Major differences in the larval anatomy of the digestive and excretory systems of three Oestridae species revealed by micro‐CT

Oestrid flies (Diptera: Oestridae) do not feed during the adult stage, so they depend on an efficient assimilation and storage of nutrients during their parasitic larval stage. We describe the general morphology and provide volumetric data for the digestive and excretory organs of the three larval instars of the nasal bot fly Oestrus ovis L., using micro‐computed tomography. The size of the digestive and excretory organs greatly increased across larval instars. In all instars, the two salivary glands were remarkably large and formed a ‘glandular band’ by coming together, but without lumina uniting, at their posterior ends. The distal region of the anterior Malpighian tubules was greatly enlarged and full of highly radio‐opaque concretions. Moreover, the anatomy of O. ovis third‐instar larva was compared to that of two species of, respectively, similar and different feeding habits: Cephenemyia stimulator (Clark) and Hypoderma actaeon Brauer. Whereas the general morphology and arrangement of the digestive and excretory systems of C. stimulator was similar to that of O. ovis, some differences were observed in H. actaeon: a swollen anterior region of the midgut, salivary glands shorter and not forming a ‘band’ and anterior Malpighian tubules narrowly uniform throughout their entire length.     

Looking into the puparium: Micro‐CT visualization of the internal morphological changes during metamorphosis of the blow fly,Calliphora vicina,with the first quantitative analysis of organ development in cyclorrhaphous dipterans

Metamorphosis of cyclorrhaphous flies takes place inside a barrel‐like puparium, formed by the shrinking, hardening and darkening of the third‐instar larval cuticle. The opacity of this structure hampers the visualization of the morphological changes occurring inside and therefore a full understanding of the metamorphosis process. Here, we use micro‐computed tomography (micro‐CT) to describe the internal morphological changes that occur during metamorphosis of the blow fly, Calliphora vicina Robineau‐Desvoidy 1830 (Diptera: Calliphoridae) at a greater temporal resolution than anything hitherto published. The morphological changes were documented at 10% intervals of the total intra‐puparial period, and down to 2.5% intervals during the first 20% interval, when the most dramatic morphological changes occur. Moreover, the development of an internal gas bubble, which plays an essential role during early metamorphosis, was further investigated with X‐ray images and micro‐CT virtual sections. The origin of this gas bubble has been largely unknown, but micro‐CT virtual sections show that it is connected to one of the main tracheal trunks. Micro‐CT virtual sections also provided enough resolution for determining the completion of the larval‐pupal and pupal‐adult apolyses, thus enabling an accurate timing of the different intra‐puparial life stages. The prepupal, pupal, and pharate adult stages last for 7.5%, 22.5%, and 70% of the total intra‐puparial development, respectively. Furthermore, we provide for the first time quantitative data on the development of two organ systems of the blow fly: the alimentary canal and the indirect flight muscles. There is a significant and negative correlation between the volume of the indirect flight muscles and the pre‐helicoidal region of the midgut during metamorphosis. The latter occupies a large portion of the thorax during the pupal stage but narrows progressively as the indirect flight muscles increase in volume during the development of the pharate adult.     

Irradiation effect on the structure of bacterial communities associated with the oriental fruit fly,Bactrocera dorsalis

The role of symbiotic microbes in insects, especially the beneficial character of this interaction for insects, has received much attention in recent years as it has been related to important aspects of the host insects' biology such as development, reproduction, survival, and fitness. Among insect hosts, tephritid fruit flies are well known to form beneficial associations with their symbionts. To control these destructive agricultural pests, environmentally friendly approaches, like the sterile insect technique as a component of integrated pest management strategies, remain most effective. In this study, changes in the bacterial profile of mass‐reared oriental fruit flies, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae), were examined in both larval and adult stages and also after irradiation by employing a 16S rRNA gene‐based Illumina sequencing approach. Proteobacteria was the prevalent bacterial phylum in non‐irradiated adults and larvae. Alphaproteobacteria was the most abundant class in larvae but almost absent in adults, which was dominated by Gammaproteobacteria. Firmicutes were present in both developmental stages but at lower relative abundance. At genus level, Acetobacter prevailed in the larval stage and members of the Enterobacteriaceae family in adults. Irradiated samples exhibited higher diversity and richness indices compared to the non‐irradiated oriental fruit flies, whereas no significant changes were observed between the two developmental stages of the non‐irradiated samples. Lactobacillus, members of the Orbacecae family, and Morganella were detected but to a lesser degree upon irradiation, whereas the relative abundance of Lactococcus and Orbus increased. The bacterial profile of larvae appeared to be different compared to that of adult B. dorsalis flies. The subsequent application of irradiation at the pupal stage led to the development of different microbiota between treated and untreated samples, affecting diversity and operational taxonomic unit composition. Irradiated samples of oriental fruit flies were characterized by higher species diversity and richness.     

Comparative study of the digestive system of three species of tinamou. I. Crypturellus tataupa,Nothoprocta cinerascens,and Nothura maculosa (Aves: Tinamidae)

The morphology and histology, as well as the cytochemistry of complex carbohydrates, of the digestive system of Crypturellus tataupa (tataupa tinamou), Nothoprocta cinerascens (brushland tinamou), and Nothura maculosa (spotted tinamou) are described. The general morphology of the digestive system of these birds follows the basic model of the avian alimentary canal, although statistical analysis shows that the lengths of the organs are significantly different among the species. From cephalic to caudal regions the alimentary tract consists of esophagus, ingluvies or crop, proventriculus or glandular stomach, ventriculus or muscular stomach, small intestine, well-developed ceca, and rectum. Histologically, each section of the tract consists of four primary tissue layers: mucosa, submucosa, muscularis, and adventitia. Variations are found in the thickness of the esophageal epithelium, which shows the highest value in C. tataupa. In the proventriculus, the depth of the compound glands is greatest in N. cinerascens. The villi of the epithelial cells in the small intestine are most extensively developed in C. tataupa. Heterogeneity of mucins is detected not only in the surface coat of the alimentary tract but in the cellular content of the glands as well. Comparisons with the morphology of the digestive system of closely related and more advanced birds are made, and the possible relationship between morphological and cytochemical variation and the diet is discussed. © 1996 Wiley-Liss, Inc.     

Symbiotic bacteria enable olive flies (Bactrocera oleae) to exploit intractable sources of nitrogen

Insects are often associated with symbiotic micro‐organisms, which allow them to utilize nutritionally marginal diets. Adult fruit flies (Diptera: Tephritidae) associate with extracellular bacteria (Enterobacteriaceae) that inhabit their digestive tract. These flies obtain nutrients by foraging for plant exudates, honeydew and bird droppings scattered on leaves and fruit – a nutritional niche which offers ample amounts of carbohydrates, but low quantities of available nitrogen. We identified the bacteria resident in the gut of the olive fly (Bactrocera oleae) – a worldwide pest of olives and examined their contribution to nitrogen metabolism in the adult insect. By suppressing bacteria in the gut and monitoring female fecundity, we demonstrate that bacteria contribute essential amino acids and metabolize urea into an available nitrogen source for the fly, thus significantly elevating egg production. In an ecological context, bacteria were found to be beneficial to females subsisting on bird droppings, but not on honeydew – two natural food sources. We suggest that a main gut bacterium (Candidatus Erwinia dacicola) forms an inseparable, essential part of this fly's nutritional ecology. The evolution of this symbiosis has allowed adult flies to utilize food substrates which are low or imbalanced in assimilable nitrogen and thereby to overcome the nitrogen limitations of their natural diet.     

Adaptive change in protective coloration in adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae): test of detectability of two colour forms by avian predators

1. Protective coloration in insects may be aposematic or cryptic, and some species change defensive strategy between instars. In Sweden, the adult striated shieldbugs Graphosoma lineatum (Heteroptera: Pentatomidae) undergo a seasonal colour change from pale brown and black striation in the pre‐hibernating adults, to red and black striation in the same post‐hibernating individuals. To the human eye the pre‐hibernating adults appear cryptic against the withered late summer vegetation, whereas the red and black post‐hibernating adults appear aposematic. This suggests a possibility of a functional colour change. However, what is cryptic to the human eye is not necessarily cryptic to a potential predator. 2. Therefore we tested the effect of coloration in adult G. lineatum on their detectability for avian predators. Great tits (Parus major) were trained to eat sunflower seeds hidden inside the emptied exoskeletons of pale or red G. lineatum. Then the detection time for both colour forms was measured in a dry vegetation environment. 3. The birds required a longer time to find the pale form of G. lineatum than the red one. The pale form appears more cryptic on withered late summer vegetation than the red form, not only to the human eye but also to avian predators. The result supports the idea that the adult individuals of G. lineatum undergo a functional change from a cryptic protective coloration to an aposematic one.     

Cathepsin D-Like Aspartic Proteinase Occurring in a Maize Weevil,Sitophilus zeamais,as a Candidate Digestive Enzyme

The maize weevil, Sitophilus zeamais, is an insect pest infesting rice and corn seeds. We identified an aspartic proteinase (AP) digesting rice glutelin in the alimentary tract of S. zeamais. The mRNA encoding the AP (SAP1) was expressed in the larvae foregut and in the adult midgut. These results indicate that SAP1 is probably digestive enzyme of S. zeamais.     

Dispersal of mass‐reared sterile,laboratory‐domesticated and wild male Queensland fruit flies

Queensland fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae) (‘Q‐flies’) were released as sexually immature adults from a point within an orchard. Marked male Q‐flies were recaptured in the trap furthest from the release point (1087 m) by 2 weeks after release, although 98.25 ± 1.04% of recaptured males were trapped <500 m from the release point. Comparison of gamma‐irradiated (sterile), laboratory‐adapted and wild male Q‐flies indicated that dispersal distance was not significantly affected by fly type. There was no significant correlation between temperature and mean dispersal distance, but total recaptures were significantly negatively correlated with increasing daily maximum, minimum and average temperature.     

Molecular phylogenetic profiling of gut‐associated bacteria in larvae and adults of flesh flies

Flesh flies of the genus Sarcophaga (Diptera: Sarcophagidae) are carrion‐breeding, necrophagous insects important in medical and veterinary entomology as potential transmitters of pathogens to humans and animals. Our aim was to analyse the diversity of gut‐associated bacteria in wild‐caught larvae and adult flesh flies using culture‐dependent and culture‐independent methods. Analysis of 16S rRNA gene sequences from cultured isolates and clone libraries revealed bacteria affiliated to Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes in the guts of larval and adult flesh flies. Bacteria cultured from larval and adult flesh fly guts belonged to the genera Acinetobacter, Bacillus, Budvicia, Citrobacter, Dermacoccus, Enterococcus, Ignatzschineria, Lysinibacillus, Myroides, Pasteurella, Proteus, Providencia and Staphylococcus. Phylogenetic analysis showed clone sequences of the genera Aeromonas, Bacillus, Bradyrhizobium, Citrobacter, Clostridium, Corynebacterium, Ignatzschineria, Klebsiella, Pantoea, Propionibacterium, Proteus, Providencia, Serratia, Sporosarcina, Weissella and Wohlfahrtiimonas. Species of clinically significant genera such as Ignatzschineria and Wohlfahrtiimonas spp. were detected in both larvae and adult flesh flies. Sequence analysis of 16S rRNA gene libraries supported culture‐based results and revealed the presence of additional bacterial taxa. This study determined the diversity of gut microbiota in flesh flies, which will bolster the ability to assess microbiological risk associated with the presence of these flies. The present data thereby establish a platform for a much larger study.     

Vibrio cholerae laboratory infection of the adult house fly Musca domestica

The present study was designed to test the hypothesis that house flies may be capable of specifically harbouring ingested Vibrio cholerae in their digestive tracts. Flies were continuously fed green fluorescent protein (GFP)‐labelled, non‐O1/non‐O139 environmental strains of V. cholerae. Bacterial burdens were quantitatively measured using plate counts and localization was directly observed using confocal microscopy. Vibrio cholerae were present in the fly alimentary canal after just 4 h, and reached a plateau of ～10⁷ colony‐forming units (CFU)/fly after 5 days in those flies most tolerant of the pathogen. However, individual flies were resistant to the pathogen: one or more flies were found to carry < 180 V. cholerae CFU at each time‐point examined. In flies carrying V. cholerae, the pathogen was predominantly localized to the midgut rather than the rectal space or crop. The proportion of house flies carrying V. cholerae in the midgut was dose‐dependent: the continuous ingestion of a concentrated, freshly prepared dose of V. cholerae increased the likelihood that fluorescent cells would be observed. However, V. cholerae may be a transient inhabitant of the house fly. This work represents the first demonstration that V. cholerae can inhabit the house fly midgut, and provides a platform for future studies of host, pathogen and environmental mediators of the successful colonization of this disease vector.     

Larvicidal potential of the polyol sweeteners erythritol and xylitol in two filth fly species

The house fly, Musca domestica (L.) (Diptera: Muscidae), and the stable fly, Stomoxys calcitrans (L.) (Diptera: Muscidae), are two filth flies responsible for significant economic losses in animal production. Although some chemical control products target adults of both species, differences in mouthpart morphology and behavior necessitates distinct modalities for each. For these reasons, larvicides are an attractive means of chemical control. We assessed the potential of the polyol sweeteners erythritol and xylitol as larvicides to the house fly and stable fly. LC₅₀ values of erythritol against 2^nd instar larvae were 34.94 mg/g media (house fly) and 22.10 mg/g media (stable fly). For xylitol, LC₅₀ values were 74.91 mg/g media (house fly) and 41.58 mg/g media (stable fly). When given a choice, neither species showed a preference for ovipositing in media treated with either sweetener at various concentrations or in media without sweetener. Significantly lower development from egg to adult was observed when the 2^nd instar LC₅₀ equivalent of each sweetener was present in the media compared to controls. Erythritol and xylitol both have larvicidal qualities, however their effective concentrations would necessitate creative product formulation and deployment methods to control all stages of developing flies.     

Molecular identification of bloodmeal sources and trypanosomes in Glossina spp., Tabanus spp. and Stomoxys spp. trapped on cattle farm settlements in southwest Nigeria

The interactions of host, vector and parasite in bovine trypanosomiasis transmission cycles in southwest Nigeria are not yet well understood. Trypanosoma (Trypanosomatida: Trypanosomatidae) species infection prevalences and bloodmeal sources were determined in transmitting vectors of the genera Glossina (Diptera: Glossinidae), Tabanus (Diptera: Tabanidae) and Stomoxys (Diptera: Muscidae) collected using Nzi traps in cattle settlements in southwest Nigeria. Sequenced cytochrome B mitochondrial DNA segments obtained from vector digestive tracts identified bloodmeal sources from eight host species, namely human, cattle, hippopotamus, giraffe, gazelle, spotted hyena, long‐tailed rat and one unidentified species. Overall, 71.1% [95% confidence interval (CI) 63.0–78.1], 33.3% (95% CI 21.9–47.0) and 22.2% (95% CI 16.2–29.9), respectively, of Glossina, Tabanus and Stomoxys flies were positive for trypanosomes. The observed trypanosome species were Trypanosoma vivax, Trypanosoma congolense, Trypanosoma brucei, Trypanosoma evansi, Trypanosoma simiae and Trypanosoma godfreyi. Trypanosome DNA was more prevalent in tsetse (34.8% Tr. vivax, 51.1% Tr. b. brucei, 5.2% Tr. congolense, 4.4% Tr. simiae and 24.4% mixed infections) than in other flies and the main determinants in all flies were seasonal factors and host availability. To the best of the present group's knowledge, this is the first report of Trypanosoma species in Tabanus and Stomoxys flies in Nigeria. It indicates that vector control programmes should always consider biting flies along with tsetse flies in the control of human and animal trypanosomiasis.     

The physiology of digestion in fish larvae

Synopsis The acquisition, digestion, and assimilation of food is critical for the growth and survival of fish larvae; a fish larva either grows or it perishes. Fish larvae are characterized by digestive systems and diets that differ from adults. Larvae undergo a pattern of trophic ontogeny, changing diet with increasing size, and these changes result in differences in digestive requirements. At first feeding, the larval alimentary canal is functional, but is structurally and functionally less complex than that of adults. The larval alimentary canal remains unchanged histologically during the larval period before transformation. During transformation, major changes that result in the development of the adult alimentary canal occur. The ontogeny of the alimentary canal differs in different taxa, and experimental evidence suggests that functional differences exist as well. Assimilation efficiency may be lower in larvae than it is in adult fishes, due to a lack of a morphological and functional stomach in larvae, but the question of improving assimilation efficiencies during larval development before transformation remains unresolved.     

Sexual development of wild and mass‐reared male Queensland fruit flies in response to natural food sources

Diet has a profound influence on the fitness of adult tephritid flies. Mass‐reared flies are provided yeast hydrolysate as a rich source of nutrition that supports rapid sexual development and mating success. In contrast, wild tephritid flies often live in environments where food may be hard to find, and these are the conditions that sexually immature mass‐reared sterile males encounter when released into the field during sterile insect technique campaigns. The effect of natural food sources (bat guano, bird droppings, citrus pollen, and wheat pollen) on the sexual development of adult mass‐reared fertile, mass‐reared sterile, and wild male Queensland fruit flies, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), was determined by measuring ejaculatory apodeme size. Inclusion of yeast hydrolysate in the adult diet was associated with faster growth of the ejaculatory apodeme in comparison with all other diets. Effects of diet were far less pronounced in mass‐reared males, which may indicate reduced nutritional requirements, whereas the ejaculatory apodeme of wild males fed on natural sources of food or sucrose alone did not increase in size over the first 20 days of adult life.     

THE ORIGIN OF AN ORGAN: PHYLOGENETIC ANALYSIS OF EVOLUTIONARY INNOVATION IN THE DIGESTIVE TRACT OF FLIES (INSECTA: DIPTERA)

The cardia, a prominent digestive tract organ consisting of several specialized cell types, occurs throughout the “higher” or muscoid flies, division Schizophora of order Diptera. Phylogenetic analysis of cellular organization in 65 insect species from 36 families indicates that this organ originated within the order Diptera from ancestrally undifferentiated tissues. “Lower” flies, suborder “Nematocera,” display little or no epithelial cell specialization at the corresponding site. Scorpionflies of the outgroup order Mecoptera are similarly unspecialized. Intermediate levels of cellular specialization occur in Tabanomorpha, Asilomorpha and Aschiza, dipteran taxa that diverge between “Nematocera” and Schizophora. The distribution of epithelial characteristics suggests that the cardia evolved through a sequence of simple tissue transformations, combining changes in epithelial configuration with local differentiation of cell structure and function. The evolution of locally specialized cell types implies the emergence of structural genes and regulatory mechanisms through the modification of an ancestral genome that had not supported such extensive differentiation. Comparison of localized gene expression in Drosophila melanogaster with that in other fly species having greater or lesser degrees of cell specialization may provide a practical model system for studying specific patterns of mutation associated with such evolutionary innovation.     

Seasonal variation of the capture and field distribution of sterile Anastrepha ludens in Tamaulipas,Mexico

Anastrepha ludens (Loew) (Diptera: Tephritidae) is one of the most important citrus pest in Mexico. The sterile insect technique (SIT) is used against pest populations of fruit flies for suppression, eradication, containment and prevention to reduce damages in fruit‐growing areas. In this study, we analyzed the seasonal variation of captures and field distribution of sterile A. ludens released in different seasons of the year in north‐eastern Mexico. Chilled releases were conducted by air at constant densities per ha on a citrus area for a period of 32 weeks that included the coldest and warmest seasons that is winter, spring and summer. Multilure traps baited with torula yeast pellets were used to capture sterile flies. Fly capture data were compared over the three seasons and correlated with climate. The lowest number of captures of the sterile insect occurred in the summer and the highest in winter and spring. High and low temperatures were negatively correlated with fly captures. Field distribution was also negatively correlated with high temperatures in summer, but no relationships were observed in winter and spring. No relationships were observed between rainfall with capture and field distribution of sterile flies. These results indicate that summer is a season involving agro‐ecological and environmental constraints for the capture and field distribution of sterile flies. This study may be useful for enhancing release strategies and optimizing economic resources in north‐eastern Mexico. Further research on the behaviour of sterile flies under stressful environments is suggested.     

Acquisition,retention and dispersal of soilborne plant pathogenic fungi by fungus gnats and moth flies

Adult fungus gnats and moth flies were experimentally demonstrated to function as potential above‐ground vectors for three soilborne plant pathogens: Verticillium dahliae, Fusarium acuminatum and Thielaviopsis basicola. The adult insects externally acquired the conidia of the pathogens after exposure to the cultures as confirmed by scanning electron microscope photography. The intestinal contents and frass deposits of larvae exposed to fungal cultures contained viable fungal propagules. Internally infested larvae developed into internally infested pupae; however, the emerging adults were free of fungal structures. Because of the maintenance of a high level of inoculum on the external body surface and the ability of these adult insects to fly, they can be a significant factor in the dispersal of soilborne fungi in greenhouse agriculture. The rate of dispersal of T. basicola by adult fungus gnats was 1.78 cm² h^?1 per insect and by adult moth flies was 1.17 cm² h^?1 per insect. The area over which the pathogen was dispersed by the adult insects increased with the increase in exposure time. The study demonstrated that adult insects are efficient distributors of soilborne plant pathogenic fungal propagules.     

Effect of conditions in sealed plastic bags on eclosion of mass‐reared Queensland fruit fly,Bactrocera tryoni

The sterile insect technique has been used for more than 50 years to control a range of insects around the world. Sterile insect technique is rapidly becoming a major component of many area‐wide fruit fly management programmes. Irradiation of immature life stages induces sterility in adults, which are then distributed over large areas to mate with wild flies, resulting in no viable offspring. However, irradiation in normal air results in declining adult quality. To optimize the quality of sterile adult flies, several techniques are available to lower the levels of oxygen in fruit fly tissues prior to irradiation. The simplest method is to seal pupae in plastic bags and allow the oxygen consumption of pupae to minimize oxygen in both the air and pupal tissue. Some fruit fly species have rapid decreases in eclosion as a result of low oxygen atmospheres. We tested the tolerance of Queensland fruit fly, Bactrocera tryoni (Froggatt) (Diptera: Tephritidae), to low oxygen for the first time. In the first two experiments, unirradiated B. tryoni pupae were tested for different periods in sealed plastic bags at 17, 21, and 26 °C. Optimum eclosion occurred at 21 °C with the lowest eclosion at 26 °C. In general, mean full eclosion declined at ca. 0.1% eclosion per hour sealed in plastic bags during the first 96 h for all temperatures. In the third and fourth experiments at 17 °C, there was a decline in average eclosion for irradiated and unirradiated pupae of about 13.4% after they were sealed in plastic bags for 192 h. In general, B. tryoni eclosion declined at 0.1% per hour inside sealed plastic bags for periods up to 192 h at 17 °C. Queensland fruit flies can tolerate long periods of conditions found inside sealed plastic bags and current practices for sterile B. tryoni release programmes will result in minimum decrease in eclosion. The possible evolution of tolerance of these conditions is discussed.     

Behavioural responses of stable flies to cattle manure slurry associated odourants

Stable flies (Stomoxys calcitrans [Diptera: Muscidae] L.) are blood‐feeding synanthropic pests, which cause significant economic losses in livestock. Stable fly antennae contain olfactory sensilla responsive to host and host environment‐associated odours. Field observation indicated that the abundance of stable flies increased significantly in grasslands or crop fields when cattle manure slurry was applied. Major volatile compounds emanating from manure slurry were collected and identified. Behavioural responses of stable flies to those compounds were investigated in laboratory bioassays and field‐trapping studies. Results from olfactometer assays revealed that phenol, p‐cresol and m‐cresol were attractive to adult stable flies. When tested individually, attraction was higher with lower dosages. Stable flies were most attracted to blends of phenol and m‐cresol or p‐cresol. Traps with binary blend lures caught more stable flies in field trials as well.