Molecular characterization of esterase E3 gene associated with organophosphorus insecticide resistance in the New World screwworm fly, Cochliomyia hominivorax

Abstract.  The New World screwworm, Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae), is one of the most important myiasis-causing flies in South America. It is responsible for severe economic losses to livestock producers, mainly because it causes mortality in newborn calves and reductions in the quality of leather and in the production of milk and meat. The economic losses caused by myiasis, along with those caused by other internal and external parasites, are the main factors limiting meat production. In Brazil, C. hominivorax has been controlled by applying insecticides, particularly organophosphate (OP)-based compounds. However, the improper and continuous use of these chemicals can lead to the selection of OP-resistant strains. This, associated with the fast development of OP resistance in other myiasis-causing flies, shows the importance of investigating resistance in C. hominivorax. Based on the findings of previous studies, the objective of the current work was to isolate and sequence the E3 gene in C. hominivorax. Mutations at the positions (Gly137 and Trp251) responsible for conferring OP resistance in Lucilia cuprina and Musca domestica L. (Muscidae) were identified in C. hominivorax . In addition, the orthologous region in C. hominivorax contained motifs that are highly conserved among carboxyl/cholinesterases and contribute to the catalytic mechanism of the active site. The characterization of this gene in natural populations of New World screwworm can be an important tool for monitoring resistance to insecticides throughout its current geographic distribution. This will provide information for the selection and implementation of more effective pest management programmes.     

Incongruent Nuclear and Mitochondrial Genetic Structure of New World Screwworm Fly Populations Due to Positive Selection of Mutations Associated with Dimethyl- and Diethyl-Organophosphates Resistance

Livestock production is an important economic activity in Brazil, which has been suffering significant losses due to the impact of parasites. The New World screwworm (NWS) fly, Cochliomyia hominivorax, is an ectoparasite and one of the most important myiasis-causing flies endemic to the Americas. The geographic distribution of NWS has been reduced after the implementation of the Sterile Insect Technique (SIT), being eradicated in North America and part of Central America. In South America, C. hominivorax is controlled by chemical insecticides, although indiscriminate use can cause selection of resistant individuals. Previous studies have associated the Gly137Asp and Trp251Leu mutations in the active site of carboxylesterase E3 to resistance of diethyl and dimethyl-organophosphates insecticides, respectively. Here, we have sequenced a fragment of the carboxylesterase E3 gene (ChαE7), comprising part of intron iII, exon eIII, intron iIII and part of exon eIV, and three mitochondrial gene sequences (CR, COI and COII), of NWS flies from 21 locations in South America. These markers were used for population structure analyses and the ChαE7 gene was also investigated to gain insight into the selective pressures that have shaped its evolution. Analysis of molecular variance (AMOVA) and pairwise FST analysis indicated an increased genetic structure between locations in the ChαE7 compared to the concatenated mitochondrial genes. Discriminant analysis of principal components (DAPC) and spatial analysis of molecular variance (SAMOVA) indicated different degrees of genetic structure for all markers, in agreement with the AMOVA results, but with low correlation to geographic data. The NWS fly is considered a panmitic species based on mitochondrial data, while it is structured into three groups considering the ChαE7 gene. A negative association between the two mutations related to organophosphate resistance and Fay & Wu’s H significant negative values for the exons, suggest that these mutations evolved under positive selection.     

The evolution of myiasis in blowflies (Calliphoridae)

Blowflies (Calliphoridae) are characterised by the ability of their larvae to develop in animal flesh. Where the host is a living vertebrate, such parasitism by dipterous larvae is known as myiasis. However, the evolutionary origins of the myiasis habit in the Calliphoridae, a family which includes the blowflies and screwworm flies, remain unclear. Species associated with an ectoparasitic lifestyle can be divided generally into three groups based on their larval feeding habits: saprophagy, facultative ectoparasitism, and obligate parasitism, and it has been proposed that this functional division may reflect the progressive evolution of parasitism in the Calliphoridae. In order to evaluate this hypothesis, phylogenetic analysis of 32 blowfly species displaying a range of forms of ectoparasitism from key subfamilies, i.e. Calliphorinae, Luciliinae, Chrysomyinae, Auchmeromyiinae and Polleniinae, was undertaken using likelihood and parsimony methods. Phylogenies were constructed from the nuclear 28S large subunit ribosomal RNA gene (28S rRNA), sequenced from each of the 32 calliphorid species, together with suitable outgroup taxa, and mitochondrial cytochrome oxidase subunit I and II (COI+II) sequences, derived primarily from published data. Phylogenies derived from each of the two markers (28S rRNA, COI+II) were largely (though not completely) congruent, as determined by incongruence-length difference and Kishino-Hasegawa tests. However, the phylogenetic relationships of blowfly subfamilies based on molecular data did not concur with the pattern of relationships defined by previous morphological analysis; significantly, molecular analysis supported the monophyly of blowflies (Calliphoridae), distinct from the bot and warble flies (Oestridae). Comparative analysis of the myiasis habit based primarily on the 28S rRNA phylogeny indicated that obligate parasitism, and the ability to initiate myiasis in higher vertebrates, has multiple independent origins across myiasis-causing flies (Calliphoridae and Oestridae) and in at least three subfamilies of blowfly (Calliphoridae). Finally, the general association of various blowfly genera and subfamily clades with particular continental and geographical regions suggests that these groups probably came into existence in the Late Cretaceous period, following the break-up of Gondwana.     

Population genetics and gene variation in screwworms (Diptera: Calliphoridae) from Brazil

Allozyme variation in New World screwworm,Cochliomyia hominivorax (Coquerel), populations from Brazil was examined. Variability was observed in 8 of 13 enzyme loci and the frequency of the most common allele was <0.95 for seven loci. Observed and expected heterozygosities were 0.159 and 0.165, respectively. Comparisons of the Brazilian populations with previously studied populations from Costa Rica resulted in Nei's genetic distances of between 0.000 and 0.006, with the greatest distance being between populations within Brazil. Comparisons with Mexican populations using only three loci resulted in genetic distances ≤0.031. Goodness-of-fit statistics for Hardy-Weinberg equilibrium and Wright'sF statistics indicated small deviations from expected equilibrium genotype frequencies and low levels of differentiation between populations within Brazil. Differentiation among screwworm populations from Brazil, Costa Rica, and Mexico was minimal.     

Characterization of the screwworm flies Cochliomyia hominivorax and Cochliomyia macellaria by PCR-RFLP of mitochondrial DNA

The primary screwworm fly Cochliomyia hominivorax (Coquerel) (Diptera: Calliphoridae) is one of the most important insect pests of livestock in neotropical regions, whereas Cochliomyia macellaria (Fabricius) (Diptera: Calliphoridae), the secondary screwworm, is of medical and sanitary importance because of its role in the dissemination of pathogens. These two species share morphological similarities and both may occur in the same myiasis, but in different developmental stages. In this work, the usefulness of PCR-RFLP (polymerase chain reaction - restriction fragment length polymorphism) of mitochondrial DNA (mtDNA) for the unambiguous identification of C. hominivorax and C. macellaria was investigated. Two specific regions of mtDNA were amplified: 870bp from Cytochrome oxidase subunit I and 2100bp from the A+T rich/12S region from C. hominivorax and C. macellaria specimens from different areas of Brazil. Reliable species-specific PCR-RFLP results were obtained for the CO I region and the A+T rich/12S region using the restriction enzymes Dra I and Ssp I. These results confirm the conservation of CO I diagnostic restriction sites previously reported and demonstrate the usefulness of the control region sequences as an efficient marker for PCR-RFLP identification of Brazilian screwworm flies. The occurrences of intraspecific polymorphic patterns are discussed based on frequencies and potential conflicts for species identification. PCR-RFLP provides a potentially useful method for identifying samples from the areas where these species are monitored.     

The molecular systematics of blowflies and screwworm flies (Diptera: Calliphoridae) using 28S rRNA, COX1 and EF-1α: insights into the evolution of dipteran parasitism

The Calliphoridae include some of the most economically significant myiasis-causing flies in the world - blowflies and screwworm flies - with many being notorious for their parasitism of livestock. However, despite more than 50 years of research, key taxonomic relationships within the family remain unresolved. This study utilizes nucleotide sequence data from the protein-coding genes COX1 (mitochondrial) and EF1α (nuclear), and the 28S rRNA (nuclear) gene, from 57 blowfly taxa to improve resolution of key evolutionary relationships within the family Calliphoridae. Bayesian phylogenetic inference was carried out for each single-gene data set, demonstrating significant topological difference between the three gene trees. Nevertheless, all gene trees supported a Calliphorinae-Luciliinae subfamily sister-lineage, with respect to Chrysomyinae. In addition, this study also elucidates the taxonomic and evolutionary status of several less well-studied groups, including the genus Bengalia (either within Calliphoridae or as a separate sister-family), genus Onesia (as a sister-genera to, or sub-genera within, Calliphora), genus Dyscritomyia and Lucilia bufonivora, a specialised parasite of frogs and toads. The occurrence of cross-species hybridisation within Calliphoridae is also further explored, focusing on the two economically significant species Lucilia cuprina and Lucilia sericata. In summary, this study represents the most comprehensive molecular phylogenetic analysis of family Calliphoridae undertaken to date.     

Random amplified polymorphic DNA markers for discriminating Cochliomyia hominivorax from C. macellaria (Diptera: Calliphoridae)

The screwworm, Cochliomyia hominivorax (Coquerel), is one of the most important pests of livestock in the Western Hemisphere. During early immature stages it is morphologically very similar (first instars are virtually indistinguishable) to the secondary screwworm, C. macellaria (Fabricius). Here, the utility of the random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was explored as a technique for developing molecular genetic markers for these two species. Of the 120 arbitrary primers screened, 21 primers produced markers that were further investigated. Seven of the 21 primers produced clear and reproducible markers that were tested with DNA of five individuals from four populations of each species; five of these primers showed 12 RAPD markers that differentiated the species in all populations. Analyses of data from these seven primers also suggested that intraspecific polymorphisms exist that could be useful in distinguishing populations of screwworms. Some population genetic tools, such as genetic distance, cluster analysis and bootstrapping, were used to statistically explore these polymorphisms. The resulting statistics showed 100% support for the ability of RAPD-PCR to discriminate between the two species. Bootstrapping with data from one of the genetic distance calculations produced a tree with all individual screwworms in the correct populations, indicating that RAPD-PCR has promise for displaying intraspecific genetic variation that could be used in establishing the general geographic origin of screwworm samples.     

Molecular characterization,relatedness of <Emphasis Type="Italic">Haematobia irritans</Emphasis> (horn fly) populations,by RAPD-PCR

Haematobia irritans is a hematophagous parasite of cattle that causes significant economic losses in many parts of the world, including Brazil. In the present work, one American, four Brazilian populations of this species were studied by Random Amplified Polymorpht DNA (RAPD) to assess basically genetic variability within and between populations. Ten different decamer random primers were employed in the genomic DNA amplification, yielding 117 fragments in the five H. irritans populations. In Drosophila prosaltans, used as an outgroup, 81 fragments were produced. Forty-three of these fragments were shared by both species. Among the H. irritans samples, that from Rio Branco (Acre State, Brazil) produced the smallest numbers of fragments, polymorphic bands. This high genetic homogenity may be ascribed to its geographic origin (in the Northwest of Brazil), which causes high isolation, low gene flow, unlike the other Brazilian populations, from the South Central region, in which cattle trade is very intensive. Marker fragments (exclusive bands) detected in every sample enabled the population origin to be characterized, but they are also potentially useful for further approaches such as the putative origin of Brazilian populations from North America. Similarity indices [Nei & Li, 1979, Proc. Natl. Acad. Sci. USA 76: 5269–5273], phylogenetic trees, rooted by using the outgroup, produced by the Phylogenetic Analysis using Parsimony (PAUP 4.0-Swofford, 2001) program showed the closest relationships between flies from São José do Rio Preto, Turiúba (both from São Paulo State, Brazil) while flies from the geographically distant Rio Branco showed the greatest differentiation relative to the others.     

The evolution of myiasis in humans and other animals in the Old and New Worlds (part I): phylogenetic analyses

Myiasis, the infestation of live vertebrates with dipterous larvae, seems to take two distinct forms that, it has been suggested, evolved from two distinct phylogenetic roots: saprophagous and sanguinivorous. However, the convergent evolution of morphological and life-history traits seems to have had a major role in simplifying this overall assessment of the evolutionary routes by which myiasis arose. Moreover, this somewhat simplistic division is further complicated by the existence of both ectoparasitic and endoparasitic species of myiasis-causing Diptera, the evolutionary affinities of which remain to be resolved. To understand how different forms of parasitism arose, the evolution of the various groups of myiasis-causing flies must be separated from the evolution of the myiasis habit per se. Until recently, evolutionary studies of myiasis-causing flies were little more than discussions of morphology-based taxonomy. Since the mid-1990s, however, several formal phylogenies - based on both morphological and, increasingly, molecular data - have been published, enabling reassessment of the hypotheses concerning myiasis evolution. In part I of this review, we focus on some recent landmark studies in this often-neglected branch of parasitology and draw together phylogenetic studies based on molecular and morphological data to provide a framework for the subsequent analysis of biochemical, immunological, behavioural, biogeographical and fossil evidence relating to the evolution of myiasis.     

Phylogeographic studies on natural populations of the South American fruit fly, <Emphasis Type="Italic">Anastrepha fraterculus</Emphasis> (Diptera: Tephritidae)

Anastrepha fraterculus is an important pest of commercial fruits in South America. The variability observed for morphological and behavioural traits as well as genetic markers suggests that A. fraterculus represents a complex of synmorphic species rather than a single biological species. We studied the correlation between geographical distribution and genetic variation in natural populations from Argentina and south Brazil. Fragments of the mitochondrial gene COII were sequenced in 28 individuals. The matrix of aligned sequences was phylogenetically analysed by parsimony, yielding a cladogram of haplotypes. Based on Templeton’s nested method, no clade showed any geographic pattern for the gene COII, indicating lack of significant association between haplotypic variability and geographic distribution. The analysis of nucleotide substitution distances by Neighbour-Joining algorithm showed that geographically distant populations exhibit low genetic distances. The corresponding trees clustered the populations without showing any geographic pattern. This result suggests that the populations studied are not reproductively isolated.     

Host specialization,genetic variability,and differentiation in three species of Tomoplagia from Brazilian rupestrian grasslands

In this study, the geographic and host plant-associated genetic structure of Tomoplagia spp. (Diptera: Tephritidae) was investigated. The study was developed in the rupestrian montane grasslands of the Espinhaço Mountain Range, Brazil, where these flies’ larvae develop within flower heads of Asteraceae. The three species of Tomoplagia investigated have different host spans within the subtribe Vernoniinae: one is strictly monophagous (Tomoplagia grandis Prado, Norrbom & Lewinsohn) and two are oligophagous [Tomoplagia incompleta (Williston) and Tomoplagia bicolor Prado, Norrbom & Lewinsohn]. The genetic variability and differentiation throughout their geographic distribution were investigated at the local scale (sympatric, between different host species in the same locality) and the regional scale (allopatric, on the same or different host species in different localities). Allele richness was greater for populations of the two oligophagous species. Moreover, population genetic structure was low in all species both on the local and regional scales, and the attribution of individuals of the three species to different genetic groups was not strongly associated with either locality or host plant. These results indicate the absence of simple geographic and host-associated genetic structure in these specialized herbivores, suggesting that females of T. bicolor and T. incompleta have low host philopatry, using various host species indiscriminately within their distribution range. Therefore, the use of host plants by fruit flies of the genus Tomoplagia is a plastic character that can shift temporally and spatially among phylogenetically related and chemically similar plants. Contrary to expectations, given the specialized and intimate nature of their host association, the studied Tomoplagia species showed no clear host-associated genetic differentiation; instead, genetic variation presents a mosaic pattern across hosts and localities.     

Old World screwworm fly, Chrysomya bezziana, occurs as two geographical races

A morphological and molecular analysis was undertaken with the objective of identifying markers for geographical populations of Old World screwworm flies, Chrysomya bezziana Villeneuve (Diptera: Calliphoridae). The morphological analysis involved 192 adult flies from 14 countries, and the molecular analysis involved 45 larvae or adults from 14 populations in 11 countries. Principal components and cluster analysis of 10 morphological characters indicated that flies from Papua New Guinea (PNG) were a distinct group and most similar to flies from nearby Asian islands (Java, Sabah). There was poor resolution of other geographical regions, but some support for clustering of flies from Africa or India. Cladistic analysis of mitochondrial DNA sequences gave strong support for recognizing two races of Old World screwworm, one from sub-Saharan Africa and the other from the Gulf region and Asia. This latter race could be further divided into two lineages, i.e. one from mainland Asia (from Iraq to the Malay Peninsula) and the other from two islands of PNG.     

Molecular approaches to the study of myiasis-causing larvae

Among arthropod diseases affecting animals, larval infections - myiases - of domestic and wild animals have been considered important since ancient times. Besides the significant economic losses to livestock worldwide, myiasis-causing larvae have attracted the attention of scientists because some parasitise humans and are of interest in forensic entomology. In the past two decades, the biology, epidemiology, immunology, immunodiagnosis and control methods of myiasis-causing larvae have been focused on and more recently the number of molecular studies have also begun to increase. The 'new technologies' (i.e. molecular biology) are being used to study taxonomy, phylogenesis, molecular identification, diagnosis (recombinant antigens) and vaccination strategies. In particular, more in depth molecular studies have now been performed on Sarcophagidae, Calliphoridae and flies of the Oestridae sister group. This review discusses the most topical issues and recent studies on myiasis-causing larvae using molecular approaches. In the first part, PCR-based techniques and the genes that have already been analysed, or are potentially useful for the molecular phylogenesis and identification of myiasis-causing larvae, are described. The second section deals with the more recent advances concerning taxonomy, phylogenetics, population studies, molecular identification, diagnosis and vaccination.     

Genetic variation and population structure of the endangered Hyacinth Macaw (<Emphasis Type="Italic">Anodorhynchus hyacinthinus</Emphasis>): implications for conservation

The Hyacinth Macaw (Anodorhynchus hyacinthinus) is one of 14 endangered species in the family Psittacidae occurring in Brazil, with an estimated total population of 6,500 specimens. We used nuclear molecular markers (single locus minisatellites and microsatellites) and 472 bp of the mitochondrial DNA control region to characterize levels of genetic variability in this species and to assess the degree of gene flow among three nesting sites in Brazil (Pantanal do Abobral, Pantanal de Miranda and Piauí). The origin of five apprehended specimens was also investigated. The results suggest that, in comparison to other species of parrots, Hyacinth Macaws possess relatively lower genetic variation and that individuals from two different localities within the Pantanal (Abobral and Miranda) belong to a unique interbreeding population and are genetically distinct at nuclear level from birds from the state of Piauí. The analyses of the five apprehended birds suggest that the Pantanal is not the source of birds for illegal trade, but their precise origin could not be assigned. The low genetic variability detected in the Hyacinth Macaw does not seem to pose a threat to the survival of this species. Nevertheless, habitat destruction and nest poaching are the most important factors negatively affecting their populations in the wild. The observed genetic structure emphasizes the need of protection of Hyacinth Macaws from different regions in order to maintain the genetic diversity of this species.     

A phylogeny of sand flies (Diptera: Psychodidae: Phlebotominae), using recent Ethiopian collections and a broad selection of publicly available DNA sequence data

Sand flies in the psychodid subfamily Phlebotominae carry important human pathogens in the trypanosomatid protozoan genus Leishmania (Cupolillo). Despite the fact that hundreds of sequences for this group are now publicly available, they constitute different sets of taxa and genetic markers. Integrating these data to construct a molecular phylogeny of the family is a significant bioinformatics challenge. We used sequences of eight markers obtained from freshly collected sand flies from Ethiopia and combined them with over 1300 publicly available sequences, performing a combined analysis after generating single terminal sequences from ancestral reconstructions for some individual markers. The resulting phylogeny had 113 terminals and recovered Phlebotominae and certain species groups as monopheletic. Although the 20 outgroups in Psychodinae were recovered as a well‐resolved clade with bootstrap support for many internal clades, Phlebotominae was recovered as several lineages with unclear relationships among them. However, phlebotomines clustered by geographic region, the most notable being all the New World species except Brumptomyia (Galati), which were recovered as monophyletic. Our phylogeny suggests a Sub‐Saharan African or South Asian origin for the subfamily, which subsequently expanded to the north and west, and eventually to the New World. Supported species groups are often composed of widespread species with overlapping ranges. This result highlights the need for a large increase in the amount and diversity of molecular sequence data, and a broad selection of terminals, to test taxonomic hypotheses and examine speciation processes in this important group of flies.     

On biology of limoniid flies of the genus Teucholabis Osten-Sacken (Diptera, Limoniidae) with a description of immature stages of Teucholabis esakii (Alexander) and T. yezoensis Alexander

The larvae and pupae of two xylobiont species of limoniid flies, Teucholabis esakii (Al.) and T. yezoensis Al., are described for the first time. The larvae live under bark among bast fibers of Maackia amurensis and Phellodendron amurense, rarely under bark of various species of oak, aspen, birch, and maple. A key to larvae and pupae of the two species is provided. Original Russian Text ? M.G. Krivosheina, 2009, published in Entomologicheskoe Obozrenie, 2009, Vol. 88, No. 1, pp. 185–193.     

Specific detection of the Old World screwworm fly,Chrysomya bezziana,in bulk fly trap catches using real‐time PCR

The Old World screwworm fly (OWS), Chrysomya bezziana Villeneuve (Diptera: Calliphoridae), is a myiasis‐causing blowfly of major concern for both animals and humans. Surveillance traps are used in several countries for early detection of incursions and to monitor control strategies. Examination of surveillance trap catches is time‐consuming and is complicated by the presence of morphologically similar flies that are difficult to differentiate from Ch. bezziana, especially when the condition of specimens is poor. A molecular‐based method to confirm or refute the presence of Ch. bezziana in trap catches would greatly simplify monitoring programmes. A species‐specific real‐time polymerase chain reaction (PCR) assay was designed to target the ribosomal DNA internal transcribed spacer 1 (rDNA ITS1) of Ch. bezziana. The assay uses both species‐specific primers and an OWS‐specific Taqman^® MGB probe. Specificity was confirmed against morphologically similar and related Chrysomya and Cochliomyia species. An optimal extraction protocol was developed to process trap catches of up to 1000 flies and the assay is sensitive enough to detect one Ch. bezziana in a sample of 1000 non‐target species. Blind testing of 29 trap catches from Australia and Malaysia detected Ch. bezziana with 100% accuracy. The probability of detecting OWS in a trap catch of 50 000 flies when the OWS population prevalence is low (one in 1000 flies) is 63.6% for one extraction. For three extractions (3000 flies), the probability of detection increases to 95.5%. The real‐time PCR assay, used in conjunction with morphology, will greatly increase screening capabilities in surveillance areas where OWS prevalence is low.     

Phylogeography of the sea urchin Paracentrotus lividus (Lamarck) (Echinodermata:Echinoidea): first insights from the South Tyrrhenian Sea

The sea urchin Paracentrotus lividus (Lamarck) (Echinodermata: Echinoidea) is an Atlanto-Mediterranean species abundant in the littoral zone, where it occurs in the sublittoral down to 20 m. The aim of our work is to investigate the genetic patterns of P. lividus along the South Tyrrhenian coasts. Five specimens were collected in six localities, from the Gulf of Naples and the Cilento coast. The nuclear rDNA ITS2 spacer and the two mitochondrial genes 16S and COI were used for the analysis. The three markers utilised did not show any structure among populations from the Gulf of Naples. All populations appear to be polyphyletic, with Cilento samples more differentiated from the others. This suggests the existence of phylogeographic structure at larger geographic scale. Absence of genetic structure has been interpreted taking into consideration theoretical dispersal of the planktotrophic larvae, which can survive for 4–8 weeks before settlement, marine current patterns and persistence of the species in the area.     

Systematics,distribution, genetics and life history of milkfish,Chanos chanos

Synopsis Chanos chanos belongs to a monotypic gonorynchiform family and is most closely related to the freshwater Ostariophysi. The earliest gonorynchiforms occurred in the Cretaceous of Brazil and west Africa. Chanos occurred in the freshwater Eocene deposits of Europe and North America, and probably invaded the circumtropical Tethys Sea during transgression episodes. At present, milkfish occurs near continental shelves and around oceanic islands throughout the tropical Indo-Pacific. Milkfish populations throughout the range show high genetic variation but low genetic divergence, similar to many other commercially important teleosts. The natural life history of milkfish is one of continual migration. Adults are relatively large (to 1.5 m or 15 kg), long-lived (to 15 years), pelagic and schooling. They spawn offshore near coral reefs or small islands. The eggs, embryos and larvae are pelagic and relatively larger than those of most marine species. Larvae ≥ 10 mm long and 2–3 weeks old move inshore via a combination of passive advection and active migration. Passing shore waters and surf zones, they settle in shallow-water depositional habitats such as mangrove swamps and coral lagoons, where they metamorphose and spend a few months as juveniles. Some juveniles may enter freshwater lakes where they grow into sub-adults but do not mature. Both small juveniles and large sub-adults go back to sea when they reach the size limit supportable by the habitat. Little else is known of the dynamics of wild populations of milkfish. A fishery on inshore larvae supports the centuries-old aquaculture of milkfish in southeast Asia. During the past ten years, milkfish have matured and spawned under various conditions of captivity, and hatcheries have produced larvae to supply the culture ponds. Much remains to be learned concerning the milkfish, particularly its ecology and physiology.     

Host range and genetic strains of leafminer flies (Diptera: Agromyzidae) in eastern Brazil reveal a new divergent clade of Liriomyza sativae

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