Characterization and Evaluation of Metarhizium spp. (Metsch.) Sorokin Isolates for Their Temperature Tolerance

A field survey was done in teak (Tectona grandis F.) forests in South India to explore the entomopathogenic effect of Metarhizium anisopliae (Ascomycota: Sordariomycetes) against teak defoliator, Hyblaea puera (Lepidoptera: Hyblaeidae). About 300 soils and infected insect samples were collected during the survey and thirty-six fungal isolates were isolated from soil and insect samples and characterized. The fungi were cultured on PDAY with dodine and antibiotics. Generally, the EPF culture was incubated at 27 °C in darkness for 15 days. Virulence of the Entomopathogenic Fungi (EPF) ability to germinate under cold and heat temperatures was assessed in a culture impregnated with conidia. In the experiment, it was found that for the first time Metarhizium quizhouense, Metarhizium robertsii, and Metarhizium majus species caused significantly higher mortality to hosts. These isolates of M. anisopliae, M. robertsii, M. majus, and M. quizhouense were all considered to be effective virulent and environmentally adaptive. The Metarhizium isolates were recommended as effective bio-control agents through the field investigation of teak defoliator Hyblaea puera from South India forest. This study paves the way to utilize the indigenous isolates of EPF for the control of teak defoliator and to combat the pests thatare resistant to insecticide.

Isolation, identification and virulence of indigenous entomopathogenic fungal strains against the peach-potato aphid, Myzus persicae Sulzer (Hemiptera: Aphididae), and the fall armyworm, Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae)

Background As different biogeographic strains and isolates of entomopathogenic fungi vary in their genetic, enzymatic and pathogenic characteristics, this study assessed the virulence of 2 indigenous strains of Beauveria bassiana (Balsam) Vuillemin and Metarhizium anisopliae (Metschn.) Sorokin (Ascomycota, Hypocreales: Clavicipitaceae), isolated from naturally infected insect cadavers, against the 3rd instar nymphs of Myzus persicae (Sulzer) (Hemiptera: Aphididae) and 3rd instar larvae of Spodoptera frugiperda (J.E. Smith) (Lepidoptera: Noctuidae) using leaf-dip and larval-dip methods, respectively. Results Both fungal isolates exhibited considerable pathogenicity against M. persicae and S. frugiperda. Mortality in all bioassays was conidial concentration and exposure time dependent and increased significantly along with both factors (R2 = 0.86–0.99 for B. bassiana and 0.82–0.94 for M. anisopliae). Moreover, M. anisopliae isolate appeared more virulent to S. frugiperda larvae than B. bassiana isolate, while the later fungal isolate was more lethal to M. persicae nymphs than the former one. At the highest conidial concentration (1.0 × 109 conidia/ml), M. anisopliae caused maximum mean mortality of S. frugiperda (88%) and M. persicae (65%) and B. bassiana exhibited maximum mean mortality of S. frugiperda (76%) and M. persicae (94%). Moreover, probit regression analyses showed LT50 values for M. persicae of 4.57 and 6.86 days at 1.0 × 109 conidia/ml for the isolates of B. bassiana and M. anisopliae, respectively, while LC50 values were 7.75 × 106 and 8.70 × 107 conidia/ml after 10th day of application, for the isolates of B. bassiana and M. anisopliae, respectively, against M. persicae. Similarly, LT50 values for S. frugiperda were 7.75 and 7.03 days for 1.0 × 109 conidia/ml concentration and LC50 values were 2.84 × 107 and 8.84 × 105 conidia/ml at 10th day data for the isolates of B. bassiana and M. anisopliae, respectively. Conclusion Overall study results demonstrated the effectiveness of B. bassiana and M. anisopliae against M. persicae and S. frugiperda, respectively. However, field evaluations of these indigenously isolated promising fungal strains against these insect pests.

Potent Ant Deterrents Emitted from Nematode-Infected Insect Cadavers

Most known species of entomopathogenic nematodes (EPNs) are generalist obligate parasites of insects. They kill their hosts within days after infection and mortality is mainly caused by toxins produced by bacteria that co-infect the hosts and serve as food for the nematodes. EPNs can infect a very broad spectrum of insects and these insects can therefore be expected to have evolved strategies to avoid infection. Indeed, ants are known to avoid feeding on EPN-infected insect cadavers, most likely because they are repelled by semiochemicals that emanate from the cadavers. The source and nature of these repellents are so far unknown. In a series of behavioral and chemical analytical experiments we identified hexadecanal and 2-heptadecanone as two compounds that are emitted by insect larva that are infected by the EPN Steinernema feltiae, but not by uninfected larvae. When spiking honey water with the two semiochemicals, they were confirmed to be highly deterrent to the ant Lasius niger. The environmentally benign hexadecanal and 2-heptadecanone could be employed to ward off ants and possibly other pests. Additional experiments are needed to fully determine their application potential.

Intracellular life cycle of Candidatus Liberibacter asisticus inside psyllid gut cells

Candidatus Liberibacter asiaticus (CLas), the devastating pathogen related to Huanglongbing (HLB), is a phloem-limited, fastidious, insect-borne bacterium. Rapid spread of HLB disease relies on CLas propagates efficiently in its vector, the Asian citrus psyllid, Diaphorina citri, in a circulative manner. Understanding the intracellular lifecycle of CLas in psyllid midgut is fundamental to improve current management strategies. Using a microscopic approach within CLas-infected insect midgut, we observed the entry of CLas into gut cells inside vesicles by endocytosis, termed Liberibacter containing vacuoles (LCVs). Endocytosis is followed by the formation of endoplasmic reticulum-related and replication permissive vacuoles (rLCVs). rLCVs then further develop into bigger double membrane autophagosome-like structure, termed autophagy-related vacuole (aLCV). Vesicles, containing CLas egress from aLCV and fuse with the cell membrane. Immunolocalization studies showed that CLas employs endo/exocytosis-like mechanisms that mediates bacterial invasion and egress. Upregulation of autophagy-related genes indicated subversion of host autophagy by CLas in psyllid vector to promote infection. These results indicate that CLas interacts with host cellular machineries to undergo a multistage intracellular cycle through endocytic, secretory, autophagic and exocytic pathways via complex machineries. Potential tactics for HLB controlling can be made depending on further investigations on the knowledge of the molecular mechanisms of CLas intracellular cycle.

Establishment, verification and application of rapid detection of baculovirus infectious titer by flow cytometry

Titer detection of baculovirus usually is time-consuming.It is important to establish a rapid detection method for baculovirus titer. In this report, Staining of cells with a fluorescently labeled anti-gp64 antibody allows for identification of infected insect cells. By inoculating cultures with a series of log dilutions of virus, and staining of the cultures 13-22 hours post inoculation, the ratio of infected to un-infected insect cells can be determined by flow cytometry. Statistical analysis of the percentage of infected cells in the virus dilution series enables accurate infectious titer determination. The culture time, cell growth state, the concentration of GP64-APC antibody and the concentration of inactivated FBS in diluent were optimized.The generality, repeatability and intermediate precision of the method were verified. The FCM method has the advantages of simplicity, accuracy, low cost and good repeatability.

Lipolytic Activity of a Carboxylesterase from Bumblebee(Bombus ignitus) Venom

Bee venom is a complex mixture composed of peptides, proteins with enzymatic properties, and low-molecular-weight compounds. Although the carboxylesterase in bee venom has been identified as an allergen, the enzyme’s role as a venom component has not been previously elucidated. Here, we show the lipolytic activity of a bumblebee (Bombus ignitus) venom carboxylesterase (BivCaE). The presence of BivCaE in the venom secreted by B. ignitus worker bees was confirmed using an anti-BivCaE antibody raised against a recombinant BivCaE protein produced in baculovirus-infected insect cells. The enzymatic activity of the recombinant BivCaE protein was optimal at 40 °C and pH 8.5. Recombinant BivCaE protein degrades triglycerides and exhibits high lipolytic activity toward long-chain triglycerides, defining the role of BivCaE as a lipolytic agent. Bee venom phospholipase A2 binds to mammalian cells and induces apoptosis, whereas BivCaE does not affect mammalian cells. Collectively, our data demonstrate that BivCaE functions as a lipolytic agent in bee venom, suggesting that BivCaE will be involved in distributing the venom via degradation of blood triglycerides.

An Unusual Cause of Facial swelling – Bot Fly Larva in an Infant – A Case Report

‘Myiasis’ is a term used to describe an infestation of humans or animals with dipterous larvae [1]. Due to increased international travel in recent times, health professionals in the UK may encounter these infections more commonly than before. We present a case of a 6 month infant who had been bitten by Dermatobia hominis Bot fly in Brazil and travelled back to the UK. The original diagnosis was of an infected insect bite, which ultimately delayed appropriate management. A detailed travel history is therefore paramount and these types of infections should be considered in differential diagnoses. Management should also involve the infectious diseases team.