Orthoptera), all included in the annexes II and IV of the Habitats Directive (HD) 92/43/EEC. One important aim is a citizen science initiative where every person may become a citizen scientist and collect faunistic data on the above species throughout Italy. Another objective of the project MIPP is the development of standard methods for monitoring the conserva- tion status of the five target beetle species. One innovative method employed is a sniffer-dog (“Osmodog”), trained to find the rare and endangered hermit beetle, Osmoderma eremita, which lives in veteran, hollow trees. The dog detects the strong smell of mature peach produced by adult males and an odor produced by the larvae. Another objective of the project MIPP is the dissemination of topics such as HD, Natura 2000, importance of dead-wood, Life projects, insect monitoring and conservation.

Background: Anopheles gambiae M and S molecular forms, the major malaria vectors in the Afro-tropical region, are ongoing a process of ecological diversification and adaptive lineage splitting, which is affecting malaria transmission and vector control strategies in West Africa. These two incipient species are defined on the basis of single nucleotide differences in the IGS and ITS regions of multicopy rDNA located on the X-chromosome. A number of PCR and PCR-RFLP approaches based on form-specific SNPs in the IGS region are used for M and S identification. Moreover, a PCR-method to detect the M-specific insertion of a short interspersed transposable element (SINE200) has recently been introduced as an alternative identification approach. However, a large-scale comparative analysis of four widely used PCR or PCR-RFLP genotyping methods for M and S identification was never carried out to evaluate whether they could be used interchangeably, as commonly assumed. Results: The genotyping of more than 400 A. gambiae specimens from nine African countries, and the sequencing of the IGS-amplicon of 115 of them, highlighted discrepancies among results obtained by the different approaches due to different kinds of biases, which may result in an overestimation of MS putative hybrids, as follows: i) incorrect match of M and S specific primers used in the allele specific-PCR approach; ii) presence of polymorphisms in the recognition sequence of restriction enzymes used in the PCR-RFLP approaches; iii) incomplete cleavage during the restriction reactions; iv) presence of different copy numbers of M and S-specific IGS-arrays in single individuals in areas of secondary contact between the two forms. Conclusions: The results reveal that the PCR and PCR-RFLP approaches most commonly utilized to identify A. gambiae M and S forms are not fully interchangeable as usually assumed, and highlight limits of the actual definition of the two molecular forms, which might not fully correspond to the two A. gambiae incipient species in their entire geographical range. These limits are discussed and operational suggestions on the choice of the most convenient method for large-scale M- and S-form identification are provided, also taking into consideration technical aspects related to the epidemiological characteristics of different study areas.