Genetic Characterizations of the Iranian Honey Bee (Apis mellifera meda Skorikov 1929) Populations Using the Microsatellite DNA Markers.

The genetic characterization of the Iranian honey bee was investigated by analyzing 10 polymorphic DNA microsatellite loci in 300 honey bee samples representative of twenty Iranian provinces. This study evaluated the heterozygosity (Ho and He), the Shannon index, the number of observed alleles, and F-statistics among tested populations as genetic parameters. Our finding demonstrated that the Iranian honey bee populations were described by low genetic diversity in terms of the number of observed alleles, Shannon index, and Heterozygosity values. Most populations had significant deviations from Hardy Weinberg equilibrium cause of heterozygote shortage. Low FST and FIS values proposed the absence or very low genetic diversity within and among A. m. meda populations in the present study. The cluster analysis has categorized the honey bee samples gathered from various regions of Iran into two main groups, including honey bees in the North-West (i.e., North, Northwest, and West) provinces and honey bees in the East-South (i.e., Eastern North, Central part, and Southern) provinces of Iran. Our results also revealed lower genetic differentiation and heterozygosity among tested honey bee populations. The results from this study are consistent with previous investigations in Iran, alarming the loss of genetic diversity in the Iranian honey bee populations, which leads to more homozygosity. This study presented new data and reports on genetic structure in investigated native Iranian honey bee populations, and it will benefit future studies on selection, native biodiversity preservation and other conservation breeding projects.

A simplified and optimized protocol for total DNA extraction from insect species: applicable for studying genetic diversity and PCR-based specimen identification via partial amplification of cytochrome oxidase I (COI) gene.

The efficient DNA extraction from insects has been suggested as a critical and main step affecting molecular entomology for taxonomic identification, the establishment of DNA barcoding library and analysis of genetic diversity relationship between insect populations. For successfully apply these molecular techniques, high-quantity and high-quality of the extracted DNA are required. Several protocols for efficient genomic DNA extraction from insects have been developed. In this research, we represent a rapid, reliable and cost-effective method that it is not reliant on poisonous and enzymatic reagents for DNA extraction from insect tissues. Results showed that high quantity and high-quality of the isolated DNA by this method is suitable and can be used directly for PCR, also is enough to do hundreds of molecular reactions. In conclusion, we described a fast, cost-effective, non-toxic and enzyme-free protocol for high yield genomic DNA extraction from green Lacewings (Chrysoperla carnea) tissues in basic equipment laboratories.

Sub-lethal concentrations of the entomopathogenic fungus, Beauveria bassiana increase fitness costs of Helicoverpa armigera (Lepidoptera: Noctuidae) offspring.

We assessed the effects of exposure of the cotton bollworm, Helicoverpa armigera (Hübner) (Lepidoptera: Noctuidae) larvae to the entomopathogenic fungus Beauveria bassiana (isolate DC2) on life history parameters of the subsequent generation. Mortality assays against second instar larvae showed B. bassiana isolates to be significantly virulent, causing high mortality. Life history parameters such as developmental time, fecundity and survivorship were affected in the offspring of survivors of exposure to LC25 and LC50 concentrations of B. bassiana DC2. Pre-adult duration of H. armigera was significantly extended and total longevity and female progeny fecundity were decreased. Oviposition duration was shortened compared to offspring of untreated controls. All population parameters including intrinsic (r) and finite (λ) rates of increase and net (R0) and gross (GRR) reproductive rates were significantly decreased in offspring derived from H. armigera larvae treated with B. bassiana DC2. The intrinsic rate of increase (r) was 0.198 d-1, 0.120 d-1 and 0.111 d-1, respectively, for the F1 generation of controls (untreated), LC25- and LC50-treated cotton bollworm. The morphogenetic effects of B. bassiana observed due to the indirect effect of sub-lethal concentrations included abnormal pupae and adults and mortality resulted from the malformations. Pupal weight was reduced in offspring of treated individuals but sex ratios did not differ. Our results revealed that B. bassiana increased fitness costs of H. armigera in both direct (mortality) and indirect (disruption of normal development) ways. Adverse effects of sub-lethal fungal treatments on the parent generation carried over to the next generation.

Microsatellite genetic diversity of Apis mellifera meda skorikov.

The genetic diversity of three Iranian honey bee populations (Apis mellifera meda) was studied using morphological and microsatellite loci in south Iran. For this purpose ten morphological characters and five microsatellite loci were studied. Morphometric analysis resulted in a distinct classification of three investigated populations but showed low diversity among them. The grouping results of the diversity study by microsatellite markers were in agreement with the results of morphometry. The cluster analysis showed that the honey bees have clustered together in one group. These populations displayed low variability estimated from both the number of alleles and heterozygosity values. Genetic differentiation within the populations is low and low heterozygosity was also presented between diverse populations. These results indicate the existence of a single population structure. The results of current research confirmed us the previous findings concerning morphological and biochemical indications of uniformity in the honey bee population of the south Iran in spite of the fact that the cities which was studied by us separated from each other by a distance of 500 km.

Genetic diversity analysis of Chrysopidae family (Insecta, Neuroptera) via molecular markers.

In entomology, improvement of molecular methods would be beneficial tools for accurate identification and detecting the genetic diversity of insect species to discover a corroborative evidence for the traditional classification based on morphology. The aim of this study was focused on RAPD-PCR method for distinguishing the genetic diversity between eight species of Chrysopidae family. In current research, many specimens were collected in different locations of Tehran province (Iran), between them 24 specimens were identified. The wing venation, male genitalia and other morphological characters were used for identification and also the sexing of species was recognized with study of external genitalia. Then, the DNA was extracted with CTAB method. The RAPD-PCR method was carried out with twenty random primers. The agarose gel electrophoresis was used for separation of the PCR products. Based on electrophoresis results, 133 bands were amplified and between them, 126 bands were poly-morph and others were mono-morph. Also, among the applied primers, the primers OPA02 with 19 bands and OPA03 with 8 bands were amplified the maximum and minimum of bands, respectively. The results showed that 80.35 and 73.21 % of genetic similarity existed between Chrysopa pallens-Chrysopa dubitans, and between the Chrysoperla kolthoffi and Chrysoperla carnea, respectively. The minimum (45.53 %) of genetic similarity was observed between C. kolthoffi and C. dubitans, and the maximum (0.80 %) was seen between C. pallens and C. dubitans.

Molecular genetic diversity within Myrmeleontidae family.

Antlions are insects which feed on ants, insect which dig a pit and lies in wait for ants and other insects. Twelve species of Myrmeleontidae family as antlions and many specimens were identified in different locations in Fars province in Iran. To unveil the genetic similarity between these species, their DNA was extracted by modified CTAB method and with the use of seventeen 10-nucleotides primers of random amplified polymorphic DNA (RAPD); the genetic analysis of them was investigated. After PCR, agarose 1.5 % was used for electrophoresis. The obtained electrophoresis bands had base pairs range between 150 and 1,000 bp. The maximum of polymorphic bands belonged to OPH5, N13, and the minimum of polymorphic bands belonged to OPA7 primers. Different genetic similarity indices were found between eight species of antlions. Possibility of use of RAPD marker together with morphological studies for classification and identification of antlions is discussed.

Study of individual and sex genetic diversity among each genus and between two genera of Chrysopa and Chrysoperla (Neuroptera, Chrysopidae) based on RAPD-PCR polymorphism.

RAPD (random amplification of polymorphic DNA) was used to distinguish the genetic diversities between two genera of Chrysopa and Chrysoperla (Neuroptera, Chrysopidae). Sixty specimens were collected in different places in Kermanshah, west of Iran. The wing venation was used for identification of each type of two genera, and the gender was determined by study of external genitalia. 20 random primers were used for polymerase chain reaction. Then, the electrophoresis was used for separation of the PCR products on agarose gel. 294 bands were amplified, which 235 bands were polymorph and others (59s) determined as monomorph. The electrophoresis results showed that the primers OPA02 with 19 bands and OPA03 with 8 bands successively amplified the maximum and minimum of bands among the applied primers. The results showed that there are maximum of genetic diversity and minimum of genetic similarity between Chrysopa male (Chrysopa-M) and Chrysoperla female)Chrysoperla-F) population, in contrast, there are maximum of genetic similarity and minimum of genetic diversity between Chrysoperla-M and Chrysoperla-F, and Chrysopa-M and Chrysopa-F. There are also more genetic similarities, between males and females of Chrysopa and Chrysoperla, than between male of Chrysopa with female of Chrysoperla or vice versa.