A cost-effective,simple and high-throughput method for DNA extraction from insects

Abstract We present a high-throughput cost-effective method to extract DNA suitable for polymerase chain reaction (PCR) from insect tissue. The method uses standard 200 μL-deep 96-well plates in which samples are ground, digested and subsequently purified. The test extraction using four different insect species and controlling for potential contamination showed that the method yields good-quantity and quality DNA. PCR with mitochondrial and nuclear primers was reliable. The proposed extraction protocol combines the speed of commercial 96-well plate methods with the economies associated with readily available and cheap laboratory chemicals, consumables and equipment. Therefore, this method is particularly suitable for low-budget research projects and for laboratories with only basic equipment present.     

Mitochondrial-driven sustained active water vapour absorption (WVA) in the firebrat, Thermobia domestica (Packard), during development and the moulting cycle

Rapid pre-functional mitochondrial biogenesis in the short-lived first-instar nymph occurs in the cells of the developing posterior rectal sacs which when mature are the organs believed to be responsible for sustained active water vapour absorption (WVA) in the firebrat Thermobia domestica (Packard). During the second instar, the mitochondria migrate apically and begin to associate with deep portasome-studded infolds of the apical plasma membrane (apm), just as WVA starts sporadically. By mid to late in the third instar the mature mitochondria-apm apical complex is fully developed, with elongated tubular mitochondria vertically packed hexagonally in transverse section and adpressed to the deeply pleated infolds of the apm, giving the greatest known concentration of mitochondria. This coincides with peak mass-specific WVA. During moults, WVA temporarily ceases as the sac cells secrete a new cuticle overlying the sac epithelium. Their apical complex fully regresses. Mitochondria migrate perinuclearly. The apm is pinched-off into numerous small portasome-studded pouches which remain sequestered within the cytoplasm. Towards the end of the moult the apm pouches reassemble into deeply pleated infolds into which the mitochondria migrate and elongate, rapidly re-establishing the hexagonal array of the apical complex. This coincides with the recommencement of WVA.     

A quantitative assay for the juvenile hormones and their precursors using fluorescent tags

Background

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors.

Methods

A sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl).

Conclusions

The method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10–20 to 1000 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.

Significance

The proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors.     

A simple method for the extraction and quantification of photopigments from Symbiodinium spp.

We have developed a simple, mild extraction procedure using methanol which, when coupled with HPLC analysis and diode array detection (DAD), can be used to quantify the major photopigments found in cultured Symbiodinium spp. Extracts were prepared by suspending, fresh or frozen (− 70 °C), wet cell pellets in methanol and sonicating or not sonicating the cell suspensions before soaking the cells for 2 h in an ice bath. To assist the soaking process, cell suspensions were vortex mixed at 30 min intervals. After soaking, 0.5 M ammonium acetate buffer was added (1 part buffer to 9 parts methanol) before suspensions were stored over night at − 20 °C. Greater than 92% the recoverable pigment was obtained in the initial extraction of the four major photopigments, chlorophyll c, peridinin, diadinoxanthin, and chlorophyll a. Neither sonication nor freezing substantially increased the recovery of photopigments extracted with methanol. Extraction by other commonly used solvents such as acetone or acetone:water with or without freezing and sonication were less effective.     

A new virus disease in the housefly, Musca domestica (Diptera)

Reovirus particles were isolated from adults in laboratory colonies of the housefly, Musca domestica. These particles were spherical in outline, 57–76 nm in diameter, and were found only in hemocyte cytoplasm, where virions have been disclosed by a new technique. Virions were present in large numbers, and viral inclusion bodies were identified. The virus particles had pentagonal and hexagonal shapes resembling a simple icosahedral structure. The virus was shown to be infectious and pathogenic to adult flies through injection or by feeding them suspensions from flies that had died of the virus. Electron micrographs of midgut sections from infected flies showed that the midgut cells were packed with dark undulating threads which were not present in uninfected flies. However, no virus particles or inclusion bodies could be seen in these cells. On the basis of their association with infected flies, and the similarity to results from other studies on reoviruses and insect viruses, it is suggested that these threads are an alternative replicative form of the reovirus. When the virus suspensions from heavily infected flies were dialyzed against weak alkaline solutions, the threads showed an inner component of coiled material, 12 nm in diameter, inside an envelope with a diameter of 50–83 nm, mean 60.3 ± 7.5, composed of subunits 7–8 nm long and 7–8 nm across.     

A rapid quantitative method for the determination of thebaine in Papaver bracteatum

A rapid method enabling a quantitative analysis of thebaine in capsules and latex of Papaver bracteatum has been devised, based on a TLC technique. This method was compared to the commonly used GLC procedure, and highly significant correlation coefficient (r = 0.90) and linear regression were found between the two methods. Values for concentrations to the nearest ±0.25% of the standard spots can be reached by this simple and rapid thebaine determination.     

Properties of olfactory conditioning in the housefly, Musca domestica

The housefly, Musca domestica, was conditioned to odours using the proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response and the properties of conditioning were investigated. Among trials including forward pairing of the conditioned stimulus (CS) with the unconditioned stimulus (US), backward pairing and isolated presentations of CS and US, only forward pairing is effective on the acquisition of conditioning. Backward pairing combined with forward pairing does not influence the effectiveness of the forward pairing. CS given overlapping with a US presentation permits only weak conditioning. The acquisition of conditioning decreases with increase of the CS-US interval. In the differential conditioning situation to two odours, discriminative responses are observed. In the flies conditioned with one antenna, the conditioned response is elicited not only by stimulation of the antenna used for conditioning but also by stimulation of the antenna not used for conditioning, although the response using the former is higher than with the latter. The ability to be conditioned is reduced immediately after fastening on a clay bed and increases with time. Ability can also be improved by transection of the ventral nerve cord.     

Multiplicity of cytochrome P-450 in microsomal membranes from the housefly, Musca domestica

The heterogeneity of cytochrome P-450 in abdominal microsomes from the CSMA, SBO, Fc, Rutgers and Baygon strains of the housefly was examined by three different methods. Examination of ‘apparent absolute absorption spectra’ indicated at least two types of cytochrome in all strains, one with an absorption maximum at about 394 nm, being present in greater quantity in the insecticide-resistant strains, while the other, with an absorption maximum at about 412 nm, predominates in the insecticide-susceptible strains.Controlled tryptic digestion of microsomes followed by spectral examination at various time intervals indicated a heterogeneous population of cytochromes P-450 in CSMA, Fc and Rutgers strains.Subfractionation of microsomes from houseflies of the CSMA and Fc strains by a two-step discontinuous sucrose gradient centrifugation method provided evidence for cytochromes P-450 of different spectral characteristics. The concentration of cytochrome P-450, as well as its spectral characteristics varied between fractions and strains.     

Storage-excretion of metallic cations in the adult housefly, Musca domestica

The objective of this investigation was to elucidate further the phenomenon of storage-excretion of metallic cations in the housefly. The sites for deposition of zinc, calcium and copper have been identified in this study. The metal intake of the flies was altered by raising one group on sucrose and tap water while in the experimental groups sucrose was supplemented with either 0.05% zinc sulphate, 0.05% calcium phosphate or 0.03% copper sulphate. There were no significant differences in the average life spans of the flies in different groups indicating physiological tolerance to the higher mineral intake. The Malpighian tubules, the midgut and the remainder of the body were analyzed for mineral content in houseflies ranging from 1 to 25 days posteclosion by atomic absorption spectrophotometry. There was a progressive age-associated increase in the total metal content of the flies with age. Zinc and calcium were primarily stored in the Malpighian tubules whereas copper was sequestered in the midgut. Microscopic examination of the epithelial cells of the Malpighian tubules and the midgut revealed a corresponding age-associated increase in mineralized concretions.     

A rapid non-destructive DNA extraction method for insects and other arthropods

Preparation of arthropods for morphological identification often damages or destroys DNA within the specimen. Conversely, DNA extraction methods often destroy the external physical characteristics essential for morphological identification. We have developed a rapid, simple and non-destructive DNA extraction technique for arthropod specimens. This technique was tested on four arthropod orders, using specimens that were fresh, preserved by air drying, stored in ethanol, or collected with sticky or propylene glycol traps. The technique could be completed in 20 min for Coleoptera, Diptera and Hemiptera, and 2 min for the subclass Acarina, without significant distortion, discolouration, or other damage to the specimens.     

Efficacy of entomopathogenic bacteria for control of Musca domestica

The aim of this study was to evaluate the larvicidal activity, and sub lethal effects of entomopathogenic bacteria Brevibacillus laterosporus, Bacillus thuringiensis var. israelensis, B. thuringiensis var. kurstaki, and a commercial formulation of Bacillus sphaericus on Musca domestica. Bacterial suspensions were prepared in different concentrations and added to the diet of newly-hatched larvae which were monitored until the adult stage. The larvae were susceptible to the B. laterosporus, B. thuringiensis var. israelensis, and B. thuringiensis var. kurstaki bacteria in varied concentration levels. These bacteria have larvicidal and sub lethal effects on the development of flies, reducing both adult size, and impairing the reproductive performance of the species.     

Gonadotrophic effect of juvenile hormone in Blattella germanica: A rapid,simple quantitative bioassay

The German cockroach is convenient for assaying gonadotrophic effects of juvenile hormone (JH) analogues. The animals can be raised synchronously from egg to adult state in 2·5 months at 30°C, a shorter time than for any of the common laboratory-reared cockroaches. Last instar larvae can be stored for up to a month at 16 to 18°C and brought to 30°C to metamorphose when needed, providing a convenient source of bioassay animals. The assay takes 2 days to complete, less time than previously published gonadotrophic assays. The growth of terminal oöcytes of the ovary responds linearly with log dose between 0·08 and 0·8 μg of Cecropia JH-I. The analogues JH-I ethyl ester and methyl-juvenate have dose response curves parallel to that for JH-I, enabling potencies, r, relative to JH-I to be calculated (r = 1·01, 0·35 respectively). There is no significant effect of the volume of hormone vehicle between 2 and 8 μl. A 30 per cent increase in the precision of the bioassay is obtained by correcting the oöcyte growth for the fed-weight of the animal. The bioassay is adequate to resolve a twofold difference in potency using as few as 36 animals per analogue.     

Classical conditioning to visual stimuli in the housefly, Musca domestica

In the housefly, Musca domestica, classical conditioning to monochromatic light was demonstrated by using a proboscis extension response to labellar stimulation with sucrose solution as an unconditioned response. Sequential and temporal relationships between the conditioned stimulus (CS) and unconditioned stimulus (UCS) to form conditioning were studied; conditioning occurred most readily when the CS was presented before the UCS without an interstimulus interval. Spectral response curves, which were obtained by examining the responsiveness of flies conditioned for one colour to other colours, were divided into two groups; one was that of 462 nm-conditioned flies and the other those of 516 nm-, 579 nm-, and 642 nm-conditioned flies.     

A method for isolating milky disease, Bacillus popilliae var. rhopaea, spores from the soil

A method based on the tyndallization procedure is described for isolation of Bacillus popilliae var. rhopaea spores from the soil. A soil suspension is diluted with a germinating medium, which promotes the germination of most spores except B. popilliae var. rhopaea, and is treated with a series of seven heat shocks (70°C for 20 min) at hourly intervals. This treatment reduced the number of contaminant spores by over 95%. The suspension is then plated out onto “J” medium which allows the germination and growth of all surviving spores including the milky disease spores. The plates are incubated anaerobically at 28°C for 7 days before the characteristic small transparent colonies of B. popilliae var. rhopaea are counted. In testing the method it was revealed that about 15% of the milky disease spores in the soil produced visible colonies, and that a spore concentration of over 1.2 × 10⁵ spores/g dry wt of soil could be quantified. This concentration of spores produces only 3% infection in Rhopaea verreauxi larvae. The method may be applicable to other varieties of B. popilliae which will grow on “J” medium.     

A quantitative description of the behaviour of wild juvenile plaice (Pleuronectes platessa L.)

The behaviour of young plaice (Pleuronectes platessa L.) as they migrate up and down sandy beaches with the tide is described. Their behaviour during this migration consists mainly of swimming and feeding interspersed with rarer behavioural acts. Over short periods swimming behaviour can be described by a random model in which the probability of a swim occurring remains constant. This probability varies markedly, however, from hour to hour. Two types of swimming movement are recognized: one of very short duration represents searching for food and the other, longer, category serves to transport the fish up and down the shore. Variations in the feeding rate and in the frequency, duration, and direction of swimming movements over the tidal cycle are described and related to the changing physical and biological conditions that the fish experience during their intertidal movements.     

Cloning,expression, and purification of a new antimicrobial peptide gene from Musca domestica larva

Musca domestica (Diptera: Muscidae), the housefly, exhibits unique immune defences and can produce antimicrobial peptides upon stimulation with bacteria. Based on the cDNA library constructed using the suppression subtractive hybridization (SSH) method, a 198-bp antimicrobial peptide gene, which we named MDAP-2, was amplified by rapid amplification of cDNA ends (RACE) from M. domestica larvae stimulated with Salmonella pullorum (Enterobacteriaceae: Salmonella). In the present study, the full-length MDAP-2 gene was cloned and inserted into a His-tagged Escherichia coli prokaryotic expression system to enable production of the recombinant peptide. The recombinant MDAP-2 peptide was purified using Ni-NTA HisTrap FF crude column chromatography. The bacteriostatic activity of the recombinant purified MDAP-2 protein was assessed. The results indicated that MDAP-2 had in vitro antibacterial activity against all of the tested Gram − bacteria from clinical isolates, including E. coli (Enterobacteriaceae: Escherichia), one strain of S. pullorum (Enterobacteriaceae: Salmonella), and one strain of Pasteurella multocida. DNA sequencing and BLAST analysis showed that the MDAP-2 antimicrobial peptide gene was not homologous to any other antimicrobial peptide genes in GenBank. The antibacterial mechanisms of the newly discovered MDAP-2 peptide warrant further study.