Towards understanding Cameraria ohridella (Lepidoptera: Gracillariidae) development: effects of microhabitat variability in naturally growing horse-chestnut tree canopy.

Dwelling intensity of horse-chestnut miner (Cameraria ohridella) larvae in various leaves insolation and temperature was measured to determine whether this pest's development follows a predictable pattern or depends more on local microenvironment conditions. Mines growing on leaves of mature host plants (Aesculus hippocastanum L.) in their natural conditions were photographed for two consecutive generations of the pest and in two separated vegetation periods. Apart from meteorological data obtained from the nearest station, the temperature of intact and mined parts of sun-exposed and shaded leaf blades was measured at various daytimes throughout the experiment. Obtained sets of digital data were analysed and combined to model mine area growth as a function of degree-days sum by adopting of Verhulst logistic equation. We showed the predictive potential of our model based on experimental data, and it may be useful in the scheduling of pest control measures in natural conditions. Our analyses also revealed that despite significant differences in microenvironment conditions depending on mines' insolation, the horse-chestnut miner larvae could partially compensate for them and complete their development at similar endpoints expressed as the cumulative sum of degree-days. We conclude that computer-aided analysis of photographic documentation of leaf-miner larval growth followed by mathematical modelling offers a noninvasive, reliable, and inexpensive alternative for monitoring local leaf-miners populations.

Set of stress biomarkers as a practical tool in the assessment of multistress effect using honeybees from urban and rural areas as a model organism: a pilot study.

A decrease among honey bee populations (Apis mellifera) in the traditional apiaries has been observed in recent years. In light of this negative phenomenon, urban beekeeping seems to be an appropriate alternative solution for the bee population in reducing the toxic effects of a large number of pesticides that are commonly used in agricultural ecosystems. Despite the rapid development of urban beekeeping, there is little information regarding the different aspects of the defense effectiveness of bees from the urban and rural areas. The study was aimed to show whether honey bees from these two locations differ in the level of the valuable biomarkers of stress exposure helpful in establishing which bees, from urban or rural areas, are under greater environmental pressure. For this purpose, foragers from an urban rooftop apiary and a traditional rural apiary were collected. The chosen biomarkers were measured in various tissues of bees. The activity of glutathione S-transferase and acetylcholinesterase, the level of total antioxidant capacity, heat shock protein 70 (Hsp70), and defensin were selected for the analyses. In our opinion, the Hsp70 and defensin levels seemed to be important in the indication of urban multistress factors. The higher level of heat shock proteins and defensins in tissues/organs of bees from the urban apiary-in the gut (an increase, respectively, 92% and 7.3%) and fat body (an increase, respectively, 130% and 7.8%), known as targets of environmental toxins, pointed out the urban environment as highly stressful at both the individual and colony levels. In turn, high total antioxidant capacity was measured in the guts of honey bees from rural area (an increase 107%). Such a situation suggests a different mechanism of defense and specificity of rural and urban environmental stressors and also honey bees foraging activity.

Elemental distribution in reproductive and neural organs of the Epilachna nylanderi (Coleoptera: Coccinellidae), a phytophage of nickel hyperaccumulator Berkheya coddii (Asterales: Asteraceae) by micro-PIXE.

The phenomenon of metal hyperaccumulation by plants is often explained by a pathogen or herbivore defense hypothesis. However, some insects feeding on metal hyperaccumulating plants are adapted to the high level of metals in plant tissues. Former studies on species that feed on the leaves of Berkheya coddii Roessler 1958 (Asteraceae), a nickel-hyperaccumulating plant, demonstrated several protective mechanisms involved in internal distribution, immobilization, and elimination of Ni from the midgut and Malpighian tubules. These species are mainly coleopterans, including the lady beetle, Epilachna nylanderi (Mulsant 1850) (Coleoptera: Coccinellidae), collected from the ultramafic ecosystem near Barberton in South Africa. By performing particle-induced X-ray emission microanalysis elemental microanalysis (PIXE), this study examined whether Ni may be harmful to internal body systems that decide on insect reactivity (central nervous system [CNS]), their reproduction, and the relationships between Ni and other micronutrients. Data on elemental distribution of nine selected elements in target organs of E. nylanderi were compared with the existing data for other insect species adapted to the excess of metals. Micro-PIXE maps of seven regions of the CNS showed Ni mainly in the neural connectives, while cerebral ganglia were better protected. Concentrations of other bivalent metals were lower than those of Ni. Testis, compared with other reproductive organs, showed low amounts of Ni. Zn was effectively regulated at physiological dietary levels. In insects exposed to excess dietary Zn, it was also accumulated in the reproductive organs. Comparison of E. nylanderii with other insects that ingest hyperaccumulating plants, especially chrysomelid Chrysolina clathrata (Clark) (Coleoptera: Chrysomelidae), showed lower protection of the CNS and reproductive organs.

Parnassius apollo last-instar larvae development prediction by analysis of weather condition as a tool in the species' conservation.

Parnassius apollo (Lepidoptera: Papilionidae) has already disappeared or is under threat of extinction in many of its former habitats. It has been documented that weather conditions--anomalies in particular--contributed to this process. In this study, we combined developmental data obtained previously for the last-instar Apollo larvae (collected in 1996, 1997, and 2003) with corresponding meteorological data to assess the effects of ambient temperature and rainfall episodes on the duration and the completion of the instar. For comparing the temperature effect, we applied the degree-day concept. We found significant positive correlation between the number of rainy days during the instar development (x) and its duration time (y): y=8.293+0.936x (±2.813) (r=0.662, P<10(-7)). Logarithmic transformation of the growth curves of the last-instar Apollo larvae revealed that there was no difference in growth among females; however, there was slower growth of males in 2003 in comparison to 1996. Growth (y) of female Apollo larvae as a function of instar duration (x) can by described by one common equation, irrespectively of the year: y=317.6+502.3 lnx (±263.3) (r=0.82, P<10(-4)).

Digestive enzymes activity in subsequent generations of Cameraria ohridella larvae harvested from horse chestnut trees after treatment with imidacloprid.

In the present study we describe the effect of chloronicotinoid pesticide (imidacloprid) on the digestive enzymes activity of the Cameraria ohridella larvae after lasting 1 year sublethal exposure to imidacloprid pesticide. Caterpillars - L4 stage (fourth instar, hyperphagic tissue-feeding phase) - were collected from chemically protected white horse chestnut trees 1 year after imidacloprid treatment, and compared with caterpillars collected from non-treated trees in a previous study. Enzymes activity of α-amylase, disaccharidases, glycosidases and proteases was assayed. The presence of pesticide in ingested food changed the digestive enzymes profile of caterpillars. The analysis of correlations between different digestive enzymes showed many significant correlations (P<0.05) among glycolytic activities like ß-glucosidase and α-galactosidase activities. Statistically significant correlations for proteolytic activity were found between trypsin and chymotrypsin activity and aminopeptidase activity that occurred only in the 1st generation. PCA distinguished five primary components with eigenvalues higher than 1, from which the first two explain almost 59% of analyzed results. Surprisingly, in the pesticide treated groups significantly higher activities of sucrase and lactase in relation to control were found. In general, glycosidase (α-glucosidase, ß-glucosidase and ß-galactosidase) activities showed a similar pattern of activity in different generations. These results contrast with those obtained with control larvae, where significant differences in activities of α-glucosidase, ß-glucosidase and ß-galactosidase may result from the different quantity and quality food intake by subsequent generations of larvae. No inter-generation differences in total proteolytic activity were observed in treated larvae. The absolute value of total proteolytic activity was higher than that in the control group. The pesticide present in the vascular system of the horse chestnut tree significantly affected some of the digestive enzymes activities and - in consequence - also interrelationships between enzymes, what may affect the food digestion.

Occurrence of sarmentosin and other hydroxynitrile glucosides in Parnassius (papilionidae) butterflies and their food plants.

Sequestration of plant secondary metabolites is a widespread phenomenon among aposematic insects. Sarmentosin is an unsaturated γ-hydroxynitrile glucoside known from plants and some Lepidoptera. It is structurally and biosynthetically closely related to cyanogenic glucosides, which are commonly sequestered from food plants and/or de novo synthesized by lepidopteran species. Sarmentosin was found previously in Parnassius (Papilionidae) butterflies, but it was not known how the occurrence was related to food plants or whether Parnassius species could biosynthesize the compound. Here, we report on the occurrence of sarmentosin and related compounds in four different Parnassius species belonging to two different clades, as well as their known and suspected food plants. There were dramatic differences between the two clades, with P. apollo and P. smintheus from the Apollo group containing high amounts of sarmentosin, and P. clodius and P. mnemosyne from the Mnemosyne group containing low or no detectable amounts. This was reflected in the larval food plants; P. apollo and P. smintheus larvae feed on Sedum species (Crassulaceae), which all contained considerable amounts of sarmentosin, while the known food plants of the two other species, Dicentra and Corydalis (Fumariaceae), had no detectable levels of sarmentosin. All insects and plants containing sarmentosin also contained other biosynthetically related hydroxynitrile glucosides in patterns previously reported for plants, but not for insects. Not all findings could be explained by sequestration alone and we therefore hypothesize that Parnassius species are able to de novo synthesize sarmentosin.

The localisation of HSP70 and oxidative stress indices in heads of Spodoptera exigua larvae in a cadmium-exposed population.

The effects of cadmium toxicity may vary between animals with different history of metal exposure. The aim of our study was to examine HSP70, protein carbonyl levels, catalase activity and total antioxidant capacity in the heads of Spodoptera exigua (Hübner) larvae originated from undergoing 1- and 44-generational cadmium treatment and in control (those that were not exposed to cadmium). We also measured the cadmium concentration and DNA damage level in the larvae. We observed higher level of heat shock proteins (HSPs) in the heads of larvae derived from multi-generational metal treatment than in the heads of those from one-generational treatment (derived from the control rearing). Analysis of HSP localisation in the larval brain suggests that these changes could be important for protecting the neural function of larval mushroom bodies for animals selected during multigenerational metal exposure. Animals from one-generational treatment had, in turn, higher total antioxidant capacity than animals from multigenerational treatment. Anyway, animals from one- and 44-generational metal treatments did not differ in metal accumulation in the heads and the whole larval bodies, catalase activity or DNA damage level. All these measurements were higher than for control larvae and cadmium accumulation in the heads was much lower than in the whole bodies.

Digestive enzymes activity in larvae of Cameraria ohridella (Lepidoptera: Gracillariidae).

This article presents the activity of carbohydratases and proteases in the midgut of Cameraria ohridella larvae--an oligophagous pest whose preferred feeding is horse chestnuts leaves. Optimal media pH of the assayed enzymes were similar to those of other Lepidopterans. Relatively high amylase activity, as well as maltase and sucrase activities, indicates that starch and sucrose are the main digested saccharides. Trehalase activity was similar to that described in other Lepidopterans. Activities of glycosidases were significantly lower than those of disaccharidases what suggests that neither cellulose nor glycosides are important for C. ohridella. Trypsin is the main endoprotease of this pest. Like in other leaf-eaters carboxypeptidase activity was higher than that of aminopeptidase. The activity of the majority of examined enzymes increased in the following successive pest generations, which could be explained by the decreased nutritional value of older leaves. Probably this phenomenon in hydrolases activity in Cameraria is a nonspecific mechanism present at this stage of co-evolution of the horse chestnut and its pest.

[Prenatal diagnosis of fetal chromosomal aneuploidies using quantitative fluorescent PCR (QF-PCR)]. / Wykorzystanie ilosciowej fluorescencyjnej techniki PCR (QF-PCR) w diagnozowaniu wybranych aneuploidii chromosomowych u plodu.

The trisomy of the 21 chromosomes is one of the most important chromosomal anomalies and is responsible for Down syndrome. It persists in Poland in 1/700 birth. The risk of childbirth with Down syndrome grows with age, especially in women after 35 years old. In this study we performed QF-PCR method with specific polymorphic sequence--D21S11 for detection of trisomy of the 21 chromosome. DNA for PCR was isolated from amniotic fluid, blood of healthy and with Down syndrome patients. PCR was performed with ProfilerPlus Kit, which permits for amplification of 9 STR loci (also D21S11) and gender marker--amelogenin gene. We detected incorrect amplification of STR sequence for D21S11 in one amniotic fluid, which was confirmed by kariotyping as trisomy of the 21 chromosome of fetus.

Midgut protease activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri.

We assayed the relative activities of midgut proteolytic enzymes in individuals of the fourth (L(4)) and fifth (L(5)) instar of Apollo larvae, inhabiting Pieniny Mts (southern Poland). The comparisons between midgut tissue with glicocalyx (MT) and liquid midgut contents with peritrophic membrane (MC) were made. Optimal media pHs of the assayed proteolytic enzymes in P. apollo midgut samples were similar to those of other lepidopteran species. Endopeptidases, as well as carboxypeptidases, digested effectively in alkaline environment, while aminopeptidases were active in a broad pH range. Trypsin is probably the main endoprotease (correlation with caseinolytic activity in MC of L(5) larvae: r=0.606; p=0.004); however, its activity was low as compared with that in other leaf-eating Lepidoptera. This suggests a minor role of trypsin and chymotrypsin in protein digestion in Apollo larvae, probably due to limited availability of the leaf proteins. Instead, due to very high carboxypeptidase A activity in midgut tissue, the larvae obtain exogenous amino acids either directly or from oligopeptides and glycoproteins. High and significant positive correlations between the enzyme activity and glucosidase as well as galactosidase activities strongly support this opinion.

Predictive value of HPV DNA in lymph nodes in surgically treated cervical carcinoma patients--a prospective study.

OBJECTIVE: High-risk types of HPV are etiological factors in cervical cancer. Lymph node involvement in cervical cancer patients reduces 5-year survival rates by 25-60%. However, the influence on survival of HPV DNA positivity in histopathologically negative lymph nodes remains unresolved. METHODS: The study included 116 of 148 patients who underwent Piver type III radical hysterectomy and pelvic lymphadenectomy and who showed HPV DNA positivity in the primary lesion. Lymph node tissues were tested for the presence of HPV DNA, using a PCR technique. RESULTS: We found the presence of HPV DNA sequences in lymph nodes dissected intraoperatively in 81 (69.83%) cases. In analysis, we compared patients from 3 groups: HPV- and metastatic-negative (LN HPV-M-); HPV-positive metastatic-negative (LN HPV+M-); and metastatic-positive (LN M+). We discovered that survival in groups LN M+ and LN HPV+M- did not differ statistically (p=0.37). However, the survival periods in these two groups differed when compared with LN HPV-M- patients (p<0.001). Using Cox's proportional hazards model, we found that the presence of lymph node HPV DNA, and FIGO stage, and primary lesion volume were independent parameters correlating with survival and mortality risk. CONCLUSION: We conclude that the presence of HPV DNA in lymph nodes is an early sign of metastasis and should be treated as such in prognostic outlook and planning the therapeutic strategy.

[Heterotopic pregnancy after in vitro fertilization]. / Przypadek ciqzy heterotopowej po zaplodnieniu pozaustrojowym.

In our paper we described a rare case of heterotopic pregnancy after in vitro fertilization (IVF-ET). Heterotopic pregnancy (coexistence of intrauterine and ectopic pregnancy) appears in 1 per 30 thousand cases of natural pregnancies and in 1% of pregnancies after IVF. The pregnancy was obtained after ICSI and transfer of 2 embryos. The pregnancy was confirmed by HCG serum presence--13 days after ET. On day 39 after IVF procedure, the patient was admitted to the hospital because of abdominal pain. At admission, ultrasound examination confirmed an 8 week intrauterine pregnancy and enlarged polycystic ovaries. After 3-day-long observation, patient status worsened and laparoscopy procedure was performed. The ectopic pregnancy was confirmed during operation and the ovum was removed a long with fallopian tube. Ultrasound examination on the 6th day after operation confirmed single intrauterine viable fetus. in order to diagnose the possible complications, women undergoing IVF procedure should be carefully observed by IVF performing physicians.

Midgut glycosidases activities in monophagous larvae of Apollo butterfly, Parnassius apollo ssp. frankenbergeri.

Parnassius apollo (Lepidoptera, Papilionidae) declines on numerous localities all over Europe. Its local subspecies frankenbergeri, inhabiting the Pieniny Mts (southern Poland) and successfully recovered from extinction, is monophagous in larval stage. In natural conditions, it completes development on the orpine Sedum telephium ssp. maximum. Since proper quality and quantity of necessary nutritional compounds of the food plant ensure developmental success, the digestive processes in the insect midgut should reflect adaptation to a specific food source. The paper presents, for the first time, the activity of detected glycolytic enzymes in midgut tissue and liquid gut contents of the L4 and L5 instars of P. apollo larvae. alpha-Amylase plays the main role in utilization of carbohydrates, contrary to cellulase activity. Saccharase seems to be the main disaccharidase, and high activity of beta-glycosidase enables hydrolysis of the plant glycosides. Trehalase activity was unexpectedly low and comparable to those of cellobiase and lactase. alpha-Amylolytic and other glycolytic activities indicate that larvae utilize starch and other carbohydrate compounds as energy sources. Possible use of some plant allelochemicals as energy sources by Apollo larvae is discussed.

Feeding preferences of the Apollo butterfly (Parnassius apollo ssp. frankenbergeri) larvae inhabiting the Pieniny Mts (southern Poland).

Parnassius apollo (Lepidoptera, Papilionidae) is considered to be typical stenophagous species. Its European forms fall generally into 'telephiophagous' or 'albophagous' trophic groups. According to some authors, 'telephiophagous' P. apollo ssp. frankenbergeri SLABY, inhabiting the Pieniny Mts, has a rather broad spectrum of food-plants. We aimed at defining its feeding preferences for successful breeding in a semi-natural colony on more than one commercially-available Sedum species. Larval development (L5) and performance were assessed in three experimental groups fed on different plant species selected in a preliminary test. Apollo larvae appeared to be quite specific in the plant choice and developed poorly in the absence of Sedum telephium. Possible reasons of this are discussed.