Magdalena Gawlak

Molecular Techniques for Detection of Tribolium confusum Infestations in Stored Products

ABSTRACT The confused flour beetle, Tribolium confusum Jacquelin du Val (Coleoptera: Tenebrionidae) is a stored-product pest that contaminates a wide range of food products, from flour and cereals to spices. The insect reduces food quality and is responsible for large economic losses every year. Although several methods for detection of stored-product pests are common and widely used, they are time-consuming and expensive. Therefore, establishing molecular methods of detection of stored-product pests could provide a useful alternative method. We have undertaken attempts to establish methods of detection of T. confusum based on molecular biology techniques of standard and real-time polymerase chain reaction (PCR). Total DNA of T. confusum and red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), used as a negative control, was isolated from insects and used as a template in standard and real-time PCR reactions. Specific primers have been designed on the basis of sequences of internal transcribed spacer (ITS) fragment of rDNA and subunit I of mitochondrial cytochrome oxidase of T. confusum available in the GenBank database. Standard PCR reactions with primers specific to the ITS fragment proved to be reliable and sensitive. Real-time PCR reactions with primers specific for mitochondrial DNA are considered to serve as a supplemental detection method for quantitative assessment of the infestation level.

Molecular techniques for the detection of granary weevil (Sitophilus granarius L.) in wheat and flour

The granary weevil (Sitophilus granarius L.) is a stored grain pest that causes major economic losses. It reduces the quantity and quality of the grain by its feeding and excretion. Sequences of S. granarius mitochondrial cytochrome oxidase subunits genes mtCOI and mtCOII were analysed and compared with mtCOI/II sequences available in GenBank. The analysed genes displayed a high level of homology between corresponding subunits. Attempts were undertaken to develop detection methods for contamination by S. granarius in wheat and wheat flour based on the molecular biology techniques: standard and real-time polymerase chain reaction (PCR) with a TaqMan® molecular probe. (TaqMan probes are dual-labelled hydrolysis probes) Specific primers designed based on available sequences for mtCOI and mtCOII genes were applied and optimal reaction conditions established. The specificity of both methods was studied by using a species closely related to S. granarius: S. oryzae and S. zeamais. It is shown that the sensitivity threshold was very high – we were able to detect the equivalent of one beetle per 100 kg of flour when the real-time PCR with TaqMan probe method was applied to model samples. The primer sets used turned out to be species specific, and the technique was rapid, reliable and very sensitive.

MORPHOLOGICAL AND MOLECULAR FEATURES OF PUNCTODERA STONEI BRZESKI, 1998 (NEMATODA: HETERODERIDAE) - SPECIES ASSOCIATED WITH ROOTS OF GRASSES

Abstract. This article provides morphological and molecular characteristics of Punctodera stonei Brzeski, 1998. Comparison of partial sequences of 18S and 28S rDNA genes from P. stonei sampled in Poland and Punctodera sp. from Canada showed their 100% similarity. This is the first report on the occurrence of P. stonei outside of Europe. We provide data on morphology of males and 2nd stage juveniles of this species and an identification key to males of the genus Punctodera Mulvey et Stone, 1976. Moreover, the paper presents evolutionary relationships of P. stonei within the family Heteroderidae.

An integrative description of a new tardigrade species Mesobiotus romani sp. nov. (Macrobiotidae: harmsworthi group) from the Ecuadorian Pacific coast

In a mixed moss and lichen sample collected in Esmeraldas Province in north-western Ecuador, 20 tardigrades and 11 eggs, belonging to a new species of the genus Mesobiotus, were found. In addition to the traditional taxonomic description with morphometrics, light and scanning microscopy imaging, we also provide nucleotide sequences of three nuclear (18S rRNA, 28S rRNA, ITS-2) and one mitochondrial (COI) DNA fragments of the new species. Based on the egg chorion morphology, Mesobiotus romani sp. nov. is the most similar to: M. binieki, M. coronatus, M. dimentmani, M. patiens, M. perfidus, M. philippinicusi, M. pseudoblocki, M. pseudocoronatus, M. pseudopatiens, M. radiatus, M. rigidus, M. simulans and M. wuzhishanensis, but differs mainly by some specific characters of both egg and adult morphology, and morphometrics.

Macrobiotus naskreckii sp. nov., a New Tardigrade (Eutardigrada: Macrobiotidae) of the Hufelandi Group from Mozambique

Abstract In this paper, Macrobiotus naskreckii sp. nov., a new species of the hufelandi group from the Gorongosa National Park in Mozambique, is described. The analysis have revealed that M. naskreckii sp. nov. is most similar to M. kristenseni, M. ramoli and M. serratus. The new species differs from M. kristenseni mainly through a different shape of egg processes and clearly dentate lunules IV; from M. ramoli mainly by smaller eggs and a smaller number of egg processes on the circumference; from M. serratus mainly by the absence of the reticulation between the egg processes. This is a third record of tardigrades from Mozambique and apart of the new species reported here, three other taxa from Gorongosa National Park are provided: Minibiotus cf. intermedius, Minibiotus sp. and Paramacrobiotus cf. richtersi.

An integrative redescription of the nominal taxon for the Mesobiotus harmsworthi group (Tardigrada: Macrobiotidae) leads to descriptions of two new Mesobiotus species from Arctic

The Mesobiotus harmsworthi group has a global distribution, with localities in polar, temperate and tropical zones. Since the first species of the harmsworthi group was described in the beginning of the 20th century, tens of new species within the group were found and named. However, the diagnosis of the nominal Mesobiotus harmsworthi is insufficient and enigmatic, thus it can be is a serious obstacle in solving the taxonomy of this group. Here, we integratively redescribe the nominal species for the genus Mesobiotus, i.e., Mesobiotus harmsworthi and clarify taxonomic statuses of the two subspecies: M. harmsworthi harmsworthi and M. harmsworthi obscurus that have been recognised as distinct taxa for more than three decades. Traditionally, egg chorion in M. harmsworthi was considered almost smooth and without any traces of areolation, however here we report many misunderstandings that accumulated across decades and we show that, in fact, the chorion in this species exhibits a partially developed areolation. We present an integrative (morphological, morphometric and molecular) diagnosis of the nominal taxon and we confirm that it differs from other species of the harmsworthi group by morphological characters of both animals and eggs. Additionally, we describe two new species of the genus Mesobiotus: M. skorackii sp. nov. from the Kyrgyz Republic (using classical morphological description) and M. occultatus sp. nov. from Svalbard Archipelago (by means of integrative taxonomy). Finally, we also provide the first genetic phylogeny of the genus Mesobiotus based on COI sequences which, together with molecular species delimitation, independently confirms the validity of the analysed taxa.