History of entomopathogenic nematology.

The history of entomopathogenic nematology is briefly reviewed. Topic selections include early descriptions of members of Steinernema and Heterorhabditis, how only morphology was originally used to distinguish between the species; descriptions of the symbiotic bacteria and elucidating their role in the nematode- insect complex, including antibiotic properties, phase variants, and impeding host defense responses. Other topics include early solutions regarding production, storage, field applications and the first commercial sales of entomopathogenic nematodes in North America. Later studies centered on how the nematodes locate insect hosts, their effects on non-target organisms and susceptibility of the infective juveniles to soil microbes. While the goals of early workers was to increase the efficacy of entomopathogenic nematodes for pest control, the increasing use of Heterorhabditis and Photorhabdus as genetic models in molecular biology is noted.

One hundred million years of chemical warfare by insects.

An important defensive strategy among animals is the use of chemical compounds with toxic or irritating properties. In this paper, we report the discovery of an Early Cretaceous soldier beetle (Coleoptera: Cantharidae) in Burmese amber that seemingly employed a chemical defense response against a potential predator. Six pairs of cuticular vesicles with associated gland reservoirs were extruded from the insect's abdomen, and a secretion released from one of these covers a portion of the antenna of a second insect species, considered to be the perpetrator of the response. This is the earliest fossil record of a putative chemical defense response and suggests that chemical defense mechanisms in beetles have been in existence for at least 100 Ma.

A fossil bee from Early Cretaceous Burmese amber.

The bee fossil record is fragmentary, making it difficult to accurately estimate the antiquity of bee-mediated pollination. Here, we describe a bee fossil [Melittosphex burmensis (new species), Melittosphecidae (new family)] from Early Cretaceous Burmese amber (approximately 100 million years before the present). The fossil provides insights into the morphology of the earliest bees and provides a new minimum date for the antiquity of bees and bee-mediated pollination.

Control of insect pests with entomopathogenic nematodes: the impact of molecular biology and phylogenetic reconstruction.

Entomopathogenic nematodes are excellent biological control agents. Utilization of these nematodes is developing rapidly with almost a doubling of newly described species in the past five years. Advances in molecular biology and phylogenetic reconstruction have revolutionized understanding of population structure, identification, genetic improvement, systematics, and the symbiosis between entomopathogenic nematodes and their bacteria. Population structure provides the most fundamental information for reliable identification of species and unique genetic variants. Such information could be further assessed for nematode potential as biological control agents. Phylogenetic reconstruction is an important approach for understanding multitrophic interactions among entomopathogenic nematodes, symbiotic bacteria, and their insect hosts. Phylogenetic reconstruction is also important for the development of a natural and stable type of systematics, which can provide guidelines for selecting appropriate entomopathogenic nematode species for particular biological control programs.

The range of life in amber: significance and implications in DNA studies.

A survey of the major fossiliferous amber deposits is provided, including ages and various categories of life forms reported from each. The frequency of occurrence of the major groups of plants and animals in these amber deposits is also given. Thus far, DNA from four insect and one plant species has been extracted from amber fossils. In the case of the stingless bee in Dominican amber, evidence of reproducibility is provided, since two independent laboratories isolated DNA from six or more different specimens of the same insect. Amber sources for DNA studies are listed together with their advantages and disadvantages. The important points are the availability of desired pieces, the proper identification of the fossil, verification of the amber deposit, the cost involved, and the feasibility of causing damage to the specimen. The availability of several types of amber (Mexican, Dominican, Baltic, Chinese, Canadian, Siberian and Lebanese) at four major sources (academic collections, commercial dealers, private collections and amber mines) is discussed. The scientific implications of obtaining DNA from amber inclusions are presented.

Bacillus DNA in fossil bees: an ancient symbiosis?

We report here the isolation of DNA from abdominal tissue of four extinct stingless bees (Proplebeia dominicana) in Dominican amber, PCR amplification of a 546-bp fragment of the 16S rRNA gene from Bacillus spp., and their corresponding nucleotide sequences. These sequences were used in basic local alignment search tool searches of nonredundant nucleic acid data bases, and the highest scores were obtained with 16S rRNA sequences from Bacillus spp. Phylogenetic inference analysis by the maximum-likelihood method revealed close phylogenetic relationships of the four presumed ancient Bacillus sequences with Bacillus pumilus, B. firmus, B. subtilis, and B. circulans. These four extant Bacillus spp. are commonly isolated from abdominal tissue of stingless bees. The close phylogenetic association of the extracted DNA sequences with these bee colonizers suggests that a similar bee-Bacillus association existed in the extinct species P. dominicana.

Amino acid racemization in amber-entombed insects: implications for DNA preservation.

DNA depurination and amino acid racemization take place at similar rates in aqueous solution at neutral pH. This relationship suggests that amino acid racemization may be useful in accessing the extent of DNA chain breakage in ancient biological remains. To test this suggestion, we have investigated the amino acids in insects entombed in fossilized tree resins ranging in age from <100 years to 130 million years. The amino acids present in 40 to 130 million year old amber-entombed insects resemble those in a modern fly and are probably the most ancient, unaltered amino acids found so far on Earth. In comparison to other geochemical environments on the surface of the Earth, the amino acid racemization rate in amber insect inclusions is retarded by a factor of >10(4). These results suggest that in amber insect inclusions DNA depurination rates would also likely be retarded in comparison to aqueous solution measurements, and thus DNA fragments containing many hundreds of base pairs should be preserved. This conclusion is consistent with the reported successful retrieval of DNA sequences from amber-entombed organisms.

The fine structure of Gastromermis sp. (Nematoda: Mermithidae) sperm.

The development and morphology of the sperm of the mermithid nematode, Gastromermis sp. appears to be different than that reported for any other nematode sperm. The mature sperm of Gastromermis are elongate with membranous organelles surrounding an apical nuclear region. The mitochondria are arranged in parallel rows and form an elongate sheath. Spermatozoa contain dense tubular structures which have not been seen in other nematodes. Spermatids lack fibrous bodies. The pseudopodia of the spermatozoa are filled with microtubules. Centrioles seen throughout sperm development are composed of 9 doublets.

Amplification and sequencing of DNA from a 120-135-million-year-old weevil.

DNA has been successfully isolated from both fossilized plant and animal tissues. The oldest material, dated as 25-40 million years old (Tertiary), was obtained from amber-entombed bees and termites. Tissues from both these insects yielded DNA of good quality, which could be amplified by the polymerase chain reaction (PCR) and subsequently sequenced, including the genes encoding 18S ribosomal RNA and 16S rRNA. We report here the extraction of DNA from a 120-135-million-year-old weevil (Nemonychidae, Coleoptera) found in Lebanese amber, PCR amplification of segments of the 18S rRNA gene and the internal transcribed spacer, and the corresponding nucleotide sequences of their 315- and 226-base-pair fragments, respectively. These sequences were used for preliminary phylogenetic analysis of the nemonychid's sequence with three extant coleopterans: Lecontellus pinicola (Nemonychidae), Hypera brunneipennis (Curculionidae) and the mealworm Tenebrio molitor (Tenebrionidae), and two extant dipterans: the fruitfly Drosophila melanogaster (Drosophilidae) and mosquito Aedes albopictus (Culicidae) for the purpose of ascertaining the origin of the extracted and amplified DNA. The results revealed that the PCR-amplified material is that of the extinct nemonychid weevil. This represents the oldest fossil DNA ever extracted and sequenced, extending by 80 million years the age of any previously reported DNA.

Terrestrial soft-bodied protists and other microorganisms in triassic amber.

Protozoa, cyanobacteria, sheathed algae, sheathed fungi, germinating pollen or spores, and fungal spores have been found in amber 220 to 230 million years old. Many of these microorganisms can be assigned to present-day groups. This discovery of terrestrial, soft-bodied protists that can be referred to modern groups indicates that morphological evolution is very gradual in many protists and that both structural and probably functional stasis extend back at least to the Upper Triassic period.

Adhesive grass spikelet with mammalian hair in Dominican amber: first fossil evidence of epizoochory.

Discovery of a female spikelet of the grass genus Pharus (Gramineae: Bambusoideae: Phareae) in association with mammalian hair in Dominican Republic amber provides the first fossil evidence of epizoochory. Hooked macrohairs on the lemma of the spikelet show that morphological modifications in grasses for dispersal by attachment to the surface of animals were present in the Late Eocene. The fossil also represents 1) the second-oldest undoubted macrofossil record of the Gramineae, 2) the earliest record of a fossil grass that can be assigned to an extant genus, 3) the earliest undoubted record of a member of the bamboo subfamily and 4) the only known fossil of Pharus.

Upper eocene gilled mushroom from the dominican republic.

Coprinites dominicana gen.nov., spec.nov. (Coprinaceae) found in amber from the Dominican Republic is the earliest known gilled mushroom, the first fossil fleshy agaric determinable, and the only kiiown fossil "mushroom" (Agaricales) from the tropics. This find is of significance with respect to the evolutionary development of the Basidiomycetes. Because the appearance of this fossil is quite modern, it suggests that mushrooms as a group probably arose much earlier than the early to mid-Tertiary.

Growth and luminescence of the bacterium Xenorhabdus luminescens from a human wound.

Xenorhabdus luminescens, a newly isolated luminous bacterium collected from a human wound, was characterized. The effects of ionic strength, temperature, oxygen, and iron on growth and development of the bioluminescent system were studied. The bacteria grew and emitted light best at 33 degrees C in a medium with low salt, and the medium after growth of cells to a high density was found to have antibiotic activity. The emission spectrum peaked at 482 nm in vivo and at 490 nm in vitro. Both growth and the development of luminescence in X. luminescens required oxygen and iron. The isolated luciferase itself exhibited a temperature optimum at about 40 degrees C; after purification by affinity chromatography, it showed two bands (52 and 41 kilodaltons) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicative of an alpha and beta subunit structure. Reduced flavin mononucleotide (Km of 1.4 microM) and tetradecanal (Km of 2.1 microM) were the best substrates for the luciferase, and the first-order decay constant under these conditions at 37 degrees C was 0.79 s-1.

Xenorhabdus luminescens (DNA hybridization group 5) from human clinical specimens.

An unusual isolate from a human leg wound was identified as Xenorhabdus luminescens. This finding led to the discovery or isolation of four additional strains, two from blood and two from wounds. Three of the five strains were from patients in San Antonio, Tex. Three strains were studied by DNA-DNA hybridization (S1 nuclease-trichloroacetic acid method) and were 77 to 100% related to each other, 34% related to the type strain of X. luminescens, 35 to 40% related to three of Grimont's other DNA hybridization groups of X. luminescens, and 9% related to the type strain of Xenorhabdus nematophilus. The new group of five strains was designated X. luminescens DNA hybridization group 5. All five strains were very inactive biochemically and fermented only D-glucose and D-mannose. The key reactions for recognizing this new organism are yellow pigment production, negative test for nitrate reduction to nitrite, weak bioluminescence (10 to 15 min of dark adaptation is required to see the weak light produced), and a unique hemolytic reaction on sheep blood agar plates incubated at 25 degrees C. Two case histories of strains from wounds are given; these suggest that X. luminescens DNA hybridization group 5 may be a new bacterial agent that causes wound infections. The two cases of wound infection, along with the two blood isolates, suggest that the new organism is clinically significant.

Preliminary observations of a bacteriophage infecting Xenorhabdus luminescens (Enterobacteriaceae).

A bacteriophage infective to Xenorhabdus luminescens, a bacterial symbiont of heterorhabditid nematodes, was recovered from insects that supported poor nematode development. Plaque tests showed the phage particles to be infective only to primary and not secondary colonies of X. luminescens. The phage was not infective to X. nematophilus primaries or secondaries. The bacteriophage particles ranged 80-90 nm in length, with the head ranging from 40 to 50 nm in diameter. Restriction analysis was performed on isolated bacteriophage DNA. This first report of a bacteriophage from Xenorhabdus species has practical implications since it could be detrimental to cultures of Heterorhabditis nematodes that are being produced throughout the world for the biological control of insects.

Mermis nigrescens (Mermithidae: Nematoda) recovered from the mouth of a child.

A gravid female Mermis nigrescens Dujardin (Mermithidae: Nematoda) was recovered from the mouth of a 1-month-old infant in Mehama, Oregon. This nematode is a parasite of grasshoppers and deposits its eggs on vegetation. The parasite was probably carried on the hair of the household dog or cat to the vicinity of the patient and was attracted to the moisture in the region of the mouth.

[Skrjabinelazia galliardi (Nematoda, Seuratoidea): morphologic complements and biological cycle]. / Skrjabinelazia galliardi (Nematoda, Seuratoidea): compléments morphologiques et cycle biologique.

Skrjabinelazia galliardi was previously known only from a description of the morphological characteristics of the female. Additional material being available, we were able to complete the morphological description and understand better the biology of the species. The male, the third stage larva and the fourth stage larva are described. Young females lay a very small number of third stage larvae, leading in a small scale to a reproduction of the atractide-type, that is a multiplication of the parasite directly inside the host. The old females are oviparous, but eggs contain a third stage infective larva. These eggs, after being ingested by field-crickets, hatch and without further development remain as larvae in the insect. The definitive entomophagous lizard host, which, in the field, has no opportunity to ingest the eggs, is infested by eating insects. So, the life history is intermediary between the cosmocercid like cycle (with three larval stages) and the spirurid-like cycle (with development of larval stages in an invertebrate).

An upper eocene frog from the dominican republic and its implication for Caribbean biogeography.

A frog of the leptodactylid genus Eleutherodactylus is reported from Eocene amber found in the Dominican Republic. It is the first described amphibian fossil in amber, and the oldest complete lissamphibian fossil from Mesoamerica (Central America and Mexico). Dating of the amber matrix indicates that by the end of the Eocene a diverse fauna was present in the Antilles, much earlier than has generally been proposed. The presence of this and other amber fossils from this same age suggests that Tertiary patterns of landmass movements were significant in determining the present distribution of species.