Oliver Stojkovic

One method of crop protection is the application of a widely used group of pesticides—pyrethroids. As xenobiotics, sub-lethal doses of insecticides cause stress in pests, resulting in a change in the shape and size of their organs or bodies. The stress caused by pesticides may lead to acute destabilization of development, but also to transgenerational canalization through the process of genetic assimilation. Fluctuating asymmetry (FA), small random deviations between the right and left sides of bilaterally symmetrical traits, is an outcome of developmental instability and is a measurable indicator of phenotypic response to stress. We exposed four populations of the seed beetle Acanthoscelides obtectus to sub-lethal doses of cypermethrin for ten generations in a laboratory evolution experiment. Using geometric morphometrics, we analyzed size and shape changes and the level of fluctuating asymmetry in untreated beetles and in samples from the fifth and tenth generation. Exposure over ten generations led to an increase in the body size of the beetles, shortening of their pronotum and elongation of their thorax and abdomen. After ten generations of exposure to cypermethrin, FA levels decreased, indicating a canalization of development. This study provides new insights into the phenotypic markers of environmental pollution from agricultural activities.

Blind mole rats (genus Nannospalax) attract a great deal of attention because of their cancer resistance and longevity. Due to the high rate of chromosome rearrangements, 74 Nannospalax chromosomal forms have been discovered. The convergence of their external morphology complicates their taxonomy, and many cryptic species remain unrecognized. Thus, the European N. leucodon supersp. is listed in the IUCN Red List of Threatened Species with “Data Deficient” status. It is crucial for the conservation of biodiversity to clarify its taxonomy, to recognize each cryptic species, and assign to them the correct conservation status. Of the more than 20 chromosomal forms described within N. leucodon, five cryptic species occur in Serbia. The most threatened among them—N. l. syrmiensis, described and named 50 years ago in the regions of Srem, Belgrade and Mačva—has been declared extinct in the literature, which may have negative consequences for the conservation of wildlife genetic diversity. Through five years of fieldwork and comparison of 16SrRNA and MT-CYTB gene segments between old, archived teeth and recently collected material, we show that N. l. syrmiensis is not extinct. However, its habitat has been fragmented and reduced, owing primarily to anthropogenic impact. Therefore, detailed surveillance, population-structure studies, risk assessment, and appropriate conservation measures are needed.

We explored the cryptic speciation of the Nannospalax leucodon species complex, characterised by intense karyotype evolution and reduced phenotypic variability that has produced different lineages, out of which 25 are described as chromosomal forms (CFs), so many cryptic species remain unnoticed. Although some of them should be classified as threatened, they lack the official nomenclature necessary to be involved in conservation strategies. Reproductive isolation between seven CFs has previously been demonstrated. To investigate the amount and dynamics of genetic discrepancy that follows chromosomal changes, infer speciation levels, and obtain phylogenetic patterns, we analysed mitochondrial 16S rRNA and MT-CYTB nucleotide polymorphism among 17 CFs—the highest number studied so far. Phylogenetic trees delineated 11 CFs as separate clades. Evolutionary divergence values overlapped with acknowledged higher taxonomic categories, or sometimes exceeded them. The fact that CFs with higher 2n are evolutionary older corresponds to the fusion hypothesis of Nannospalax karyotype evolution. To participate in conservation strategies, N. leucodon classification should follow the biological species concept, and proposed cryptic species should be formally named, despite a lack of classical morphometric discrepancy. We draw attention towards the syrmiensis and montanosyrmiensis CFs, estimated to be endangered/critically endangered, and emphasise the need for detailed monitoring and population survey for other cryptic species.

Physiological adaptations to various types of prolonged and intensive physical activity, as seen in elite athletes from different sports, include changes in blood pressure (BP) response to acute exercise. Also, functional polymorphisms of the angiotensin I converting enzyme (ACE) and alfa-actinin-3 (ACTN3) genes are shown to be associated with BP parameters changes, both in athletes and sedentary population. In this study, an Alu insertion (I)/deletion (D) polymorphism in ACE gene, as well as nonsense mutation in the gene encoding ACTN3 have been scored in 107 elite Serbian athletes classified according to their sporting discipline to power/sprint (short distance runners/swimmers), endurance (rowers, footballers, middle-distance swimmers) or mixed sports (water polo, handball, volleyball players). Presence of nonfunctional allele in ACTN3 is associated with significantly increased maximal systolic BP (SBPmax, p = 0.04). Athletes with Alu insertion in ACE had significantly (p = 0.006) larger decline of systolic BP after 3 minutes of recovery (SBPR3), calculated as the percentage of maximal SBP response during exercise stress testing. Concomitant presence of non-functional variant in ACTN3 gene decreased this beneficiary effect of ACE mutation on SBPR3. Long term enrollment in power/sprint sports significantly increased resting diastolic BP (DBPrest: 74 mmHg) and SBPmax (197 mmHg) and improved SBPR3 (74.8%) compared to enrolment in endurance (72 mmHg; 178 mmHg; 81.1%) and mixed sports (69 mmHg; 185 mmHg; 80.0%). Lack of the effect of genotype by sport interaction on BP parameters suggests that the long-term effects of different disciplines on BP are not mediated by these two genes.

Methylenetetrahydrofolate reductase (MTHFR) plays a critical role in the folate metabolism. The polymorphism 677C>T of the MTHFR gene, producing thermolabile enzyme with decreased function, is widely studied and associated with many conditions. Additionally, it has been shown that another polymorphism, 1298A>C, also reduces the activity of this enzyme, although to a lesser extent. The aim of this study is to evaluate the clinical informativeness of testing both MTHFR polymorphisms. Genomic DNA, were extracted from peripheral blood of 180 female patients with pregnancy complications and 183 healthy female controls, and genotyped for MTHFR 677C>T and 1298A>C loci, using TaqMan assays. Our study found similar frequency of alleles and genotypes between two groups. Based on MTHFR 677C>T genotype, 11.7% of patients homozygous for this mutation were under the possible risk. When the position 1298 was included in the testing, 22.8% of the patients were heterozygous for both polymorphisms. Additionally, 8.9% of the patients were homozygous only for the MTHFR 1298 mutation. Although,s there was no differences compared to healthy control (p>0.05), 43% of patients were found to have elevated risk which is about four time highers than results with only MTHFR 677C>T genotyping. After obtaining information for the 677 position, testing for the second polymorphism (1298A>C) should be considered, since we have shown that it dramatically increases the rate of detection of patients who are potentially at risk for MTHFR associated conditions.

Reproductive failure (recurrent foetal loss, unexplained infertility and IVF implantation failure) may be, in a number of cases, explained by thrombophilia, either acquired or inherited. Several genes contribute to thrombophilia, some with major effect (Factor V, Factor II), and some with minor effect (MTHFR, PAI-1, ATIII, etc.). The aim of this study was to estimate frequency of thrombophilia-associated genotypes (FII20210G > A, FV1691G > A, MTHFR677C > T and PAI-1 -675 4G/5G) in a group of 1631 Serbian women experiencing reproductive failure, and compare it with a healthy, female control group. Our results showed marginally significant (p = 0.050) differences in allele frequencies between patients and controls for the FV1691 mutations. For the FII20210G > A, although the statistical significance was not achieved (p = 0.076), we found higher frequency of variant allele in patients compared to controls (1.87% vs. 0.38%, respectively) which may point to a possible role of this polymorphism in thrombotic events. For the MTHFR677C > T and PAI-1 -675 4G/5G, we found no difference in distributions of genotype or allele frequencies between these two groups (p > 0.05). For three subjects with very rare genotypes (two patients homozygous for FV1691G > A and one patient homozygous for FII20210G > A) we performed additional biochemical analyses for haemostasis, as well as genotyping of two polymorphisms (MTHFR1298A > C and ATIII786G > A).