John K. Moulton

Evolution and phylogenetic utility of CAD (rudimentary) among Mesozoic-aged Eremoneuran Diptera (Insecta)

We sequenced nearly the entire carbomoylphosphate synthase (CPS) domain of CAD, or rudimentary, (ca. 4 kb) from 29 species of flies representing all major clades within Eremoneura, or higher flies, and several orthorrhaphous brachyceran outgroups. We compared these sequences with orthologs from Anopheles gambiae and Drosophila melanogaster to assess structure, compositional bias, and phylogenetic utility. CAD is large (6.6+ kb), complex (comprised of three major and myriad minor functional domains) and relatively free of introns, extreme nucleotide bias (except third codon positions), and large hypervariable regions. The CPS domain possesses moderate levels of nonsynonymous divergence among taxa of intermediate evolutionary age and conveys considerable phylogenetic signal. Phylogenetic analysis of CPS sequences under varying methods and assumptions resulted in well-resolved, strongly supported trees concordant with many traditional ideas about higher dipteran phylogeny and with prior inferences from 28S rDNA. The most robustly supported major eremoneuran clades were Cyclorrhapha, Platypezoidea, Eumuscomorpha, Empidoidea, Atelestidae, Empidoidea exclusive of Atelestidae, Hybotidae s.l., Microphoridae+Dolichopodidae, and Empididae s. str. Because CAD is ubiquitous, apparently single copy (at least within holometabolous insects), readily obtained from several insect orders using primers described herein, and exhibits considerable phylogenetic utility, it should have wide applicability in insect molecular systematics.

Comparison of Aflatoxigenic and Nonaflatoxigenic Isolates of Aspergillus flavus using DNA Amplification Fingerprinting Techniques

Aspergillus flavus is a filamentous fungus that produces mycotoxins in many food and feed crops, such as maize (Zea mays L.). Isolates were analyzed for toxin production by nucleic acid profiles in an attempt to differentiate aflatoxigenic from nonaflatoxigenic isolates. A total of 41 aflatoxigenic and 34 nonalfatoxigenic isolates were included in the study. The isolates were evaluated initially using DNA amplification fingerprinting (DAF) without clear resolution of the groups. A weak association of aflatoxigenic isolates was observed, as evidenced by their clustering in 18 of 81 trees recovered from maximum parsimony analysis of binary characters derived from arbitrary signatures from amplification profiles (ASAP) data; nonaflatoxigenic isolates exhibited a pattern of paraphyletic laddering. Up to five markers unambiguously supported the aflatoxigenic isolate grouping, but the presence of alternative conflicting topologies in equally parsimonious trees precluded the observation of meaningful statistical support. With additional markers for genome of A. flavus, this method could be used to resolve toxigenic from nontoxigenic strains. This additional work could resolve aflatoxigenic isolates of A. flavus present on maize plants using ASAP, which would reduce labor intense costs and potentially lead to faster determination of resistant cultivars in breeding efforts.

Juvenile hormone and insulin suppress lipolysis between periods of lactation during tsetse fly pregnancy

Tsetse flies are viviparous insects that nurture a single intrauterine progeny per gonotrophic cycle. The developing larva is nourished by the lipid-rich, milk-like secretions from a modified female accessory gland (milk gland). An essential feature of the lactation process involves lipid mobilization for incorporation into the milk. In this study, we examined roles for juvenile hormone (JH) and insulin/IGF-like (IIS) signaling pathways during tsetse pregnancy. In particular, we examined the roles for these pathways in regulating lipid homeostasis during transitions between non-lactating (dry) and lactating periods. The dry period occurs over the course of oogenesis and embryogenesis, while the lactation period spans intrauterine larvigenesis. Genes involved in the JH and IIS pathways were upregulated during dry periods, correlating with lipid accumulation between bouts of lactation. RNAi suppression of Forkhead Box Sub Group O (FOXO) expression impaired lipolysis during tsetse lactation and reduced fecundity. Similar reduction of the JH receptor Methoprene tolerant (Met), but not its paralog germ cell expressed (gce), reduced lipid accumulation during dry periods, indicating functional divergence between Met and gce during tsetse reproduction. Reduced lipid levels following Met knockdown led to impaired fecundity due to inadequate fat reserves at the initiation of milk production. Both the application of the JH analog (JHA) methoprene and injection of insulin into lactating females increased stored lipids by suppressing lipolysis and reduced transcripts of lactation-specific genes, leading to elevated rates of larval abortion. To our knowledge, this study is the first to address the molecular physiology of JH and IIS in a viviparous insect, and specifically to provide a role for JH signaling through Met in the regulation of lipid metabolism during insect lactation.

Variability of United States Isolates of Macrophomina phaseolina Based on Simple Sequence Repeats and Cross Genus Transferability to Related Genera Within Botryosphaeriaceae

Twelve simple sequence repeat (SSRs) loci were used to evaluate genetic diversity of 109 isolates of Macrophomina phaseolina collected from different geographical regions and host species throughout the United States (US). Genetic diversity was assessed using Nei’s minimum genetic distance, and the usefulness of each locus was determined by calculating the polymorphism information content (PIC). A total of 98 alleles were detected and of these 31 were unique to individual genotypes. Eight of twelve loci were highly informative with PIC values greater than 0.50. The majority of pairwise comparisons of genetic distance were greater than 0.60 indicating moderate to high genetic diversity. Dendrograms based on the genetic dissimilarities were created for the 109 isolates of which 79 were from soybean. Some clustering by host and geography was noted, but, the dendrograms generally grouped isolates independent of host or geography. Additionally, sequencing of the internal transcribed spacer region (ITS) for 10 isolates revealed that all of these isolates were 99% similar. Three SSR loci from M. phaseolina were cross amplified in other genera in the Botryosphaeriaceae. This was the first study of genotyping and assessing genetic diversity of M. phaseolina isolates collected from a widespread host and geographic range across the US with SSRs. With an additional 34 loci publically available for M. phaseolina, the results indicate that previously developed SSRs from one species can be used in future population, ecological, and genetic studies of M. phaseolina and other genera within the Botryosphaeriaceae.

Phylogeny of psychodid subfamilies (Diptera: Psychodidae) inferred from nuclear DNA sequences with a review of morphological evidence for relationships

Psychodidae is a diverse family of flies with approximately 3000 described species in six subfamilies, including Phlebotominae vectors of human disease. Psychodidae has been the subject of few phylogenetic investigations and development of a stable classification has been hampered by poor understanding of the morphology of larvae, pupae and adults. Specimens were collected, and we analysed DNA sequence data from two nuclear genes for one or more representatives of all subfamilies. The subfamilies with multiple representatives included were resolved as monophyletic with good support. Placement of Horaiellinae, Sycoracinae and Trichomyiinae remains unclear, whereas Bruchomyiinae is hypothesized as the sister group to (Phlebotominae + Psychodinae). Representatives of some psychodine tribes were resolved in agreement with previous hypotheses. Relationships among and within subfamilies are discussed, and morphological characters supporting these relationships are reviewed. One compelling synapomorphy of the male genitalia supporting a relationship between Phlebotominae and Psychodinae is the presence of articulated surstyli with apical retinacula. Only cerci are present and sometimes developed into clasping structures in males of other subfamilies.

Survey of Pogonognathellus Börner (Collembola: Tomoceridae) in the Southern Appalachians Based on Morphological and Molecular Data

ABSTRACT Pogonognathellus Börner is the most common genus of tomocerid Collembola in the southern Appalachian region of the United States. Scale pattern, cuticle color, and molecular data were used with morphology and chaetotaxy to reappraise the members of this genus. P. bidentatus and P. elongatus are confirmed as well-marked species; P. nigritus Maynard is removed from synonymy with P. elongatus and reestablished as a valid species, and a neotype is designated. Two new species are described. Pogonognathellus danieli n. sp. from a cave in Great Smoky Mountains National Park resembles the California cave species P. celsus but possesses clubbed tenent hairs on all tibiotarsi; in P. celsus, the tenent hairs are pointed. P. mystax n. sp. is related to the “P. flavescens complex” but differs in having a purple clypeus and a prominent band of light scales along the posterior edge of each tergite. Many collections of P. flavescens-like and P. dubius-like specimens were made but molecular analysis indicated that these specimens consisted of four P. dubius-like taxa and four P. flavescens-like taxa. True P. flavescens from Sweden (type locality) were molecularly distinct from the putative American P. flavescens included in the analysis. A tentative phylogenetic tree indicated three clades of southern Appalachian Pogonognathellus: one clade containing P. bidentatus; another clade containing species with posterior cephalic macrochaetae but without anterior macrochaetae on the fourth abdominal tergite (Abd. IV; P. elongatus, P. nigritus, and an undescribed species); and a third clade without posterior cephalic macrochaetae but with one pair of anterior macrochaetae on Abd. IV (P. danieli, P. mystax, and eight undescribed species).

Impact of Phlebotomine Sand Flies on U.S. Military Operations at Tallil Air Base, Iraq: 4. Detection and Identification of Leishmania Parasites in Sand Flies

ABSTRACT Sand flies collected between April 2003 and November 2004 at Tallil Air Base, Iraq, were evaluated for the presence of Leishmania parasites using a combination of a real-time Leishmania-generic polymerase chain reaction (PCR) assay and sequencing of a 360-bp fragment of the glucose- 6-phosphate-isomerase (GPI) gene. A total of 2,505 pools containing 26,574 sand flies were tested using the real-time PCR assay. Leishmania DNA was initially detected in 536 pools; however, after extensive retesting with the real-time PCR assay, a total of 456 pools were considered positive and 80 were considered indeterminate. A total of 532 samples were evaluated for Leishmania GPI by sequencing, to include 439 PCR-positive samples, 80 PCR-indeterminate samples, and 13 PCR-negative samples. Leishmania GPI was detected in 284 samples that were sequenced, to include 281 (64%) of the PCR-positive samples and 3 (4%) of the PCR-indeterminate samples. Of the 284 sequences identified as Leishmania, 261 (91.9%) were L. tarentolae, 18 (6.3%) were L. donouam-complex parasites, 3 (1.1%) were L. tropica, and 2 were similar to both L. major and L. tropica. Minimum field infection rates were 0.09% for L. donovani-complex parasites, 0.02% for L. tropica, and 0.01% for the L. major/tropica-like parasite. Subsequent sequencing of a 600-bp region of the “Hyper” gene of 12 of the L. donovani-complex parasites showed that all 12 parasites were L. infantum. These data suggest that L. infantum was the primary leishmanial threat to U.S. military personnel deployed to Tallil Air Base. The implications of these findings are discussed.

Description of the egg of Ochlerotatus japonicus japonicus (Diptera: Culicidae) using variable pressure scanning electron microscopy.

ABSTRACT The egg of Ochlerotatus japonicus japonicus (Theobald) is described with the aid of variable pressure scanning electron micrographs. The egg is black, cigar shaped, and tapers ventrally. The length is ≈591 µm and the width is ≈172 µm. The outer chorionic cells are irregular in shape, either hexagonal or pentagonal, and decrease in size toward the anterior and posterior poles. Ventral tubercles typically range from three to six and contact the chorionic reticulum. Dorsal tubercles contain two large tubercles with small oval-shaped tubercles grouped around them. A large thread-shaped tubercle extends from this grouping either as a single tubercle or as a series of connected tubercles. The micropylar collar is low and discontinuous and is seldom complete. This description will aid researchers in the identification of this invasive vector species.

New state and host plant records for metallic woodboring beetles (Coleoptera: Buprestidae) in Tennessee, U.S.A.

ABSTRACT The following 41 metallic woodboring beetle species (Coleoptera: Buprestidae) from Tennessee are reported as new state records: Acmaeodera texana LeConte, Actenodes simi Fisher, Agrilus acutipennis Mannerheim, Agrilus arcuatus Say, Agrilus bilineatus (Weber), Agrilus cephalicus LeConte, Agrilus defectus LeConte, Agrilus geminatus (Say), Agrilus lecontei celticola Fisher, Agrilus masculinus Horn, Agrilus obsoletoguttatus Gory, Agrilus otiosus Say, Agrilus parvus parvus Saunders, Agrilus pseudofallax Frost, Agrilus putillus putillus Say, Agrilus quadriguttatus quadriguttatus Gory, Agrilus subcinctus Gory, Anthaxia cyanella Gory, Anthaxia dichroa Bílý, Anthaxia quercata (F.), Anthaxia quercicola Wellso, Brachys aerosus (Melsheimer), Brachys ovatus (Weber), Buprestis consularis Gory, Buprestis decora F., Buprestis fasciata F., Buprestis maculipennis Gory, Buprestis rufipes Olivier, Chalcophora virginiensis (Drury), Chrysobothris cribraria Mannerheim, Chrysobothris dentipes (Germar), Chrysobothris rotundicollis Gory and Laporte, Chrysobothris scabripennis Gory and Laporte, Chrysobothris sexsignata Say, Dicerca divaricata Say, Dicerca lepida LeConte, Eupristocerus cogitans (Weber), Phaenops aeneola (Melsheimer), Phaenops drummondi (Kirby), Phaenops obtusa (Horn), and Xenorhipis brendeli LeConte. New larval host plant records are reported for Actenodes acornis (Say), A. cephalicus, A. cyanella, A. quercicola, Chrysobothris azurea LeConte, Chrysobothris chlorocephala Gory, Chrysobothris femorata (Olivier), C. sexsignata, and X. brendeli, and biological notes for five species are presented.

Development of microsatellite loci in Pityophthorus juglandis, a vector of thousand cankers disease in Juglans spp.

Using next-generation sequencing, 18 microsatellite loci were developed and characterized for walnut twig beetle, Pityophthorus juglandis, a vector of thousand cankers disease (TCD) affecting Juglans spp. Although all Juglans species are susceptible to TCD infection, native populations of J. nigra and J. cinerea, which is endangered in Canada, are most susceptible and threatened by habitat loss. Novel primers amplified di-, tri-, and tetra nucleotide repeats and detected 4–14 alleles per locus. Averaged observed and expected heterozygosity was 0.22 and 0.67, respectively. Our results indicate that P. juglandis microsatellite loci can be used to investigate genetic diversity and population structure of this vector across a widespread geography. These markers will be useful tools for evaluating genetic structure of P. juglandis population outbreaks and developing appropriate conservation strategies. Microsatellite loci obtained in this study can also be utilized to determine relationships of P. juglandis to other closely related Pityophthorus spp.