Khalid Ali Khan

In vitro evaluation of the effects of some plant essential oils on Paenibacillus larvae, the causative agent of American foulbrood

ABSTRACT Paenibacillus larvae is one of the major bacterial pathogens of honey bee broods and the causative agent of American foulbrood disease. The factors responsible for the pathogenesis of American foulbrood disease are still not fully understood, and the increasing resistance of P. larvae to commonly used antibiotics necessitates a search for new agents to control this disease. The in vitro antibacterial activities of 28 plant essential oils against P. larvae ATCC 9545 were evaluated. Out of the 28 plant essential oils tested, 20 were found to be effective in killing P. larvae. Based on their minimum bactericidal concentration (MBC) values, the effective oils were grouped into three categories: highly effective, moderately effective and minimally effective. Jamaica pepper oil, mountain pepper oil, ajwain oil, corn mint oil, spearmint oil, star anise oil, nutmeg oil and camphor oil were highly effective, with MBC values between 162.0 and 375.0 µg/mL. Jamaica pepper oil was the most effective essential oil, with an MBC value of 162.0 µg/mL. The results of the time-response effect assays showed that no viable P. larvae cells were observed after 24 h of treatment with Jamaica pepper oil (162.0 µg/mL), 36 h of treatment with mountain pepper oil (186.0 µg/mL), 48 h of treatment with ajwain oil (224.8 µg/mL) or 48 h of treatment with oxytetracycline (5.89 µg/mL). The tested essential oils exhibited significant antimicrobial activities against P. larvae, and they may contain compounds that could play an important role in the treatment or prevention of American foulbrood disease.

In vitro evaluation of the effects of some plant essential oils on Ascosphaera apis, the causative agent of Chalkbrood disease

Ascosphaera apis is one of the major fungal pathogens of honey bee broods and the causative agent of Chalkbrood disease. The factors responsible for the pathogenesis of Chalkbrood disease are still not fully understood, and the increasing resistance of A. apis to commonly used antifungal agents necessitates a search for new agents to control this disease. The in vitro antifungal activities of 27 plant essential oils against two isolates of A. apis (Aksu-4 and Aksu-9) were evaluated. Out of the 27 plant essential oils tested, 21 were found to be effective in killing both isolates of A. apis. Based on their minimum fungicidal concentration (MFC) values, the effective oils were grouped into three categories: highly effective, moderately effective and minimally effective. Mountain pepper oil, Kala Bhangra oil, spearmint oil, babuna oil, betel leaf oil, carrot seed oil, cumin seed oil and clove bud oil were highly effective, with MBC values between 50.0 μg/mL and 600.0 μg/mL. Mountain pepper was the most effective essential oil, with an MBC value of 50.0 μg/mL. Citral and caryophyllene containing oils were the most effective with MIC 50 ppm. The essential oils tested exhibited significant antimicrobial activities against both strains of A. apis, and they may contain compounds that could play an important role in the treatment or prevention of Chalkbrood disease of honeybee.

Investigation of gut microbial communities associated with indigenous honey bee (Apis mellifera jemenitica) from two different eco-regions of Saudi Arabia

The microbial communities associated with the alimentary tract of honey bees are very important as they help with food digestion, provide essential nutrients, protect the host from pathogens, detoxify harmful molecules, and increase host immunity. In this study, the structural diversity of the gut microbial communities of native honey bees, Apis mellifera jemenitica from two different geographical regions (Riyadh and Al-Baha) of Saudi Arabia was analyzed by culture-dependent methods and 16S ribosomal RNA (rRNA) gene sequencing. In this study, 100 bacterial isolates were cultivated and phylogenetic analyses grouped them into three phyla: Proteobacteria, Firmicutes, and Actinobacteria. Bacteria in the phylum Proteobacteria were the most dominant (17 species), followed by Firmicutes (13 species) and Actinobacteria (4 species). Some of the identified bacteria (Citrobacter sp., Providencia vermicola, Exiguobacterium acetylicum, and Planomicrobium okeanokoites) were reported for the first time in the genus Apis, while others identified bacteria belonged to the genera Proteus, Enterobacter, Bacillus, Morganella, Lactobacillus, and Fructobacillus. To the best of our knowledge, this is the first study on the gut microbiota of the local honey bees in Saudi Arabia.

Geographical distribution and molecular detection of Nosema ceranae from indigenous honey bees of Saudi Arabia

The aim of the study was to detect the infection level of honey bees with Nosema apis and/or Nosema ceranae using microscopic and molecular analysis from indigenous honeybee race of eight Saudi Arabian geographical regions. A detailed survey was conducted and fifty apiaries were chosen at random from these locations. Infection level was determined both by microscope and Multiplex-PCR and data were analyzed using bioinformatics tools and phylogenetic analysis. Result showed that N. ceranae was the only species infecting indigenous honeybee colonies in Saudi Arabia. As determined by microscope, Nosema spores were found to be in 20.59% of total samples colonies, while 58% of the samples evaluated by PCR were found to be positive for N. ceranae, with the highest prevalence in Al-Bahah, a tropical wet and dry climatic region, whereas low prevalence was found in the regions with hot arid climate. Honeybees from all eight locations surveyed were positive for N. ceranae. This is the first report about the N. ceranae detection, contamination level and distribution pattern in Saudi Arabia.

Effect of gut bacterial isolates from Apis mellifera jemenitica on Paenibacillus larvae infected bee larvae

The probiotic effects of seven newly isolated gut bacteria, from the indegenous honey bees of Saudi Arabia were investigated. In vivo bioassays were used to investigate the effects of each gut bacterium namely, Fructobacillus fructosus (T1), Proteus mirabilis (T2), Bacillus licheniformis (T3), Lactobacillus kunkeei (T4), Bacillus subtilis (T5), Enterobacter kobei (T6), and Morganella morganii (T7) on mortality percentage of honey bee larvae infected with P. larvae spores along with negative control (normal diet) and positive control (normal diet spiked with P. larvae spores). Addition of gut bacteria to the normal diet significantly reduced the mortality percentage of the treated groups. Mortality percentage in all treated groups ranged from 56.67% up to 86.67%. T6 treated group exhibited the highest mortality (86.67%), whereas T4 group showed the lowest mortality (56.67%). Among the seven gut bacterial treatments, T4 and T3 decreased the mortality 56.67% and 66.67%, respectively, whereas, for T2, T6, and T7 the mortality percentage was equal to that of the positive control (86.67%). Mortality percentages in infected larval groups treated with T1, and T5 were 78.33% and 73.33% respectively. Most of the mortality occurred in the treated larvae during days 2 and 3. Treatments T3 and T4 treatments showed positive effects and reduced mortality.

Diagnosis and molecular detection of Paenibacillus larvae, the causative agent of American foulbrood in honey bees in Saudi Arabia

Apis mellifera jemenitica, the only indigenous honey bee race of Saudi Arabia, is well adapted to the harsh local environmental conditions. A large-scale feld survey was conducted to screen major Saudi Arabian beekeeping locations for infection by Paenibacillus larvae. Paenibacillus larvae is one of the major bacterial pathogens of honey bee broods and is the causative agent of American foulbrood disease. Larvae from samples suspected of infection were collected from different apiaries and homogenized in phosphate-buffered saline. Bacteria were isolated on MYPGP agar medium. Two bacterial isolates, ksuPL3 and ksuPL5 (16S rRNA GenBank accession numbers, KR780760 and KR780761, respectively), were subjected to molecular identifcation using P. larvae-specifc primers. A BLAST sequence analysis revealed that the two isolates were P. larvae with more than 98% sequence identity. This detection of P. larvae in the indigenous honey bee is the frst recorded incidence of this pathogen in Saudi Arabia. This study emphasizes the need for the relevant authorities to take immediate steps towards treating and limiting the spread of this disease throughout the country.

Comparative insecticidal activity of different plant materials from six common plant species against Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae)

Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae) is one of the major insect pests of stored grains. Due to export legislation and zero-tolerance for live insect in trade commodities, extensive use of synthetic insecticides is in practice in order to eliminate pest infestations from the lots. Currently, the one and only acceptable chemical to be used in stored grain is phosphine but due to its excessive usage the stored grain pests are becoming resistant against it. Hence discovery of alternative compounds is much needed. In this study we have compared insecticidal efficacy of different plant materials from six commonly grown plants of Pakistan, viz. Allium sativum (Alliaceae), Zingiber officinale (Zingiberaceae), Cymbopogon citratus (Poaceae), Eucalyptus globulus (Myrtaceae), Nicotiana tabacum (Solanaceae), and Azadirachta indica (Meliaceae) against T. castaneum infesting stored wheat, rice, corn and gram pulse. Various plant parts were dried, powdered, and used as admixtures to the stored commodities in the experiments. The results have suggested that A. sativum (garlic) and Z. officinale (ginger) were more effective resulting into 15 times higher adult mortality and 4 to 5 times reduction in grain weight losses when mixed with rice grains. Similarly, A. indica when admixture with wheat checked the population growth in the resources resulting into 3.5 times less adult production compared to controls. A subsequent experiment was conducted to study the dose response of neem seed powder against the beetle pest infesting milled products. Surprisingly, better control was observed either at lowest (1% w/w) or the highest doses (5% w/w). This finding is of great interest to understand the underlying phenomenon which we assume is the ability of T. castaneum to feed selectively in flour mediums, however, further research on this aspect is required to be investigated. The results of this study support the use of botanicals for stored product pest management.

Insect pollinator diversity in four forested ecosystems of southern Punjab, Pakistan

This study investigated pollinator assemblage diversity and richness in four forested ecosystems of southern Punjab, Pakistan, with different landscape types. Pirowal is situated in the plains of irrigated Punjab, Lal Suhanra is part of a sandy desert ecosystem, Ghazi Ghat is part of the Indus River delta, and Fort Munro is located in dry hilly mountains. A yearlong survey of pollinator populations was carried out in these four forested ecosystems from January to December of 2010. Fortnightly hand netting was performed for collecting flower-visiting insects whereas, pan traps of three colors (white, blue, and yellow) were deployed for collecting the data. A total of 8,812 individuals from two orders (Lepidoptera and Diptera) were observed, including 22 families and 154 species. Bees were the most abundant, with 4,502 individuals, and the most species-rich taxa, with 70 species in five families, followed by flies having 2,509 individuals and 51species in 10 families. Wasps were the least abundant with 1,801 individuals and 33 species in seven families. The assemblage structure of pollinator communities as visualized through rank abundance curves showed that there were many species with low abundance and only a few species with a much higher abundance. The most abundant species among the bees, in order, were Nomia sp.3, Megachile bicolor, and Colletes sp.3; among flies, Syrphus sp.2, Calliphoridae sp.1, and Empididae sp.4; and among wasps, Tiphiidae sp.1, Myzininae sp.2, and Scelionidae sp.1.

Validation of botanical origins and geographical sources of some Saudi honeys using ultraviolet spectroscopy and chemometric analysis

This study aims at distinguishing honey based on botanical and geographical sources. Different floral honey samples were collected from diverse geographical locations of Saudi Arabia. UV spectroscopy in combination with chemometric analysis including Hierarchical Cluster Analysis (HCA), Principal Component Analysis (PCA), and Soft Independent Modeling of Class Analogy (SIMCA) were used to classify honey samples. HCA and PCA presented the initial clustering pattern to differentiate between botanical as well as geographical sources. The SIMCA model clearly separated the Ziziphus sp. and other monofloral honey samples based on different locations and botanical sources. The results successfully discriminated the honey samples of different botanical and geographical sources validating the segregation observed using few physicochemical parameters that are regularly used for discrimination.

Yearlong association of insect pollinator, Pseudapis oxybeloides with flowering plants: Planted forest vs. agricultural landscape

The yearlong association of a native bee, Pseudapis oxybeloides (Halictidae: Hymenoptera) was studied with 72 plant species in a sub-tropical planted forest and some adjacent agricultural landscapes at Multan, Pakistan. The study resulted in 66 interactions of P. oxybeloides with only 24 plant species in 15 families while other 48 plant species were not visited by this bee. The maximum abundance of P. oxybeloides (7–9 individuals) was recorded on Achyranthes aspera and Launaea procumbens followed by Ageratum conyzoides, Trianthema portulacastrum and Cleome viscosa (5–6 individuals). Majority (19) of plant species were visited by only 1–4 individuals. The bee activity was started in the month of March which attained its peak in May followed by a gradual decline until September. No bees were observed during the months of January and February. There was a significant positive relationship between bee abundance and number of flowering plant species. Bee abundance had a strong positive relationship with temperature while it had a strong negative relationship with relative humidity (%). Floral abundance increased with the number of flowering plant species while it was not influenced by floral span of plant species. Besides giving the floral host plants of P. oxybeloides, the current study also gives a better understanding of its seasonality along with its relationships with different biotic and abiotic factors under local conditions. These findings can help in maintaining and managing P. oxybeloides population particularly and other native bees in general at local scale.