Cyclorrhaphan Flies Research Articles

Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom.

Extraembryonic tissues: exploring concepts, definitions and functions across the animal kingdom.

Porcupine/Wntless‐dependent trafficking of the conserved WntA ligand in butterflies

Wnt ligands are key signaling molecules in animals, but little is known about the evolutionary dynamics and mode of action of the WntA orthologs, which are not present in the vertebrates or in Drosophila. Here we show that the WntA subfamily evolved at the base of the Bilateria + Cnidaria clade, and conserved the thumb region and Ser209 acylation site present in most other Wnts, suggesting WntA requires the core Wnt secretory pathway. WntA proteins are distinguishable from other Wnts by a synapomorphic Iso/Val/Ala216 amino-acid residue that replaces the otherwise ubiquitous Thr216 position. WntA embryonic expression is conserved between beetles and butterflies, suggesting functionality, but the WntA gene was lost three times within arthropods, in podoplean copepods, in the cyclorrhaphan fly radiation, and in ensiferan crickets and katydids. Finally, CRISPR mosaic knockouts (KOs) of porcupine and wntless phenocopied the pattern-specific effects of WntA KOs in the wings of Vanessa cardui butterflies. These results highlight the molecular conservation of the WntA protein across invertebrates, and imply it functions as a typical Wnt ligand that is acylated and secreted through the Porcupine/Wntless secretory pathway.

Characterizing the embryonic development of B. hygida (Diptera: Sciaridae) following enzymatic treatment to permeabilize the serosal cuticle

Understanding the evolution of the developmental programs active during dipteran embryogenesis depends on comparative studies. As a counterpoint to the intensively investigated and highly derived cyclorrhaphan flies that include the model organism Drosophila melanogaster, we are studying the basal Diptera Bradysia hygida, a member of the Sciaridae family that is amenable to laboratory cultivation. Here we describe the B. hygida embryogenesis, which lasts 9 days at 22 °C. The use of standard fixation D. melanogaster protocols resulted in embryos refractory to DAPI staining and to overcome this, a new enzyme-based method was developed. Calcofluor-White staining of enzimatically-treated embryos revealed that this method removes chitin from the serosal cuticle surrounding the B. hygida embryo. Chitin is one of the main components of serosal cuticles and searches in a B. hygida embryonic transcriptome database revealed conservation of the chitin synthesis pathway, further supporting the occurrence of chitin biosynthesis in B. hygida embryos. Combining the enzymatic treatment protocol with the use of both DIC and fluorescence microscopy allowed the first complete description of the B. hygida embryogenesis. Our results constitute an important step towards the understanding of early development of a basal Diptera and pave the way for future evo-devo studies.

Leg deformation during imaginal ecdysis in the downy emerald, Cordulia aenea (Odonata, Corduliidae)

A dragonfly larva migrates from the water to the shore, perches on a plant stem and grasps it with strongly flexed legs. Adult legs inside the larval exoskeleton fit to the larval legs joint-to-joint. The adult emerges with stretched legs. During the molt, an imaginal leg must follow all the angles in exuvial joints. In turn, larval apodemes are withdrawn from imaginal legs. We visualized transient shapes of the imaginal legs by the instant fixation of insects at different moments of the molt, photographed isolated exuvial legs with the imaginal legs inside and then removed the exuvial sheath. Instant shapes of the imaginal tibia show sharp intrapodomere bends copying the angle in the larval femoro-tibial joint. The site of bending shifts distad during the molt. This is possible if the imaginal leg is pliable. The same problem of leg squeezing is also common in hemimetabolous insects as well as in other arthropods, whereas holometabolous insects overcome problems of a tight confinement either by using leg pliability in other ways but not squeezing (cyclorrhaphan flies, mosquitoes) or by pulling hardened legs out without change of their pupal zigzag configuration (moths, ants, honey bees). The pupal legs are not intended to grasp any external substrate.

Antarctotrechus balli sp. n. (Carabidae, Trechini): the first ground beetle from Antarctica.

Fossil elytra of a small trechine carabid are reported from the Oliver Bluffs on the Beardmore Glacier at lat. 85°S. They were compared with counterparts from the extant genera Trechisibus, Tasmanorites, Oxytrechus and Pseudocnides. The fossils share some characters but are sufficiently different to be described as a new genus and species. We named the new species Antarctotrechus balli in honour of George E. Ball who made major contributions to the study of carabids through his own research and the training of students while at the University of Alberta, Edmonton, Alberta, Canada. The closest extant relatives to the extinct Antarctotrechus balli are species of Trechisibus, which inhabit South America, the Falkland Islands and South Georgia, and Tasmanorites, which inhabit Tasmania, Australia. Plant fossils associated with Antarctotrechus balli included Nothofagus (southern beech), Ranunculus (buttercup), moss mats and cushion plants that were part of a tundra biome. Collectively, the stratigraphic relationships and the growth characteristics of the fossil plants indicate that Antarctotrechus balli inhabited the sparsely-vegetated banks of a stream that was part of an outwash plain at the head of a fjord in the Transantarctic Mountains. Other insects represented by fossils in the tundra biome include a listroderine weevil and a cyclorrhaphan fly. The age of the fossils, based on comparison of associated pollen with 40Ar/39Ar dated pollen assemblages from the McMurdo Dry Valleys, is probably Early to Mid-Miocene in the range 14–20 Ma. The tundra biome, including Antarctotrechus balli, became extinct in the interior of Antarctica about 14 Ma and on the margins of the continent by 10–13 Ma. Antarctotrechus balli confirms that trechines were once widely distributed in Gondwana. For Antarctotrechus balli and other elements of the tundra biome it appears they continued to inhabit a warmer Antarctica for many millions of years after rifting of Tasmania (45 Ma) and southern South America (31 Ma).

Incidence of Myiasis and bionomics of cyclorrhaphan flies in small ruminants of Jammu

A study was conducted to determine the incidence of myiasis and bionomics of cyclorrhaphan flies in small ruminants of Jammu. Sheep and goats (10 each) having cutaneous wounds were examined monthly (total 240 animals) for the presence/absence of maggots. The incidence of active maggot wound myiasis was 37.1%. The larvae of Lucilia cuprina were recovered from both sheep and goats whereas Calliphora erythrocephala and Phormia spp. larvae were found only in sheep and Hypoderma spp. larvae only in goats. There were marked seasonal fluctuations in the occurrence of infestation rate of maggots with a significantly (P<0.05) maximum incidence (71.7%) in monsoon season. To study the bionomics of cyclorrhaphan flies, meat pieces of sheep and goats were kept in open field as well as in dairy farm. Eight species viz. Lucilia cuprina, Calliphora erythrocephala, Chrysomyia megacephala, Sarcophaga rufifaciens, Phormia regina, Musca domestica and Stomoxys calcitrans of cyclorrhaphan flies were recorded. The study would help to formulate an appropriate control strategy against cutaneous myiasis causing flies and to minimize the perpetuating financial losses to animal owners in the region.

The complete mitochondrial genomes of Musca domestica and Scathophaga stercoraria (Diptera: Muscoidea: Muscidae and Scathophagidae)

The complete mitochondrial genomes of Musca domestica (Muscidae) and Scathophaga stercoraria (Scathophagidae) are circular molecules of 16,108 bp and 16,223 bp in length, respectively. The first complete mitochondrial genome of Scathophagidae is reported here. Both genomes contain all 37 genes, including 13 protein-coding genes (PCGs), 22 tRNA genes, 2 rRNA genes, and a large control region, with conserved arrangement pattern reported in all cyclorrhaphan flies. All PCGs start with standard ATN codons except for the CO1 which starts with TCG in both species. All PCGs terminate with the common stop codons TAA or TAG, except for the CO2 and ND5 in both species and ND4 in S. stercoraria which end with a single T.

Comparative gene expression analysis of Dtg, a novel target gene of Dpp signaling pathway in the early Drosophila melanogaster embryo

In the early Drosophila melanogaster embryo, Dpp, a secreted molecule that belongs to the TGF-β superfamily of growth factors, activates a set of downstream genes to subdivide the dorsal region into amnioserosa and dorsal epidermis. Here, we examined the expression pattern and transcriptional regulation of Dtg, a new target gene of Dpp signaling pathway that is required for proper amnioserosa differentiation. We showed that the expression of Dtg was controlled by Dpp and characterized a 524-bp enhancer that mediated expression in the dorsal midline, as well as, in the differentiated amnioserosa in transgenic reporter embryos. This enhancer contained a highly conserved region of 48-bp in which bioinformatic predictions and in vitro assays identified three Mad binding motifs. Mutational analysis revealed that these three motifs were necessary for proper expression of a reporter gene in transgenic embryos, suggesting that short and highly conserved genomic sequences may be indicative of functional regulatory regions in D. melanogaster genes.Dtg orthologs were not detected in basal lineages of Dipterans, which unlike D. melanogaster develop two extra-embryonic membranes, amnion and serosa, nevertheless Dtg orthologs were identified in the transcriptome of Musca domestica, in which dorsal ectoderm patterning leads to the formation of a single extra-embryonic membrane. These results suggest that Dtg was recruited as a new component of the network that controls dorsal ectoderm patterning in the lineage leading to higher Cyclorrhaphan flies, such as D. melanogaster and M. domestica.

Evolution and expression of BMP genes in flies

Bone morphogenetic proteins (BMPs) play key roles in development. In Drosophila melanogaster, there are three BMP-encoding genes: decapentaplegic (dpp), glass bottom boat (gbb) and screw (scw). dpp and gbb are found in all groups of insects. In contrast, the origin of scw via duplication of an ancestral gbb homologue is more recent, with new evidence placing it within the Diptera. Recent studies show that scw appeared basal to the Schizophora, since scw orthologues exist in aschizan cyclorrhaphan flies. In order to further localise the origin of scw, we have utilised new genomic resources for the nematoceran moth midge Clogmia albipunctata (Psychodidae). We identified the BMP subclass members dpp and gbb from an early embryonic transcriptome and show that their expression patterns in the blastoderm differ considerably from those seen in cyclorrhaphan flies. Further searches of the genome of C. albipunctata were unable to identify a scw-like gbb duplicate, but confirm the presence of dpp and gbb. Our phylogenetic analysis shows these to be clear orthologues of dpp and gbb from other non-cyclorrhaphan insects, with C. albipunctata gbb branching ancestrally to the cyclorrhaphan gbb/scw split. Furthermore, our analysis suggests that scw is absent from all Nematocera, including the Bibionomorpha. We conclude that the gbb/scw duplication occurred between the separation of the lineage leading to Brachycera and the origin of cyclorrhaphan flies 200–150 Ma ago.Electronic supplementary materialThe online version of this article (doi:10.1007/s00427-013-0445-9) contains supplementary material, which is available to authorized users.

BMP-dependent serosa and amnion specification in the scuttle fly Megaselia abdita

Bone morphogenetic protein (BMP) signaling is an essential factor in dorsoventral patterning of animal embryos but how BMP signaling evolved with fundamental changes in dorsoventral tissue differentiation is unclear. Flies experienced an evolutionary reduction of extra-embryonic tissue types from two (amniotic and serosal tissue) to one (amnionserosal tissue). BMP-dependent amnioserosa specification has been studied in Drosophila melanogaster. However, the mechanisms of serosal and amniotic tissue specification in less diverged flies remain unknown. To better understand potential evolutionary links between BMP signaling and extra-embryonic tissue specification, we examined the activity profile and function of BMP signaling in serosa and amnion patterning of the scuttle fly Megaselia abdita (Phoridae) and compared the BMP activity profiles between M. abdita and D. melanogaster. In blastoderm embryos of both species, BMP activity peaked at the dorsal midline. However, at the beginning of gastrulation, peak BMP activity in M. abdita shifted towards prospective amnion tissue. This transition correlated with the first signs of amnion differentiation laterally adjacent to the serosa anlage. Marker-assisted analysis of six BMP signaling components (dpp, gbb, scw, tkv, sax, sog) by RNA interference revealed that both serosa and amnion specification of M. abdita are dependent on BMP activity. Conversely, BMP gain-of-function experiments caused sharpened expression boundaries of extra-embryonic target genes indicative of positive feedback. We propose that changes in the BMP activity profile at the beginning of gastrulation might have contributed to the reduction of extra-embryonic tissue types during the radiation of cyclorrhaphan flies.

Command and Compensation in a Neuromodulatory Decision Network

The neural circuits that mediate behavioral choices must not only weigh internal demands and environmental circumstances, but also select and implement specific actions, including associated visceral or neuroendocrine functions. Coordinating these multiple processes suggests considerable complexity. As a consequence, even circuits that support simple behavioral decisions remain poorly understood. Here we show that the environmentally sensitive wing expansion decision of adult fruit flies is coordinated by a single pair of neuromodulatory neurons with command-like function. Targeted suppression of these neurons using the Split Gal4 system abrogates the fly's ability to expand its wings in the face of environmental challenges, while stimulating them forces expansion by coordinately activating both motor and neuroendocrine outputs. The arbitration and implementation of the wing expansion decision by this neuronal pair may illustrate a general strategy by which neuromodulatory neurons orchestrate behavior. Interestingly, the decision network exhibits a plasticity that is unmasked under conducive environmental conditions in flies lacking the function of the command-like neuromodulatory neurons. Such flies can often expand their wings using a motor program distinct from that of wild-type animals and controls. This compensatory program may be the vestige of an ancestral, environmentally insensitive program used for wing expansion that existed before the evolution of the environmentally adaptive program currently used by Drosophila and other cyclorrhaphan flies.

Postgastrular zen expression is required to develop distinct amniotic and serosal epithelia in the scuttle fly Megaselia

The amnioserosa is an extraembryonic epithelium that evolved in higher cyclorrhaphan flies from distinct serosal and amniotic epithelia. The underlying genetic mechanism of this evolutionary transition is unknown. Amnioserosa development of Drosophila correlates with novel expression characteristics of the homeobox gene zerknüllt ( zen), including a broad zen expression domain in the syncytial blastoderm and the complete absence of postgastrular zen expression. Here we examine the functional significance of these features by altering the activity profile of zen in Megaselia (a lower cyclorrhaphan fly with distinct serosal and amniotic epithelia) and Drosophila, and by examining in Megaselia the function of u-shaped group (ush-group) genes, which in Drosophila maintain the amnioserosa after gastrulation when zen is no longer expressed. In Megaselia, loss of postgastrular zen expression abrogates serosa development but allows amnion development. Ectopic expression of zen in early Megaselia embryos allows serosa formation but perturbs amnion development. Megaselia homologues of u-shaped group genes are not essential for serosa formation but mediate germband retraction and dorsal closure. Finally, ectopic postgastrular zen expression in Drosophila causes an enlargement of amnioserosa cells and interferes with the morphogenetic functions of the amnioserosa. Our results suggest that the origin of the amnioserosa involved the loss of postgastrular zen expression from extraembryonic tissue, that the early broad expression domain of Drosophila zen evolved afterwards, and that the ush-group genes ancestrally played a role in morphogenetic functions of the amnion.

Evidence for a composite anterior determinant in the hover flyEpisyrphus balteatus(Syrphidae), a cyclorrhaphan fly with an anterodorsal serosa anlage

Most insect embryos develop from a monolayer of cells around the yolk, but only part of this blastoderm forms the embryonic rudiment. Another part forms extra-embryonic serosa. Size and position of the serosa anlage vary between species, and previous work raises the issue of whether such differences co-evolve with the mechanisms that establish anteroposterior (AP) polarity of the embryo. AP polarity of the Drosophila embryo depends on bicoid, which is necessary and sufficient to determine the anterior body plan. Orthologs of bicoid have been identified in various cyclorrhaphan flies and their occurrence seems to correlate with a mid-dorsal serosa or amnioserosa anlage. Here, we introduce with Episyrphus balteatus (Syrphidae) a cyclorrhaphan model for embryonic AP axis specification that features an anterodorsal serosa anlage. Current phylogenies place Episyrphus within the clade that uses bicoid mRNA as anterior determinant, but no bicoid-like sequence could be identified in this species. Using RNA interference (RNAi) and ectopic mRNA injection, we obtained evidence that pattern formation along the entire AP axis of the Episyrphus embryo relies heavily on the precise regulation of caudal, and that anterior pattern formation in particular depends on two localized factors rather than one. Early zygotic activation of orthodenticle is separated from anterior repression of caudal, two distinct functions which in Drosophila are performed jointly by bicoid, whereas hunchback appears to be regulated by both factors. Furthermore, we found that overexpression of orthodenticle is sufficient to confine the serosa anlage of Episyrphus to dorsal blastoderm. We discuss our findings in a phylogenetic context and propose that Episyrphus employs a primitive cyclorrhaphan mechanism of AP axis specification.

Morphology versus molecules: the phylogenetic relationships of Sepsidae (Diptera: Cyclorrhapha) based on morphology and DNA sequence data from ten genes.

The Sepsidae is, with approximately 300 described species, a relatively small family of cyclorrhaphan flies whose behaviour, morphology, and development have been extensively studied. However, currently the only available tree for Sepsidae is more than 10 years old and was based entirely on morphological characters. Here, we present the results of parsimony and Bayesian analyses based on 75 species, ten genes, and morphology. Parsimony and Bayesian analyses produce largely congruent and well-supported topologies regardless of whether indels are coded as 5th character states, as missing values, or all sites with indels are removed. The tree confirms the monophyly of Sepsidae and identifies the Ropalomeridae as its sister group. With regard to higher-level relationships, we identify widespread conflict between the morphological and the DNA sequence data. The proposed hypothesis based on both partitions largely reflects the signal in the molecular data. Particularly surprising is the rejection of two relationship hypotheses with strong morphological support, namely the sister group relationship between Orygma and the remaining Sepsidae and the monophyly of the Sepsis species group. Our partitioned Bremer support (PBS) analyses imply that indel coding has a stronger effect on the relative performance of individual gene partitions than the exclusion of alignment-ambiguous sequences or the location of a gene on the mitochondrial or nuclear genome. However, these analyses also reveal unexpectedly strong fluctuations in PBS values given that indel treatment has only a minor effect on tree topology and jacknife support. These unexpected fluctuations highlight the need for a comparative study across multiple data sets that investigates the influence of conflict and indel treatment on PBS values. © The Willi Hennig Society 2008.

Public health importance of non-biting cyclorrhaphan flies

This study was carried out to determine the role of non-biting cyclorrhaphan flies as carriers of human intestinal parasites at Woreta, northwestern Ethiopia. In total, 6530 flies were collected from four breeding sites and then examined for human intestinal parasites, mainly using the formol-ether concentration method. Fly species identified were Musca domestica (32.9%), Chrysomya rufifacies (32.6%), Musca sorbens (23%), Lucina cuprina (4.7%), Calliphora vicina (2.8%), Chrysomya bezziana (2.3%) and Wohlfahrtia magnifica (1.7%). Intestinal parasites such as Ascaris lumbricoides (36.9%), Trichuris trichiura (38.8%), hookworm (13.0%), Hymenolepis nana (0.6%), Taenia spp. (8.4%), Strongyloides stercoralis (1.7%), Entamoeba histolytica/dispar (48.1%), Entamoeba coli (24.7%), Cryptosporidium spp. (16.7%) and Giardia lamblia (10.4%) were isolated from both external and gut contents of the flies. Trichuris trichiura and A. lumbricoides among the helminths and E. histolytica/dispar and E. coli among the protozoans were the dominant parasites identified. It was observed that more parasites were isolated from gut contents than the external surfaces of the flies examined (P<0.001). Chrysomya rufifacies were found to carry more helminths than M. sorbens and M. domestica. Musca sorbens were the highest carriers of protozoan parasites followed by M. domestica and C. rufifacies. The significance of filth flies as carriers of human intestinal parasites has been highlighted.

Bicoid occurrence and Bicoid‐dependent hunchback regulation in lower cyclorrhaphan flies

The homeobox gene bicoid functions as an anterior pattern organizer of the Drosophila embryo, but other than in higher flies (Cyclorrhapha), bicoid orthologues appear to be absent from insect genomes. In Drosophila, bicoid is expressed in an anterior-to-posterior protein gradient and regulates spatially restricted expression domains of segmentation genes in a concentration-dependent manner. hunchback, a direct transcriptional target of Bicoid, complements the "morphogen" activity of Bicoid. hunchback is activated by Bicoid throughout the anterior half of the blastoderm and a Bicoid-binding cis-regulatory element has been identified immediately upstream of the proximal hunchback promoter P2 of Drosophila and other higher Cyclorrhapha (Schizophora). bicoid and Bicoid-dependent hunchback regulation are thought to have originated during or before the radiation of extant Cyclorrhapha, although the precise occurrence of these traits in lower Cyclorrhapha remains unknown. Previously, we have described a bicoid orthologue in Megaselia, a species of the lower cyclorrhaphan family Phoridae. Here, we report the occurrence of bicoid in two additional lower cyclorrhaphan families, Lonchopteridae and Platypezidae. We show that Megaselia Bicoid is required for anterior expression of Megaselia hunchback, and binds upstream of its P2 promoter. Furthermore, we report the expression of lacZ reporter constructs under the control of hunchback regulatory sequences from a range of lower cyclorrhaphan and non-cyclorrhaphan flies in transgenic Drosophila embryos. Our results are consistent with a cyclorrhaphan origin of bicoid and suggest that a Bicoid-binding enhancer upstream of the hunchback P2 promoter evolved at the latest in the last common ancestor of Megaselia and Schizophora.

Expression and regulation of caudal in the lower cyclorrhaphan fly Megaselia

The homeobox gene caudal (cad) regulates posterior development in Drosophila. In early embryos, the cad protein (CAD) is expressed in a posterior-to-anterior concentration gradient, which contributes polarity to the developing embryo. The CAD gradient is complementary to and dependent on the anterior pattern organizer Bicoid (BCD), which represses the translation of ubiquitous maternal cad transcripts in the anterior embryo through a direct interaction with the cad 3′ untranslated region (UTR). Here, we show that early embryos of the lower cyclorrhaphan fly Megaselia express the putative cad orthologue Mab-cad throughout the posterior three quarters of the blastoderm but lack maternal transcripts. In transgenic blastoderm embryos of Drosophila, Mab-cad cis-regulatory DNA drives the expression of a reporter gene in a similar pattern, while Mab-cad 3′ UTR fails to mediate translational repression of a ubiquitously transcribed reporter. For another lower cyclorrhaphan fly (Lonchoptera) and two related outgroup taxa of Cyclorrhapha (Empis, Haematopota), we report maternal cad expression in ovarian follicles. Together, our results suggest that BCD is not required for the translational repression of Mab-cad, and that maternal cad expression was lost in the Megaselia lineage.

Evolutionary origin of the amnioserosa in cyclorrhaphan flies correlates with spatial and temporal expression changes of zen

Higher cyclorrhaphan flies including Drosophila develop a single extraembryonic epithelium (amnioserosa), which closes the germband dorsally. In most other insects two extraembryonic epithelia, serosa and amnion, line the inner eggshell and the ventral germband, respectively. How the two extraembryonic epithelia evolved into one is unclear. Recent studies have shown that, in the flour beetle Tribolium and in the milkweed bug Oncopeltus, the homeobox gene zerknüllt (zen) controls the fusion of the amnion with the serosa before dorsal closure. To understand the origin of the amnioserosa in evolution, we examined the expression and function of zen in the extraembryonic tissue of lower Cyclorrhapha. We show that Megaselia abdita (Phoridae) and Episyrphus balteatus (Syrphidae) develop a serosa and a dorsal amnion, suggesting that a dorsal amnion preceded the origin of the amnioserosa in evolution. Using Krüppel (Kr) and pannier (pnr) homologues of Megaselia as markers for serosal and amniotic tissue, respectively, we show that after zen RNAi all extraembryonic tissue becomes indistinguishable from amniotic cells, like in Tribolium but unlike in Drosophila, in which zen controls all aspects of extraembryonic development. Compared with Megaselia and Episyrphus, zen expression in Drosophila is extended to cells that form the amnion in lower Cyclorrhapha and is down-regulated at the developmental stage, when serosa cells in lower Cyclorrhapha begin to expand. These expression differences between species with distinct extraembryonic tissue organizations and the conserved requirement of zen for serosa development suggest that the origin of an amnioserosa-like epithelium was accompanied by expression changes of zen.

Non-biting cyclorrhaphan flies (Diptera) as carriers of intestinal human parasites in slum areas of Addis Ababa, Ethiopia

A study was conducted to determine the role of non-biting cyclorrhaphan flies as carriers of intestinal parasites in slum areas of Addis Ababa from January 2004 to June 2004. A total of 9550 flies, comprising of at least seven species were collected from four selected sites and examined for human intestinal parasites using the formol–ether concentration method. The dominant fly species was Chrysomya rufifacies (34.9%) followed by Musca domestica (31%), Musca sorbens (20.5.%), Lucina cuprina (6.8%), Sarcophaga sp. (2.8%), Calliphora vicina (2.2%) and Wohlfahrtia sp. (1.8%). Six intestinal helminths ( Ascaris lumbricoides, Trichuris trichiura, hookworms, Hymenolepis nana, Taenia spp. and Strongyloides stercoralis) and at least four protozoan parasites ( Entamoeba histolytica/ dispar, Entamoeba coli, Giardia lamblia and Cryptosporidium sp.) were isolated from both the external and gut contents of the flies. A. lumbricoides and T. trichiura among the helminths and E. histolytica/ dispar and E. coli among the protozoans were the dominant parasites detected both on the external and in the gut contents of the flies, but occurring more in the latter. Among the flies, C. rufifacies and M. sorbens were the highest carriers of the helminth and protozoan parasites, respectively. The public health significance of these findings is highlighted.

The major yolk proteins of higher Diptera are homologs of a class of minor yolk proteins in lepidoptera.

In most oviparous animals, including insects, vitellogenin (Vg) is the major yolk protein precursor. However, in the higher Diptera (cyclorrhaphan flies), a class of proteins homologous to lipoprotein lipases called yolk polypeptides (YP) are accumulated by oocytes instead of Vg, which is not produced at all. Lepidopterans (moths) produce Vg as the major yolk protein precursor, but also manufacture a class of minor yolk proteins referred to as egg-specific proteins (ESP) or YP2s. Although the lepidopteran ESP/YP2s are related to lipoprotein lipases, previous attempts to directly demonstrate their homology with higher-dipteran YPs were unsuccessful. In this paper, a multiple alignment of amino acid sequences was constructed using a shared lipid binding motif as an anchor, to demonstrate that lepidopteran ESP/YP2s, higher-dipteran YPs, and lipoprotein lipases are indeed homologous. Phylogenetic analyses of the aligned sequences were performed using both distance-based and parsimony strategies. It is apparent that the higher dipterans did not requisition a lipoprotein lipase to replace Vg as a yolk protein precursor, but instead utilize a class of proteins with an evolutionary history of use as minor constituents of yolk in other insects.