Protophormia Terraenovae Research Articles

Oviposition Restraint and Developmental Alterations in the Ectoparasitic Wasp,Nasonia vitripennis, When Utilizing Puparia Resulting From Different Size Maggot Masses ofLucilia illustris,Protophormia terraenovae, andSarcophaga bullata

Adult females of the ectoparasitoid Nasonia vitripennis (Walker) are capable of distinguishing between hosts of different quality, and then correspondingly adjust clutch sizes and sex ratios of the offspring. In this study, we examined whether the size of the maggot mass, and presumably the developmental temperature, influenced the suitability of the resulting fly pupal and pharate adult stages as hosts for N. vitripennis. Three sizes of maggot masses (100; 500; and 1,000 individuals per mass) were selected for use to generate hosts based on previous studies characterizing developmental and heat shock response differences for the flies. For all host species tested (Lucilia illustris, Protophormia terraenovae, and Sarcophaga bullata), the rate of parasitism by N. vitripennis decreased with increasing maggot mass size. When successful parasitism did occur, parasitoid development increased in duration, clutch sizes decreased, mortality from egg hatch to adult emergence elevated, male biased sex ratios were produced, and adult wasp body sizes were truncated with increasing fly larval density. These wasp life history features are consistent with reductions in host quality. Host quality reductions corresponded to production of heat shock proteins 23, 60, and 70. Heat shock protein synthesis appeared to occur at the expense of normal protein production because total hemolymph protein concentrations decreased with increased larval density in maggot masses. These observations argue that use of N. vitripennis in criminal investigations to estimate periods of insect activity or a minimum post mortem interval must take into account the maggot mass history of the hosts used by the wasp.

Morphology and identification of first instars of the European and Mediterranean blowflies of forensic importance. Part I: Chrysomyinae

Light microscopy photographs, line illustrations and scanning electron micrographs are provided for first instars of Chrysomya albiceps (Wiedemann), Chrysomya megacephala (Fabricius) and Protophormia terraenovae (Robineau-Desvoidy) (all: Diptera: Calliphoridae). The following morphological structures are documented: pseudocephalon; antennal complex; maxillary palpus; facial mask; thoracic and abdominal spinulation; spiracular field; posterior spiracles, and cephaloskeleton. New diagnostic features of the cephaloskeleton and the spinulation of abdominal segments are described. Verification of earlier descriptions revealed major discrepancies among published data. The present results allow for the clarification, correction and, especially, complementation of existing information provided by numerous authors. Finally, an identification key for first-instar larvae of European and Mediterranean Chrysomyinae of forensic importance is presented.

Transgenerational Effects of Heavy Metal Pollution on Immune Defense of the Blow Fly Protophormia terraenovae

Recently environmental conditions during early parental development have been found to have transgenerational effects on immunity and other condition-dependent traits. However, potential transgenerational effects of heavy metal pollution have not previously been studied. Here we show that direct exposure to heavy metal (copper) upregulates the immune system of the blow fly, Protophormia terraenovae, reared in copper contaminated food. In the second experiment, to test transgenerational effects of heavy metal, the parental generation of the P. terraenovae was reared in food supplemented with copper, and the immunocompetence of their offspring, reared on uncontaminated food, was measured. Copper concentration used in this study was, in the preliminary test, found to have no effect on mortality of the flies. Immunity was tested on the imago stage by measuring encapsulation response against an artificial antigen, nylon monofilament. We found that exposure to copper during the parental development stages through the larval diet resulted in immune responses that were still apparent in the next generation that was not exposed to the heavy metal. We found that individuals reared on copper-contaminated food developed more slowly compared with those reared on uncontaminated food. The treatment groups did not differ in their dry body mass. However, parental exposure to copper did not have an effect on the development time or body mass of their offspring. Our study suggests that heavy metal pollution has positive feedback effect on encapsulation response through generations which multiplies the harmful effects of heavy metal pollution in following generations.

Plausible neural circuitry for photoperiodism in the blow fly,Protophormia terraenovae

Photoperiodism is important for seasonal adaptation in insects. Although photoreceptors and endocrine outputs for photoperiodism have been investigated, its neural mechanisms are less studied. This paper proposes three groups of neurons involved in photoperiodic control of adult diapause in the blow fly, Protophormia terraenovae. Ablation experiments showed that pars lateralis neurons in the dorsal protocerebrum are important for diapause induction under short-days and low temperature, the pars intercerebralis neurons for ovarian development under long-days and high temperature. When regions containing pigment-dispersing factor and PERIOD immunoreactive s-LNvs were bilaterally ablated, flies became arrhythmic in locomotor activities, and did not discriminate photoperiod for diapause induction, suggesting that s-LNvs are important for circadian rhythm and photoperiodism. In the s-LNvs, PERIOD-immunoreactivity in the nucleus was highest at 12 h after lights-off and lowest 12 h after lights-on regardless of photoperiod. Thus, as in D. melanogaster, it is possible that PERIOD nuclear translocation entrains to photoperiod, and day-length information seems to be encoded in s-LNvs. Immunoelectronmicroscopy revealed synaptic connections from s-LNvs to the pars lateralis neurons, suggesting that circadian clock neurons, s-LNvs, are involved in time measurements and may synaptically signal day-length information to the pars lateralis neurons.

Phenotypic plasticity in temperature stress resistance is triggered by photoperiod in a fly

Temperature variation poses a substantial challenge for individual survival and reproductive success, warranting effective means to counter negative effects of temperature extremes. Phenotypic plasticity offers a particularly powerful mechanism to cope with short-time temperature fluctuations. While temperature-induced plasticity in thermal tolerance has been widely explored, effects of other environmental factors have received much less attention. Using a full-factorial design we here show that variation in temperature stress resistance can be triggered by photoperiod (and temperature) in the fly Protophormia terraenovae, with shorter day lengths inducing more cold- and longer day lengths more heat-tolerant phenotypes. Such plastic changes were not related to different developmental pathways (reproductive activity or reproductive diapause), and can be induced during development but also in the adult stage (at least for cold tolerance). We suggest that short-term, photoperiod-mediated changes in insect thermal tolerance represent a mechanism of adaptive seasonal plasticity. Photoperiod further affected development time and body size, the significance of which is currently unclear.

The use of COI barcodes for molecular identification of forensically important fly species in Germany

Deoxyribonucleic acid (DNA)-based insect identification has become a routine and accurate tool in forensic entomology. In the present study, we demonstrate the utility of the mitochondrial DNA cytochrome oxidase I gene "barcoding region" as a universal marker for molecular identification of forensically important Diptera. We analyzed 111 specimens belonging to 13 species originating from Frankfurt am Main, Germany (Calliphoridae: Calliphora vicina, Calliphora vomitoria, Lucilia ampullacea, Lucilia caesar, Lucilia illustris, Lucilia sericata, Lucilia silvarum, Phormia regina, Protophormia terraenovae; Piophilidae: Parapiophila vulgaris; Muscidae: Hydrotaea dentipes, Hydrotaea ignava, Hydrotaea similis). Intraspecific variation ranged from 0 to 1.17% and interspecific variation occurred between 1.17% and 15.21%. Although differences within species were generally less than among species, divergence percentages overlapped due to low interspecific nucleotide divergence of the recently separated sister species L. caesar and L. illustris. However, all species formed distinct monophyletic clades and thus the cytochrome oxidase 1 (COI) barcode has been shown suitable for clear differentiation and identification of forensically relevant Diptera in Germany.

Blow flies (Diptera: Calliphoridae) survive burial: Evidence of ascending vertical dispersal

This study was undertaken to determine if immature blow flies could complete development following burial and emerge from the soil as adults. Two species of blow flies, Cochliomyia macellaria and Protophormia terraenovae, were placed at three depths and at three different life stages, in a simulated burial to evaluate the impact of soil on ascending vertical dispersal and fly survival. In soil columns, immature stages of each species were covered with 5, 25 and 50cm of soil. Emerging adult flies of both species reached the surface from all depths at all three immature stages (2nd instar, 3rd instar and pupae). At the 50-cm depth, flies were least successful in reaching the surface when buried as pupae and most successful as late 3rd instar larvae (prepupae). Collectively, more adult flies emerged from the soil if buried as 3rd instars (79.6%) than either 2nd instars or pupae (59.6% and 59.3%, respectively (F(2,159)=14.76, P<0.0001)). Similarly, at shallow burial depths of 5 and 25cm, 75.6% and 70.4% of the adults successfully reached the surface, compared with 52.6% at the 50-cm depth (F(2,159)=15.95, P<0.0001). Second instars demonstrated ascending vertical dispersal behaviours in the soil column by pupating closer to the surface. Nearly half (46.6%) of the C. macellaria 2nd instars buried in 25cm of soil pupated nearer to the surface. Similarly, 45.4% of the P. terraenovae 2nd instars pupated nearer to the surface. When buried at 50cm, approximately 25% of 2nd instars of both species pupated nearer to the surface. When 3rd instars of C. macellaria and P. terraenovae were buried at 120cm, 40% and 4.3% of the adults, respectively, successfully reached the soil surface.

Comparison of the effect of constant light on the circadian rhythm of white-eye mutant and wild-type blow fly Protophormia terraenovae

Compound eyes of white-eye mutant flies are considered to be more sensitive than those of the wild type because the former lack the screening pigment. The effects of constant light conditions on the circadian rhythm were examined in wild-type and white-eye mutant Protophormia terraenovae to identify plausible photoreceptors mediating constant light to clocks, which affect the free-running period and rhythmicity. Both the wild type and white-eye mutant showed clear rhythmicity under constant light conditions at 0.5 lx and 500 lx. Although the incidence of arrhythmia increased as light intensity increased, more than half of the flies were rhythmic at 500 lx. P. terraenovae seems to have a high threshold for light causing arrhythmia. Incidence of rhythmic flies and the free-running period under constant light conditions were not significantly different between the wild type and white-eye mutant. This suggests that photoreceptors operating under constant light conditions are extraretinal.

Sensilla on the antennal funiculus of the blow fly, Protophormia terraenovae (Diptera: Calliphoridae)

The morphology of the antennal funiculus and the external morphological characteristics and distribution of sensilla of blow fly, Protophormia terraenovae, have been studied using light and scanning electron microscopy. Cross section of the funiculus is roughly triangular in shape, with an anterior-medial, anterior-lateral, and posterior surface. The latter presents some large-size pits on restricted lateral and median areas of the proximal funiculus, and several smaller-size ones close to the pedicel–funiculus joint. The entire surface of the antennal sub-segment appears densely populated by microtrichia and is inhabited by seven types of sensilla: one trichoid, two basiconic, one auriculate, one coeloconic, and two basiconic-like pit sensilla. Trichoid, basiconic, auriculate and basiconic-like types display a multiporous wall, a feature characteristic of insect olfactory sensilla. It remains to be verified whether or not the coeloconic structure type has wall pores. The most abundant sensilla are the trichoid ones, which are followed by the basiconic, coeloconic and auriculate types in a decreasing density order. The basiconic-like pit sensilla are present only on the posterior funicular surface, unlike the remaining ones which populate the entire sub-segment. The blow fly’ funiculus displays a significant, even though moderate sexual dimorphism, the female sub-segment being bigger and presenting a higher number of trichoid and auriculate sensilla. The presence of multiple wall pores in most of sensilla types implies an olfactory modality for sensory neurons they accomodate, thus indicating that the blow fly’ funiculus is a plain olfactory organ.

Purification and Characterization of an Antimicrobial Peptide, Insect Defensin, From Immunized House Fly (Diptera: Muscidae)

The house fly, Musca domestica L. (Diptera: Muscidae), is involved in phoretic movement of pathogenic agents, but it has a very efficient defense mechanism against infection. It is believed that antimicrobial peptides play a significant role in the defense system of the house fly. Here, we isolated a peptide from the immunized house fly pupae, measured its molecular mass (3987.6 Da) by matrix-assisted laser desorption ionization/time of flight-mass spectrometry, and determined its amino acid sequence by using the Procise Protein Sequencing System (Applied Biosystems, Foster City, CA). The peptide was confirmed as a member of the insect defensin family. It displayed high activity against gram-positive bacteria but lower activity against gram-negative bacteria and fungi. Reverse transcription-polymerase chain reaction showed that the house fly defensin gene was constitutively expressed in naive pupae and strongly up-regulated after immunization. House fly defensin is an amphiphilic peptide with a structure similar to that of the CSalphabeta scaffold of insect defensin A from the flesh fly, Phormia terraenovae Robineau-Desvoidy. To our knowledge, this is the first isolated and characterized house fly antimicrobial peptide, and our work may provide useful information for developing pharmacologically active antimicrobial agents.

Comparison of Decomposition Rates and Faunal Colonization of Carrion in Indoor and Outdoor Environments

Decomposition and insect colonization of pig (Sus scrofa L.) carcasses were observed over a 42-day period inside and outside a house in a suburban region of Edmonton, Alberta. Three freshly killed pig carcasses were placed outdoors on grass and three carcasses were placed in separate rooms inside a house in a suburban residential area. The carcasses were examined and sampled regularly. Outside carcasses were colonized rapidly by Calliphora vicina (R-D), Lucilia sericata (Meigen), Lucilia illustris (Meigen), Eucalliphora latifrons (Hough), Cynomya cadaverina (R-D), Phormia regina (Meigen), and Protophormia terraenovae (R-D). There was a delay of 5 days before inside carcasses were first colonized but all species except E. latifrons and L. illustris readily colonized at this time. Many more insects colonized the outside carcasses, and these were rapidly skeletonized. Inside, much fewer insects were present and decomposition was slowed and colonization extended. Dispersal patterns of postfeeding larvae inside the house on hard substrate were greatly expanded compared with those from carcasses on grass.

The sense of water in the blowfly Protophormia terraenovae

The gustatory system of the blowfly, Protophormia terraenovae, is a relatively simple biological model for studies on chemosensory input and behavioral output. It appears to have renewed interest as a model for studies on the role of water channels, namely aquaporins or aquaglyceroporins, in water detection. To this end, we investigated the presence of water channels, their role in “water” and “salt” cell responsiveness and the transduction mechanism involved. For the first time our electrophysiological results point to the presence of an aquaglyceroporin in the chemoreceptor membrane of the “water” cell in the blowfly taste chemosensilla whose transduction mechanism ultimately involves an intracellular calcium increase and consequently cell depolarization. This hypothesis is also supported by calcium imaging data following proper stimulation. This mechanism is triggered by “water” cell stimulation with hypotonic solutions and/or solutes such as glycerol which crosses the membrane by way of aquaglyceroporins. Behavioral output indicates that the “sense” of water in blowflies is definitely not dependent on the “water” cell only, but also on the “salt” cell sensitivity. These findings also hypothesize a new role for aquaglyceroporin in spiking cell excitability.

Changes in Development and Heat Shock Protein Expression in Two Species of Flies (Sarcophaga bullata [Diptera: Sarcophagidae] and Protophormia terraenovae [Diptera: Calliphoridae]) Reared in Different Sized Maggot Masses

AbstractDevelopment of two species of necrophagous flies, Sarcophaga bullata Parker (Sarcophagidae) and Protophormia terraenovae (Robineau-Desvoidy) (Calliphoridae), was examined in different size maggot masses generated under laboratory conditions. Larvae from both species induced elevated mass temperatures dependent on the number of individuals per mass. The relationship was more evident for S. bullata, as larvae generated higher temperatures in every size maggot mass than P. terraenovae. Several development events were altered with increasing maggot mass size of flesh flies, and to a lesser extent blow flies, which corresponded with elevated temperatures. Duration of development of all feeding larval stages decreased with increased size of maggot mass. However, the length of development during puparial stages actually increased for these same flies. Puparial weights also declined with maggot mass size, as did the ability to eclose. The altered fly development was attributed to the induction of heat stress conditions, which was evident by the expression of heat shock proteins (23, 60, 70, and 90) in larval brains of both fly types.

Changes in Development and Heat Shock Protein Expression in Two Species of Flies (&lt;I&gt;Sarcophaga bullata&lt;/I&gt; [Diptera: Sarcophagidae] and &lt;I&gt;Protophormia terraenovae&lt;/I&gt; [Diptera: Calliphoridae]) Reared in Different Sized Maggot Masses

Development of two species of necrophagous flies, Sarcophaga bullata Parker (Sarcophagidae) and Protophormia terraenovae (Robineau-Desvoidy) (Calliphoridae), was examined in different size maggot masses generated under laboratory conditions. Larvae from both species induced elevated mass temperatures dependent on the number of individuals per mass. The relationship was more evident for S. bullata, as larvae generated higher temperatures in every size maggot mass than P. terraenovae. Several development events were altered with increasing maggot mass size of flesh flies, and to a lesser extent blow flies, which corresponded with elevated temperatures. Duration of development of all feeding larval stages decreased with increased size of maggot mass. However, the length of development during puparial stages actually increased for these same flies. Puparial weights also declined with maggot mass size, as did the ability to eclose. The altered fly development was attributed to the induction of heat stress conditions, which was evident by the expression of heat shock proteins (23, 60, 70, and 90) in larval brains of both fly types.

First record of Thinoseius spinosus (Acari: Eviphididae) from the high Arctic island of Spitsbergen (Svalbard) including a key to deutonymphs of genus Thinoseius

ABSTRACT Thinoseius spinosus is reported from the high Arctic island of Spitsbergen, Svalbard, for the first time. The deutonymphs were phoretic on the calliphorid dipteran Protophormia terraenovae. Mites were observed on 20.4% of adult flies. The number of mites per fly varied between 0 up to a maximum of 17 (median number of mites per fly with mites = 1). Although mites were found attached all over the body it seems likely that during dispersal the mites are usually found intercalated between the abdominal plates. An overview of the currently known species of Thinoseius, their occurrence and a key to the deutonymphs are presented.

Effect of photoperiod on clock gene expression and subcellular distribution of PERIOD in the circadian clock neurons of the blow fly Protophormia terraenovae

We examined the effect of photoperiod on the expression of circadian clock genes period (per) and timeless (tim), using quantitative real-time polymerase chain reaction (PCR), and the effect of photoperiod on subcellular distribution of PERIOD (PER), using immunocytochemistry, in the blow fly, Protophormia terraenovae. Under both short-day and long-day conditions, the mRNA levels of per and tim in the brain oscillated, and their peaks and troughs occurred around lights-off and lights-on, respectively. The oscillations persisted even under constant darkness. In the large ventral lateral neurons (l-LN(v)s), small ventral lateral neurons (s-LN(v)s), dorsal lateral neurons (LN(d)s), and medial dorsal neurons (DN(m)s), the subcellular distribution of PER-immunoreactivity changed with time. The number of cells with PER-immunoreactivity in the nucleus was highest 12 h after lights-off and lowest 12 h after lights-on, regardless of photoperiod, suggesting that PER nuclear translocation entrains to photoperiod. When temporal changes in the nuclear localization of PER were compared, the neurons could be classified into 2 groups: the l-LN(v)s were similar to the s-LN(v)s, and the LN(d)s were similar to DN(m)s. In LN(d)s and DN(m)s, decreasing rates of the number of cells with PER immunoreactivity in the nucleus per brain from the maximum were large as compared with those in l-LN(v)s and s-LN(v)s under short-day conditions. These results suggest that photoperiodic information is reflected in the expression patterns of circadian clock genes per and tim and in the subcellular distribution of PER. This observation suggests that the 2 different groups of clock neurons respond to photoperiod in slightly different manners.

Indoors forensic entomology: Colonization of human remains in closed environments by specific species of sarcosaprophagous flies

Fly species that are commonly recovered on human corpses concealed in houses or other dwellings are often dependent on human created environments and might have special features in their biology that allow them to colonize indoor cadavers. In this study we describe nine typical cases involving forensically relevant flies on human remains found indoors in southern Finland. Eggs, larvae and puparia were reared to adult stage and determined to species. Of the five species found the most common were Lucilia sericata Meigen, Calliphora vicina Robineau-Desvoidy and Protophormia terraenovae Robineau-Desvoidy. The fleshfly Sarcophaga caerulescens Zetterstedt is reported for the first time to colonize human cadavers inside houses and a COI gene sequence based DNA barcode is provided for it to help facilitate identification in the future. Fly biology, colonization speed and the significance of indoors forensic entomological evidence are discussed.

IDENTIFICATION OF HOST MATERIAL FROM CROPS AND WHOLE BODIES OF Protophormia terraenovae (R-D) (DIPTERA) LARVAE, PUPAE, AND ADULTS, AND THE IMPLICATIONS FOR FORENSIC STUDIES

ABSTRACTLarvae of Diptera are used routinely in forensic entomology to estimate accurately the post mortem interval. These applications require a detailed understanding of insect growth rates and insect succession profiles under different climactic conditions and geographical locations. There may be times when larvae may have been removed from a single food source or the corpse has been moved from one location to another, confounding the interpretation of the entomological data. We report here a study that evaluates the time period during which a food source can be detected in larvae of Protophormia terraenovae. We could detect the food source (pork or chicken liver) that larvae had ingested from early second instar to late third instar larvae. No detection was possible in pupae or adults. When larvae were transferred from one food source to another, the primary source was detectable for only 8–12 hours after transfer, whereas it was detectable for 12 hours in starved larvae. In most studies that use insects to estimate PMI, investigators collect larvae directly from the corpse. In cases where there have been significant disturbances, investigators must understand the changes in insect dieting and development that may take place.

Transduction mechanism(s) of Na-saccharin in the blowfly Protophormia terraenovae: evidence for potassium and calcium conductance involvement

The study on transduction mechanisms underlying bitter stimuli is a particularly intriguing challenge for taste researchers. The present study investigates, in the labellar chemosensilla of the blowfly Protophormia terraenovae, the transduction mechanism by which saccharin evokes the response of the "deterrent" cell, with particular attention to the contribution of K(+) and Ca(2+) current and the role of cyclic nucleotides, since second messengers modulate Ca(2+), Cl(-) and K(+) currents to different extents. As assessed by extracellular single-sensillum recordings, our results show that the addition of a Ca(2+) chelator such as EGTA or the Ca(2+) current blockers SK&F-96365, Mibefradil, Nifedipine and W-7 decrease the response of the "deterrent" cell to saccharin. A similar decreasing effect was also obtained following the addition of 4-aminopyridine, a K(+) current blocker. On the contrary, the membrane-permeable cyclic nucleotide 8-bromoguanosine 3',5'-cyclic monophosphate (8Br-cGMP) activates this cell and shows an additive effect when presented mixed with saccharin. Our results are consistent with the hypothesis that in the labellar chemosensilla of the blowfly both Ca(2+) and K(+) ions are involved in the transduction mechanism of the "deterrent" cell in response to saccharin. Our results also suggest a possible pathway common to saccharin and 8Br-cGMP.

Mass spectrometric analysis of peptides in brain neurosecretory cells and neurohemal organs in the adult blowfly, Protophormia terraenovae

Neuropeptides in neurosecretory cells of the pars intercerebralis (PI) and pars lateralis (PL) in the brain, and those in the corpus cardiacum-hypocerebral ganglion complex (CC-HG) and corpus allatum (CA) were examined by mass spectrometry and immunocytochemistry in adult females of the blowfly, Protophormia terraenovae. By using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and electrospray ionization quadrupole orthogonal acceleration time-of-flight mass spectrometry (ESI-Q-Tof MS) and MS/MS, 4 peptides (including myosuppressin and SIFamide) were detected in the PI, 12 peptides (including [Arg(7)]-corazonin and [Arg(7)]-corazonin(3)(-)(11)) in the PL, 13 peptides (including myosuppressin, [Arg(7)]-corazonin and [Arg(7)]-corazonin(3-11)) in the CC-HG, and 6 peptides in the CA. MALDI-TOF MS analysis of each tissue or organ was made in about 20 flies under diapause-inducing (LD 12:12 at 20 degrees C) and diapause-averting conditions (LD 18:6 at 25 degrees C). These molecular ion peaks did not distinctively differ between diapause-inducing and diapause-averting conditions. A peptide with an m/z value at 1395.1 was purified from 240 brains and the 2nd-10th amino acids were sequenced as -YRKPPFNGS-, corresponding to a partial sequence of SIFamide. Only two pairs of somata in the PI were immunoreactive to antisera against SIFamide, which were local neurons widely extending fibers throughout the brain neuropils.