Ant Tissue Research Articles

Bacillus predominates in the Ophiocordyceps pseudolloydii-infected ants, and it potentially improves protection and utilization of the host cadavers

The bacterial communities that colonize the cadaver environment of insects infected and killed by parasitic fungi can be selected by the sympatric fungi and provide novel impacts. In this study, we found that Bacillus cereus/thuringiensis predominate the bacterial community in Dolichoderus thoracicus ant cadavers colonized by O. pseudolloydii. The most predominant bacterial strains in these ant cadavers were hemolytic and able to produce hydrolytic enzymes for digesting the ant tissue. A relatively intense lethal effect on the co-cultured nematode was displayed by a hemolytic strain. Moreover, the antagonistic effect against pathogenic fungi detected in the bacteria sympatric with O. pseudolloydii was reported here. Naphthoquinones have been shown to confer antibacterial activities and produced by the ant-pathogenic Ophiocordyceps fungi. However, our results did not show the naphthoquinone tolerance we expected to be detected in the bacteria from the ant infected by O. pseudolloydii. The bacterial diversity in the samples associated with O. pseudolloydii infected ants as revealed in this study will be a step forward to the understanding of the roles playing by the microbial community in the native habitats of O. pseudolloydii.

Development of a Rapid, Accurate, and On-Site Detection Protocol for Red Imported Fire Ants, Solenopsis invicta (Hymenoptera: Formicidae).

A rapid, accurate, and on-site molecular diagnostic protocol for red imported fire ants (Solenopsis invicta, Si) was developed using loop-mediated isothermal amplification (LAMP) assays. Si11977 (GenBank accession no. MK986826) was confirmed to be a Si-specific gene. Four-primer Si11977-LAMP (4pSi-LAMP) and six-primer Si11977-LAMP (6pSi-LAMP) assays specifically detected Si. The reaction time of 6pSi-LAMP assays was reduced by 5 min compared with 4pSi-LAMP assays. The optimal amount of polymerase and the detection limit for the 6pSi-LAMP assays were 0.1 unit/μL and 5 fg/μL, respectively. In addition, a method for extracting genomic DNA from ant tissues within 2 to 3 min and a protocol for performing on-site LAMP assays using a car heating mug and a LAMP observation box were described. The on-site Si detection protocol used in this study was possible within 30 min from DNA extraction to species identification.

Chemical warfare between leafcutter ant symbionts and a co-evolved pathogen

Acromyrmex leafcutter ants form a mutually beneficial symbiosis with the fungus Leucoagaricus gongylophorus and with Pseudonocardia bacteria. Both are vertically transmitted and actively maintained by the ants. The fungus garden is manured with freshly cut leaves and provides the sole food for the ant larvae, while Pseudonocardia cultures are reared on the ant-cuticle and make antifungal metabolites to help protect the cultivar against disease. If left unchecked, specialized parasitic Escovopsis fungi can overrun the fungus garden and lead to colony collapse. We report that Escovopsis upregulates the production of two specialized metabolites when it infects the cultivar. These compounds inhibit Pseudonocardia and one, shearinine D, also reduces worker behavioral defenses and is ultimately lethal when it accumulates in ant tissues. Our results are consistent with an active evolutionary arms race between Pseudonocardia and Escovopsis, which modifies both bacterial and behavioral defenses such that colony collapse is unavoidable once Escovopsis infections escalate.

3D virtual histology at the host/parasite interface: visualisation of the master manipulator, Dicrocoelium dendriticum, in the brain of its ant host

Some parasites are able to manipulate the behaviour of their hosts to their own advantage. One of the most well-established textbook examples of host manipulation is that of the trematode Dicrocoelium dendriticum on ants, its second intermediate host. Infected ants harbour encysted metacercariae in the gaster and a non-encysted metacercaria in the suboesophageal ganglion (SOG); however, the mechanisms that D. dendriticum uses to manipulate the ant behaviour remain unknown, partly because of a lack of a proper and direct visualisation of the physical interface between the parasite and the ant brain tissue. Here we provide new insights into the potential mechanisms that this iconic manipulator uses to alter its host’s behaviour by characterising the interface between D. dendriticum and the ant tissues with the use of non-invasive micro-CT scanning. For the first time, we show that there is a physical contact between the parasite and the ant brain tissue at the anteriormost part of the SOG, including in a case of multiple brain infection where only the parasite lodged in the most anterior part of the SOG was in contact with the ant brain tissue. We demonstrate the potential of micro-CT to further understand other parasite/host systems in parasitological research.

Lead bioaccumulation in Texas Harvester Ants (Pogonomyrmex barbatus) and toxicological implications for Texas Horned Lizard (Phrynosoma cornutum) populations of Bexar County, Texas.

Uptake of lead from soil was examined in order to establish a site-specific ecological protective concentration level for the Texas Horned Lizard (Phrynosoma cornutum) at the Former Humble Refinery in San Antonio, Texas. Soils, harvester ants, and rinse water from the ants were analyzed at 11 Texas Harvester Ant (Pogonomyrmex barbatus) mounds. Soil concentrations at the harvester ant mounds ranged from 13 to 7474mg/kg of lead dry weight. Ant tissue sample concentrations ranged from < 0.82 to 21.17mg/kg dry weight. Rinse water concentrations were below the reporting limit in the majority of samples. Two uptake models were developed for the ants. A bioaccumulation factor model did not fit the data, as there was a strong decay in the calculated value with rising soil concentrations. A univariate natural log-transformed regression model produced a significant regression (p < .0001) with a high coefficient of determination (0.82), indicating a good fit to the data. Other diagnostic regression statistics indicated that the regression model could be reliably used to predict concentrations of lead in harvester ants from soil concentrations. Estimates of protective levels for P. cornutum were developed using published sub-chronic toxicological findings for the Western Fence Lizard that were allometricly adapted and compared to the Doseoral equation, which estimated lead consumed through ants plus incidental soil ingestion. The no observed adverse effect level toxicological limit for P. cornutum was estimated to be 5500mg/kg.

Fungi Associated withSolenopsis invictaBuren (Red Imported Fire Ant, Hymenoptera: Formicidae) from Mounds in Mississippi

In 2004, we determined baseline data on fungal-community assemblages from Solenopsis invicta (Red Imported Fire Ant) mounds in 3 counties (Hinds, Leake, and Madison) within the Natchez Trace Parkway, MS. We assayed mound soil, plant debris within the mounds, and ants obtained from mounds on 3 sampling dates (March, July, and November). We processed samples based on standard microbiological protocols, and used traditional morphological and molecular techniques to identify fungal taxa. We documented a total of 1445 isolates consisting of 50 fungal taxa and calculated a diversity index value (H′) of 3.11 across all substrates, which was indicative of a variable fungal community within the mounds. The taxa with the highest percent isolation frequencies included Hypocrea lixii (12.8%), Fusarium sp. 1 (12.3%), Fusarium equiseti (7.9%), Purpureocillium lilacinum (= Paecilomyces lilacinus) (6.5%), Fusarium oxysporum 2 (5.8%), and Mortierella alpina (5.4%). We isolated 2 common parasitic (entomopathogenic) fungi, Purpureocillium lilacinum and Metarhizium anisopliae var. anisopliae (9.4%), from mound soil, plant debris, and ant external tissues. Hypocrea lixii, the teleomorphic reproductive stage of Trichoderma harzianum, is noted as a natural biological control of some soil-borne microbes, possibly limiting important natural entomopathogenic activity within the mounds. Species richness and diversity values from mound soils across locations were significantly greater (P ≤ 0.05) than those from the plant debris and ant body-tissue substrates. Species richness values between locations were similar. Species richness of samples collected in November (47) was significantly greater (P ≤ 0.05) than that of the March (41) and July (39) samples. Community coefficient values ranged from 0.79 to 0.87 between substrates, 0.85 to 0.91 between locations, and 0.85 to 0.86 between sampling dates, indicating that taxa were similar.

Thermal adaptation and phosphorus shape thermal performance in an assemblage of rainforest ants.

We studied the Thermal Performance Curves (TPCs) of 87 species of rainforest ants and found support for both the Thermal Adaptation and Phosphorus-Tolerance hypotheses. TPCs relate a fitness proxy (here, worker speed) to environmental temperature. Thermal Adaptation posits that thermal generalists (ants with flatter, broader TPCs) are favored in the hotter, more variable tropical canopy compared to the cooler, less variable litter below. As predicted, species nesting in the forest canopy 1) had running speeds less sensitive to temperature; 2) ran over a greater range of temperatures; and 3) ran at lower maximum speeds. Tradeoffs between tolerance and maximum performance are often invoked for constraining the evolution of thermal generalists. There was no evidence that ant species traded off thermal tolerance for maximum speed, however. Phosphorus-Tolerance is a second mechanism for generating ectotherms able to tolerate thermal extremes. It posits that ants active at high temperatures invest in P-rich machinery to buffer their metabolism against thermal extremes. Phosphorus content in ant tissue varied three-fold, and as predicted, temperature sensitivity was lower and thermal range was higher in P-rich species. Combined, we show how the vertical distribution of hot and variable vs. cooler and stable microclimates in a single forest contribute to a diversity of TPCs and suggest that a widely varying P stoichiometry among these ants may drive some of these differences.

Bacteria Associated with Red Imported Fire Ants (Solenopsis invicta) from Mounds in Mississippi

Abstract A study was conducted to determine microbial community structure and baseline information of cultural bacteria taxa within Solenopsis invicta (Red Imported Fire Ant) mounds from 3 locations along the roadside of Natchez Trace Parkway in Mississippi. At each location, samples consisting of mound soils, plant debris of primarily grass stem and leaves (control), and ant body tissues were obtained from replicate mounds during March, July, and November 2004. Bacteria isolate frequencies from soil were significantly greater than from plant or ant body tissues. Using 16S sequence data, 68 taxa from 2324 isolates were obtained from the 3 substrate types. The 7 most common bacteria following in order of greatest isolation frequencies were Bacillus sp. (5) (species complex), Achromobacter xylosoxidans, Bacillus cereus (complex), Lysininibacillus boronitolerans, Serratia liquefaciens, Pseudomonas protegens, and Lysinibacillus sphaericus. Richness, diversity, and evenness values varied between the locations,...

Metabolism and the rise of fungus cultivation by ants.

Most ant colonies are comprised of workers that cooperate to harvest resources and feed developing larvae. Around 50 million years ago (MYA), ants of the attine lineage adopted an alternative strategy, harvesting resources used as compost to produce fungal gardens. While fungus cultivation is considered a major breakthrough in ant evolution, the associated ecological consequences remain poorly understood. Here, we compare the energetics of attine colony-farms and ancestral hunter-gatherer colonies using metabolic scaling principles within a phylogenetic context. We find two major energetic transitions. First, the earliest lower-attine farmers transitioned to lower mass-specific metabolic rates while shifting significant fractions of biomass from ant tissue to fungus gardens. Second, a transition 20 MYA to specialized cultivars in the higher-attine clade was associated with increased colony metabolism (without changes in garden fungal content) and with metabolic scaling nearly identical to hypometry observed in hunter-gatherer ants, although only the hunter-gatherer slope was distinguishable from isometry. Based on these evolutionary transitions, we propose that shifting living-tissue storage from ants to fungal mutualists provided energetic storage advantages contributing to attine diversification and outline critical assumptions that, when tested, will help link metabolism, farming efficiency, and colony fitness.

Unique soil microbial assemblages associated with grassland ant species with different nesting and foraging strategies

Ants are important ecosystem engineers and can be abundant in extensively managed grassland ecosystems. Different ant species create nests varying in structure and size, and tend to have a variety of feeding strategies. Differences in food imported to the nest and contrasting nesting behaviour may control soil microbial community structure in nest soil, with cascading effects on nutrient cycling, but this has not been tested in grassland ants. Soil and ants were sampled from nests of three ant species: two formicines; Lasius flavus (aphid farmer/scavenger, mound builder) and Formica lemani (scavenger/hunter, non-mound builder), and a myrmicine; Myrmica sabuleti (hunter/scavenger, non-mound builder), in an extensively grazed temperate grassland and compared to similar soils without ants. Microbial assemblages were determined using molecular approaches (terminal restriction length polymorphism and automated ribosomal intergenic spacer analysis). Both aboveground (vegetation diversity) and belowground (soil physico-chemical properties) components were measured to assess the potential of the different ant species to modify the environment. Stable isotope ratios (δ13C and δ15N) of ant tissues and nest soil organic matter confirmed differences in trophic distances. Significant changes in soil pH, moisture content, total C and total N, and in vegetation composition, demonstrated ant ecosystem engineering effects. In turn, nests of L. flavus, M. sabuleti and F. lemani had different microbial activities and harboured significantly different microbial assemblages (total bacteria, total fungi, ammonia-oxidising bacteria and nitrogen-fixing bacteria), but the diversity was similar. These findings suggest that grassland ants can control microbial assemblages via changes in physical and biological soil characteristics in their nests, and as such, different ant species harbour unique microbial assemblages in nests.

Arsenic Speciation of Terrestrial Invertebrates

The distribution and chemical form (speciation) of arsenic in terrestrial food chains determines both the amount of arsenic available to higher organisms, and the toxicity of this metalloid in affected ecosystems. Invertebrates are part of complex terrestrial food webs. This paper provides arsenic concentrations and arsenic speciation profiles for eight orders of terrestrial invertebrates collected at three historical gold mine sites and one background site in Nova Scotia, Canada. Total arsenic concentrations, determined by inductively coupled plasma mass spectrometry (ICP-MS), were dependent upon the classification of invertebrate. Arsenic species were determined by high-performance liquid chromatography (HPLC) ICP-MS and X-ray absorption spectroscopy (XAS). Invertebrates were found by HPLC ICP-MS to contain predominantly arsenite and arsenate in methanol/water extracts, while XAS revealed that most arsenic is bound to sulfur in vivo. Examination of the spatial distribution of arsenic within an ant tissue highlighted the differences between exogenous and endogenous arsenic, as well as the extent to which arsenic is transformed upon ingestion. Similar arsenic speciation patterns for invertebrate groups were observed across sites. Trace amounts of arsenobetaine and arsenocholine were identified in slugs, ants, and spiders.

An oral bioassay for the toxicity of hydramethylnon to individual workers and queens of Argentine ants, Linepithema humile.

We have developed an oral bioassay to determine the toxicity of hydramethylnon to individual workers and queens of the Argentine ant, Linepithema humile. We fed seven concentrations of hydramethylnon in suspension to individual workers or queens, determined the amount of hydramethylnon ingested and evaluated the individual ants for mortality 14 days later. At concentrations > or = 0.37 g liter-1, the amount of liquid the queens ingested decreased dramatically, indicating that Argentine ant queens may detect hydramethylnon. Significantly larger volumes of the two highest concentrations of the hydramethylnon suspension were ingested by the workers, compared to the lower concentrations, suggesting that hydramethylnon may act as a feeding stimulant for the workers. Worker mortality was higher than queen mortality at the highest concentrations tested. The highest worker mortality resulted when the ants ingested 1.03 micrograms of hydramethylnon per mg of ant tissue. At the highest concentration (1.0 g liter-1) tested, workers ingested almost 12 times as much active ingredient per mg of body weight as did queens, suggesting that, in order to increase mortality of queens, multiple feedings must occur.

A rapid field technique for preparing ant chromosomes for karyotypic analysis.

This technique for chromosomal preparation of ant tissues for karyotypic analysis is advantageous under field conditions because it reduces processing time and can be used under humid conditions. The cerebral ganglia from prepupae or early pupae are incubated 20 minutes in a hypotonic citrate solution, minced in a fixative solution of 3:3:4 glacial acetic acid: absolute methanol: distilled HOH, rinsed in a fixative solution of 1:1 glacial acetic acid: methanol followed by Carnoy's fixative, then immediately flame dried. The resulting metaphase chromosomes are well spaced and usually show banding characteristics.

Ultrastructural comparison of freeze-drying and freeze-substitution in preservation of the frozen state

Correlation of cellular ultrastructure before freezing, while frozen, and after thawing provides a requisite basis for evaluation of functional ‘alterations which are induced during the formation and dissolution of ice (9). Th e respective roles of intracellular and extracellular ice figure prominently in theories of freezing injury (2, 3, 6, ll), often without correlative observations on the frozen state, especially in tissue cells. Freeze-drying and freeze-substitution are the techniques most commonly used by investigators, almost interchangeably, to preserve the areas in which ice forms in and around cells during freezing at various rates. Occasional statements have been made which suggest that these techniques may yield the same results in representation of the frozen state (4, 9). Results with muscular tissue indicate no difference in the size of intracellular ice spaces with both methods ( 12), as well as the unreliability of freeze-drying (5). There is little or no information on direct experimental comparisons of these methods with epithelial cells, however, especially on the ultrastructural level. Such information is required for a better interpretation of studies on the frozen state of import’ant tissues and organs. Freeze-etching, which theoretically may represent an even more valid picture of actual structure, but which pre--.Rcwivcd June 28, 1976. sents similar results in practice ( 12), has not “suplemented or replaced freeze-substitution and freeze-drying” in this cryobiological application ( 1, 9). The purpose of this brief communication is to present the results of a comparative evaluation of freeze-drying and freezesubstitution in the electron microscopic representation of ice in the frozen state in pancreatic cells.

Bioenergetics of the Western Harvester Ant1 in the Shortgrass Plains Ecosystem2

The bioenergetics of Pogonomyrmex occidentalis (Cresson) was studied on the IBP grassland intensive study site in northeast Colorado. Energy flow values were determined by estimating the energy required per colony for production of ant tissue and for respiratory energy loss. Extrapolation of the energy values per colony to colony densities in the differentially grazed pastures resulted in seasonal estimates of population energy flow values for western harvester ant populations in ungrazed, lightly grazed, moderately grazed, and heavily grazed pastures. Excavation of 11 colonies resulted in estimates of 2032 worker ants and 315 winged reproductives produced per colony each year. This resulted in an estimate of 46.81 kcal per colony required for secondary production per year. Respiratory energy losses were estimated from measurements of CO2 production in relation to field temperatures, as determined from measurements conducted on 2 colonies established in the laboratory. Annual energy requirements for respiration were estimated at 418.42 kcal per colony. Energy flow values (tissue production + heat production) ranged from 1.45 kcal per m3 per year in the moderately grazed pasture to 0.14 kcal per m2 per year under heavy grazing pressure. The energy flow values for P. occidentalis are much lower than those calculated for P. badius (Latreille). The major area of difference occurs in estimates made of energy required for respiration.

A new method to obtain cellular ant tissue components

A new method to obtain cells and tissue ingredients almost unalterated is described. The tissue is emulsified in an indifferent oil in which process the different histological structures are separated into the different droplets. Separating similar droplets, it is possible to gain single cells and tissue components.