Australis Larvae Research Articles

Expression network analysis of bovine skin infested with Rhipicephalus australis identifies pro-inflammatory genes contributing to tick susceptibility

The skin is the primary feeding site of ticks that infest livestock animals such as cattle. The highly specialised functions of skin at the molecular level may be a factor contributing to variation in susceptibility to tick infestation; but these remain to be well defined. The aim of this study was to investigate the bovine skin transcriptomic profiles of tick-naïve and tick-infested cattle and to uncover the gene expression networks that influence contrasting phenotypes of host resistance to ticks. RNA-Seq data was obtained from skin of Brangus cattle with high (n = 5) and low (n = 6) host resistance at 0 and 12 weeks following artificial tick challenge with Rhipicephalus australis larvae. No differentially expressed genes were detected pre-infestation between high and low resistance groups, but at 12-weeks there were 229 differentially expressed genes (DEGs; FDR < 0.05), of which 212 were the target of at least 1866 transcription factors (TFs) expressed in skin. Regulatory impact factor (RIF) analysis identified 158 significant TFs (P < 0.05) of which GRHL3, and DTX1 were also DEGs in the experiment. Gene term enrichment showed the significant TFs and DEGs were enriched in processes related to immune response and biological pathways related to host response to infectious diseases. Interferon Type 1-stimulated genes, including MX2, ISG15, MX1, OAS2 were upregulated in low host resistance steers after repeated tick challenge, suggesting dysregulated wound healing and chronic inflammatory skin processes contributing to host susceptibility to ticks. The present study provides an assessment of the bovine skin transcriptome before and after repeated tick challenge and shows that the up-regulation of pro-inflammatory genes is a prominent feature in the skin of tick-susceptible animals. In addition, the identification of transcription factors with high regulatory impact provides insights into the potentially meaningful gene–gene interactions involved in the variation of phenotypes of bovine host resistance to ticks.

Larvicidal activity of the black pepper, Piper nigrum (Fam: Piperaceae) extracts on the cattle tick, Rhipicephalus australis (Acari: Ixodidae)

BackgroundThe cattle farming parasite Rhipicephalus australis is the main tick and one of the most important in the world from an economic point of view. Various studies have been developed in order to find plant extracts with effective acaricidal properties and environmentally friendly. Studies involving plant extracts for parasite control on commercial animal herds is a developing area in New Caledonia. Bioactive natural products play an important role as lead compounds in the development of new pesticides.ResultsThe ethanolic extract of Piper nigrum L. dried fruits as well as the ethyl acetate extract and the methanolic extract of stems exhibited 100% larvicidal activity (50 mg/mL) against Rh. australis larvae, the cattle tick, an hematophagous parasite. Bioguided fractionation of the ethanolic extract of dried mature fruits using the same assay led to the isolation of five compounds belonging to piperamide family. The structures of isolated compounds were elucidated using spectroscopic methods: ESI-HRMS, 1H- and 13C-NMR spectral data, including DEPT and 2D-NMR experiments (COSY, HSQC, HMBC, and NOESY). These include 1 compound described for the first time in P. nigrum, homopellitorine (2) and 4 known compounds, namely pellitorine (1), pipyaqubine (3), 2-methylpropylamide (4), and N-isobutyl-2,4-eicosadienamide (5).ConclusionThis first report on the larvicidal activity of P. nigrum extract and pure compounds on this tick species suggests that P. nigrum could be a natural biosourced alternative for the control of the larval stage of Rh. australis.Graphical

Transcriptional changes in the peripheral blood leukocytes from Brangus cattle before and after tick challenge with Rhipicephalus australis

BackgroundDisease emergence and production loss caused by cattle tick infestations have focused attention on genetic selection strategies to breed beef cattle with increased tick resistance. However, the mechanisms behind host responses to tick infestation have not been fully characterised. Hence, this study examined gene expression profiles of peripheral blood leukocytes from tick-naive Brangus steers (Bos taurus x Bos indicus) at 0, 3, and 12 weeks following artificial tick challenge experiments with Rhipicephalus australis larvae. The aim of the study was to investigate the effect of tick infestation on host leukocyte response to explore genes associated with the expression of high and low host resistance to ticks.ResultsAnimals with high (HR, n = 5) and low (LR, n = 5) host resistance were identified after repeated tick challenge. A total of 3644 unique differentially expressed genes (FDR < 0.05) were identified in the comparison of tick-exposed (both HR and LR) and tick-naive steers for the 3-week and 12-week infestation period. Enrichment analyses showed genes were involved in leukocyte chemotaxis, coagulation, and inflammatory response. The IL-17 signalling, and cytokine-cytokine interactions pathways appeared to be relevant in protection and immunopathology to tick challenge. Comparison of HR and LR phenotypes at timepoints of weeks 0, 3, and 12 showed there were 69, 8, and 4 differentially expressed genes, respectively. Most of these genes were related to immune, tissue remodelling, and angiogenesis functions, suggesting this is relevant in the development of resistance or susceptibility to tick challenge.ConclusionsThis study showed the effect of tick infestation on Brangus cattle with variable phenotypes of host resistance to R. australis ticks. Steers responded to infestation by expressing leukocyte genes related to chemotaxis, cytokine secretion, and inflammatory response. The altered expression of genes from the bovine MHC complex in highly resistant animals at pre- and post- infestation stages also supports the relevance of this genomic region for disease resilience. Overall, this study offers a resource of leukocyte gene expression data on matched tick-naive and tick-infested steers relevant for the improvement of tick resistance in composite cattle.

Two Epichloë festucae var. lolii endophytes in Lolium perenne reduce larval populations of the wheat sheath miner, Cerodontha australis

AbstractThe wheat sheath miner, Cerodontha australis Malloch (Diptera: Agromyzidae), is a small leaf‐ and stem‐mining fly that is native to New Zealand and eastern Australia where it feeds on grasses and cereals. Although not considered to be a major insect pest of pastures in New Zealand, it can be abundant during spring and summer in Waikato, New Zealand, and infests tillers of the introduced pasture plant, perennial ryegrass, Lolium perenne L. (Poaceae). Cerodontha australis is not deterred from feeding on L. perenne infected with commercially available strains of the fungal endophyte Epichloë festucae var. lolii, which have been shown to provide protection from a range of other insect pests. In this study, three‐pot trials and periodic examination over 18 months of field‐collected L. perenne tillers showed that two Epichloë endophyte strains, AR47 and AR48, reduced the survival and feeding damage by C. australis larvae, but there was no evidence of an effect on adult female feeding or oviposition. Compared to other strains of E. festucae var. lolii endophytes tested, plants infected with AR47 and AR48 had fewer and smaller larvae and less larval mining. The number of tillers with mining was reduced in AR47 relative to endophyte‐free (Nil) by 57.1% (ranging from 25.0 to 92.5% across trials) and in AR48 by 67.1% (26.3–100%). Larvae in AR47 and AR48 were found further from the base of the tiller where they were less likely to damage the meristem and cause tiller death. The number of larvae and pupae in pseudostems was reduced in AR47 and AR48 compared with Nil by 78.4 and 82.0%, respectively, in the field trial. Very few larvae survived to pupation in AR47 and AR48 plants, with a reduction compared to Nil of over 81.5% in AR47 and over 97% in AR48 in each of the four trials presented here.

Distribution of anguillid leptocephali and possible spawning areas in the South Pacific Ocean

Seven South Pacific anguillid eel species live from New Guinea to French Polynesia, but their spawning areas and life histories are mostly unknown despite previous sampling surveys. A July–October 2016 research cruise was conducted to study the spawning areas and times, and larval distributions of South Pacific anguillid eels, which included a short 155°E station-line northeast of New Guinea and five long transects (5–25°S, 160°E–140°W) crossing the South Equatorial (SEC) and other currents. This survey collected nearly 4000 anguilliform leptocephali at 179 stations using an Isaacs-Kidd Midwater Trawl accompanied by 104 CTD casts. Based on morphometric observations and DNA sequencing, 74 anguillid leptocephali were collected, which in the southern areas included 29 larvae of six species: Anguilla bicolor pacifica, A. marmorata, A. australis, A. reinhardtii, A. megastoma,and A. obscura (all anguillid species of the region were caught except A. dieffenbachii). Small A. australis (9.0–16.8 mm) and A. reinhardtii (12.4, 12.5 mm) leptocephali were collected south of the Solomon Islands, other A. australis (10.8–12.0 mm) larvae were caught northwest of Fiji along with an A. obscura (20.0 mm) larva, and an A. marmorata (7.8 mm) larva was collected near Samoa. Considering collection sites, larval ages from otolith analysis, and westward SEC drift, multiple spawning locations occurred from south of the Solomon Islands and the Fiji area (16–20 days old larvae) to near Samoa (19 days old larva) during June and July in areas where high-salinity Subtropical Underwater (STUW, ~150 m depth) and the warm, low-salinity surface Fresh Pool were present. Five long hydrographic sections showed the strong Fresh Pool in the west and the STUW formation area in the east.

A combination of antibodies against Bm86 and Subolesin inhibits engorgement of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae in vitro

BackgroundRhipicephalus microplus is a hard tick species that has a high impact on cattle health and production in tropical and subtropical regions. Recently, ribosomal DNA and morphological analysis resulted in the reinstatement of R. australis as a separate species from R. microplus. Both feed on cattle and can transmit bovine pathogens such as Anaplasma and Babesia species. The current treatment with acaricides is becoming increasingly less effective due to the emergence of resistant tick strains. A promising alternative can be found in the form of anti-tick vaccines. The available commercial vaccines can be used to control tick infestation, but the lack of a knockdown effect (> 90% reduction in tick numbers as seen with effective acaricides) hampers its widespread use, hence higher efficacious vaccines are needed. Instead of searching for new protective antigens, we investigated the efficacy of vaccines that contain more than one (partially) protective antigen. For screening vaccine formulations, a previously developed in vitro feeding assay was used in which R. australis larvae are fed sera that were raised against the candidate vaccine antigens. In the present study, the efficacy of the Bm86 midgut antigen and the cytosolic Subolesin (SUB) antigen were evaluated in vitro.ResultsAntiserum against recombinant Bm86 (rBm86) partially inhibited larval engorgement, whereas antiserum against recombinant SUB (rSUB) did not have any effect on feeding of larvae. Importantly, when larvae were fed a combination of antiserum against rBm86 and rSUB, a synergistic effect on significantly reducing larval infestations was found. Immunohistochemical analysis revealed that the rBm86 antiserum reacted with gut epithelium of R. australis larvae, whereas the antiserum against rSUB stained salivary glands and rectal sac epithelium.ConclusionsCombining anti-Bm86 and anti-subolesin antibodies synergistically reduced R. australis larval feeding in vitro. Rhipicephalus australis is a one host tick, meaning that the larvae develop to nymphs and subsequently adults on the same host. Hence, this protective effect could be even more pronounced when larvae are used for infestation of vaccinated cattle, as the antibodies could then affect all three developmental stages. This will be tested in future in vivo experiments.

A new method for in vitro feeding of Rhipicephalus australis (formerly Rhipicephalus microplus) larvae: a valuable tool for tick vaccine development

BackgroundRhipicephalus microplus is a hard tick that has a major impact on cattle health in tropical and subtropical regions because it feeds on cattle and is implicated in the transmission of pathogens that cause diseases such as bovine anaplasmosis and babesiosis. Presently, acaricides are used to control tick infestation but this is becoming increasingly less effective due to the emergence of tick strains that are resistant to one or more classes of acaricides. Anti-tick vaccines are a promising alternative to control tick infestation in cattle. The life-cycle and host preference of R. microplus, however, makes vaccine research in cattle costly and would therefore greatly benefit from an in vitro screening system.MethodsTo this aim, a stacked 24-well in vitro feeding system was designed in which the blood meal was administered in a chamber on top of the compartment containing the ticks, exploiting their anti-gravitational tendency. Both compartments were separated by a special feeding membrane, which was made by applying a silicone mixture to a gold beater’s skin (baudruche membrane) with a paint roller to create a slightly uneven surface of 17–40 μm variable thickness. To further stimulate feeding, the membrane was treated with bovine hair extract and the unit was placed at 37 °C with 90% RH and 5% CO2.ResultsUsing this set-up with Rhipicephalus australis (formerly Rhipicephalus microplus), a larval engorgement rate of up to 71% could be achieved. The larvae could successfully feed on blood, but also on serum. The latter allows easy screening of the effect of sera that are raised against tick proteins on feeding. As an example, serum from cattle that were vaccinated with the Bm86 midgut protein of R. microplus significantly reduced larval engorgement rates by 42%.ConclusionThe in vitro feeding system’s high throughput design and its ability to measure statistically significant anti-tick effects in sera from immunized cattle enables screening of multiple vaccine candidates in a cost-effective manner.

Repellent effects of Melaleuca alternifolia (tea tree) oil against cattle tick larvae (Rhipicephalus australis) when formulated as emulsions and in β-cyclodextrin inclusion complexes

Rhipicephalus australis (formerly Boophilus microplus) is a one host tick responsible for major economic loss in tropical and subtropical cattle production enterprises. Control is largely dependent on the application of acaricides but resistance has developed to most currently registered chemical groups. Repellent compounds that prevent initial attachment of tick larvae offer a potential alternative to control with chemical toxicants. The repellent effects of Melaleuca alternifolia oil (TTO) emulsions and two β-cyclodextrin complex formulations, a slow release form (SR) and a modified faster release form (FR), were examined in a series of laboratory studies.Emulsions containing 4% and 5% TTO applied to cattle hair in laboratory studies completely repelled ascending tick larvae for 24h whereas 2% and 3% formulations provided 80% protection. At 48h, 5% TTO provided 78% repellency but lower concentrations repelled less than 60% of larvae. In a study conducted over 15 days, 3% TTO emulsion applied to cattle hair provided close to 100% repellency for 2 days, but then protection fell to 23% by day 15. The FR formulation gave significantly greater repellency than the emulsion and the SR formulation from day 3 until the end of the study (P<0.05), providing almost complete repellency at day 3 (99.5%), then decreasing over the period of the study to 49% repellency at day 15. Proof of concept is established for the use of appropriately designed controlled-release formulations to extend the period of repellency provided by TTO against R. australis larvae.

An original description of the larval stages of Phoronis australis Haswell, 1883 and an analysis of the world fauna of phoronid larvae

Phoronida is a phylum of marine invertebrates; they have a worldwide distribution and form huge benthic aggregations in some areas. The taxonomy of Phoronida remains very poorly developed; only 13 species of phoronids have been described worldwide, while approximately 40 varieties of competent larvae are known. Morphological description of phoronid larvae and the search for correspondence between the larval and adult forms make an important contribution to the taxonomy of this enigmatic group. We present an original description of the larval forms of Phoronis australis Haswell, 1883. The larvae and adults of P. australis were collected in the plankton of Nha Trang Bay in the South China Sea. For the first time, the external morphology of larval forms of P. australis was described at the stages of 4, 8, and 20 tentacles. The basic distinguishing features of Phoronis australis larvae were determined: opaque integuments, the presence of a characteristic dark pigmentation of the oral field epidermis, of the unpaired ventral stomach diverticulum, and more than 20 tentacles in the competent larvae. A molecular-genetic analysis based on comparison of the DNA sequences of the 18S rDNA and 28S rDNA nuclear genes confirmed the identity of the phoronid larvae that were found in the planktonic samples from Nhatrang Bay and adult P. australis that were collected in the same area. The problems of Phoronida taxonomy and correspondence of larval and adult forms can be solved only with a complex approach that involves both morphological and molecular-genetic information.

Investigations into the Toxic and Repellent Effects of Propiconazole on the Wool-Digesting Carpet Beetle Larvae Anthrenocerus australis (Coleoptera: Dermestidae)

Previous work had shown propiconazole applied to wool gave a protective effect against the keratinophagous insect larvae of the Australian carpet beetle Anthrenocerus australis. To elucidate the mode of action of propiconazole on Anthrenocerus australis, repellency trials were carried out using untreated control and propiconazole-treated wool. A reversible choice experiment using Petri dishes, and an irreversible olfactometer choice experiment were carried out. Treated surface repellency trials were also carried out. No repellency effect was detected with any of these experiments. Direct contact experiments were carried out by application of propiconazole solutions directly to Anthrenocerus australis larvae and by feeding larvae propiconazole-treated wool. No short or long-term toxic effects were detected, and subsequent feeding on untreated wool was not reduced. These results suggest an anti-feeding and/or gut-specific mode of action of propiconazole on Anthrenocerus australis that is likely to be different to that of the commonly used wool insecticides. This provides an opportunity to research a new approach to the control of this wool pest.

The efficacy of antifungal azole and antiprotozoal compounds in protection of wool from keratin-digesting insect larvae

The seven azole compounds, econazole nitrate, sulconazole nitrate, miconazole nitrate, thiabendazole, epoxiconazole, propiconazole, and tebuconazole, were applied to wool fabrics at 3.0% on mass of wool and the protection from wool-digesting Tineola bisselliella (common clothes moth) larvae was assessed in bioassays. Econazole nitrate gave the greatest protection according to Wools of New Zealand Test Method 25, easily passing the bioassay. Other good results with sulconazole and epoxiconazole led to further bioassays being performed with Australian carpet beetle larvae Anthrenocerus australis. Propiconazole provided the most effective protection of wool from this species, with a concentration of approximately 0.4% on mass of wool predicted to give adequate insect resistance. A combination of propiconazole and isoniazid was trialed against Anthrenocerus australis, without any synergistic effect. The antiprotozoal compound pentamidine isethionate had no measurable effect on Anthrenocerus australis larvae, but provided a moderate anti-feeding effect on Tineola bisselliella. Antimicrobial compounds could affect the digestive process of wool-digesting insects either directly or by their effects on any gut flora and/or fauna. Azoles are antifungal compounds, and are likely to disrupt the insect utilisation of cholesterol by inhibiting the cytochrome P450 enzymes. Propiconazole was trialed as a dyebath-applied insect-proofing agent on piece-dyed carpet, showing inefficient uptake onto wool, but good fastness to carpet shampooing and light exposure.

Larval fish assemblages and particle back-tracking define latitudinal and cross-shelf variability in an eastern Indian Ocean boundary current

MEPS Marine Ecology Progress Series Contact the journal Facebook Twitter RSS Mailing List Subscribe to our mailing list via Mailchimp HomeLatest VolumeAbout the JournalEditorsTheme Sections MEPS 460:127-144 (2012) - DOI: https://doi.org/10.3354/meps09730 Larval fish assemblages and particle back-tracking define latitudinal and cross-shelf variability in an eastern Indian Ocean boundary current D. Holliday1,*, L. E. Beckley1, N. Millar1, M. P. Olivar2, D. Slawinski3, M. Feng3, P. A . Thompson4 1School of Environmental Science, Murdoch University, 90 South St, Murdoch, 6150, Western Australia, Australia 2Institut de Ciènces del Mar (CSIC), Passeig Marítim 37-49, Barcelona 08003, Spain 3CSIRO Marine and Atmospheric Research, Private Bag 5, Wembley, Western Australia, 6913, Australia 4CSIRO Marine and Atmospheric Research, GPO Box 1538, Hobart, Tasmania 7701, Australia *Email: d.holliday@murdoch.edu.au ABSTRACT: Empirical and modelled data were used to examine the influence of the Leeuwin Current on larval fish assemblages along the Western Australian continental shelf and adjacent eastern Indian Ocean (22°S−34°S) during the late austral autumn. Larval fish assemblages, comprising >200 taxa from 114 neritic and oceanic teleost families, displayed significantly distinct latitudinal and cross-shelf variability, which was linked to meso-scale features of the Leeuwin Current, specifically anticyclonic eddies. Results of Lagrangian particle back-tracking showed good connection between the shelf and a developing eddy situated at 27°S; connectivity was highest in the 0−20 m depth stratum where >80% of the particles entering the eddy were derived from the shelf. This was supported by the high abundance (mean: 84 larvae m−2) of larval neritic taxa, especially small anchovy Engraulis australis larvae in the eddy. The lack of a significant difference in the size structure of these larvae between the shelf and eddy indicated continuous connection between these waters. In contrast, connectivity between the shelf and an older eddy further south was much lower (<40% of particles in the 0−20 m stratum) and the larval fish assemblage was dominated by meso-pelagic species. Further, the distribution of Myctophidae larvae (Diaphus spp.) highlighted areas of enhanced onshore transport in the south. The study has shown that, in the alongshore dominated Leeuwin Current system, cross-shelf transport can be similarly important in dispersal processes. KEY WORDS: Leeuwin Current · Western Australia · Eddy · Lagrangian · Myctophidae · Engraulidae Full text in pdf format Supplementary material PreviousNextCite this article as: Holliday D, Beckley LE, Millar N, Olivar MP, Slawinski D, Feng M, Thompson PA (2012) Larval fish assemblages and particle back-tracking define latitudinal and cross-shelf variability in an eastern Indian Ocean boundary current. Mar Ecol Prog Ser 460:127-144. https://doi.org/10.3354/meps09730 Export citation RSS - Facebook - Tweet - linkedIn Cited by Published in MEPS Vol. 460. Online publication date: July 24, 2012 Print ISSN: 0171-8630; Online ISSN: 1616-1599 Copyright © 2012 Inter-Research.

First artificial hybrid of the eel species Anguilla australis and Anguilla anguilla

BackgroundStudies on artificial hybridization of different Anguilla species were conducted recently, i.e. female A. australis with male A. dieffenbachii, and female A. japonica with male A. anguilla. The existence of these artificial hybrids was however not demonstrated by independent genetic methods. Two species - A. anguilla and A. australis - that are phylogenetically close but have different sexual maturation times (12-25 weeks and 6-8 weeks, respectively), were expected to produce favourable hybrids for reproduction studies.ResultsA modification of the protocol for the reproduction of Anguilla japonica was used to produce eight-day Anguilla australis larvae, with a success rate of 71.4%. Thus ten out of 14 females produced eggs that could be fertilized, and three batches resulted in mass hatching. Hybrid larvae from female A. australis x male A. Anguilla survived for up to seven days post fertilization (dpf). The early development of the hybrid showed typical characteristics of A. anguilla tail pigmentation at 50 hours post fertilization (hpf), indicating expression of genes derived from the father.ConclusionsIn this paper we describe the first production of hybrid larvae from male A. anguilla and female A. australis and their survival for up to 7 dpf. A species-specific nucleotide difference in the 18 S rDNA gene confirmed that genes from both A. australis and A. anguilla were present in the hybrids. The developmental stages of the hybrid eel embryos and larvae are described using high resolution images. Video footage also indicated a heart beat in 5-dpf larva.

Identification of the Optimal Fluorochrome for Marking Larvae of the Pulmonate LimpetSiphonaria australis

This study identified the most suitable fluorochrome to mark prodissoeonch of larval Siphonaria australis, a common pulmonate gastropod commonly found on rocky shores throughout New Zealand. The pulmonate habit of laying attached larval egg masses makes them an excellent candidate for studies of larval connectivity using chemical staining methods. Larval S. australis egg masses were immersed in 200 mg/L of either alizarin red S (ARS), calcein, or Oxytetracycline (OTC) for 24 h before viewing under ultraviolet light to allow quantification of mark brightness using image analysis software. Counts of pre- and postimmersion mortality were also made to identify any fluorochrome toxicity to S. australis larvae. Calcein was found to be the most suitable fluorochrome to use when fluorescently marking larval S. australis, because average brightness was significantly greater than either ARS or OTC, with negligible mortality levels recorded for calcein. The results show that fluorochromes can penetrate the gelatinous egg matrix and the larval capsules, thus providing an opportunity for staining large numbers of these species for alongshore connectivity studies.

Effects of temperature and lipid droplet adherence on mortality of hatchery-reared southern hake Merluccius australis larvae

Effects of exogenous (water temperature) and endogenous (lipid droplet adherence) factors were experimentally tested on early survival of southern hake Merluccius australis reared under controlled conditions. Experiments to determine the effect of temperature (10, 12 and 14 °C) on larval growth rates and yolk-sac absorption rates of unfed southern hake were carried out under laboratory conditions. There was no significant differences in growth rates at the temperature range tested (ANCOVA, F = 0.164, p > 0.25), but yolk-sac absorption rates and mortality increased with temperature (ANCOVA, F = 53.84, p < 0.001). A high percentage (between 31 and 81%) of hake eggs showed a lipid droplet not adhered (i.e., freely moving in the yolk, and not located in the posteriormost portion of the yolk-sac). In a second experiment, fed southern hake larvae with the lipid droplet not adhered during embryonic development did not survive after yolk-sac absorption. This study provides the first data on the influence of the lipid droplet absorption on larval survival of cultured hake, and can be used as an early indication of the quality of the batch.

Factors affecting settlement and early post‐settlement survival of the New Zealand abalone Haliotis australis

This paper describes studies on the settlement of the abalone Haliotis australis Gmelin. Ten‐day‐old H. australis larvae had a slower rate of settlement than 5‐day‐old larvae. A high proportion of veliger larvae had the ability to prolong the searching phase of settlement and delay metamorphosis, even over prepared settlement surfaces but radula development continued at a similar rate in both settled and swimming larvae. In any one batch of larvae settlement was slow and asynchronous and post‐settlement survival was low. Light was shown to influence the swimming behaviour of larvae in settlement tanks, with more larvae swimming in the dark than in the light. The implications of these behaviours are discussed in relation to larval and post‐larval energetics and feeding.

A light and electron microscope study of the gills of larval lampreys (Geotria australis) with particular reference to the water‐blood pathway.

The gills of ammocoetes of the Southern Hemisphere lamprey Geotria australis have been studied using light and electron microscopy. Emphasis has been placed on describing the structures and vessels involved in gaseous exchange, and on providing quantitative data for the water‐blood barrier, including diffusion distance, diffusing capacity and the relative volumes of the component tissues. Although lamprey gills lie inside rather than outside the branchial skeleton as in gnathostomatous fishes. the morphology and ultrastructure of the gill filaments and secondary lamellae of G. australis larvae are very similar to those of teleost fishes. The extensive blood spaces within the secondary lamellae are enclosed by pillar cell bodies and pillar cell flanges which support two layers of epithelial cells. The outer surfaces of the epithelial cells are ridged and covered in a flocculent material which probably represents mucus. Differences were observed in the components of the water‐blood barrier at the distal edges and at the surface of the secondary lamellae. At the distal edge, the lining of the marginal channel consisted of an endothelial cell rather than the pillar cell flanges which line the blood spaces of other regions. Based on light micrograph measurements, these differences result in a reduction in the arithmetic mean thickness of the water‐blood barrier from 3.62 μm over the pillar cells to 2.22 μm over the marginal channel. Using values for the water‐blood barrier obtained from light micrographs, the arithmetic and harmonic mean diffusing capacities were calculated as 1.1046 and 1.7589 ml O2min/mm Hg/Kg.

Coelomomyces opifexiPillai &amp; Smith (Coelomomycetaceae: Blastocladiales) VI. Observations on the mode of entry intoAedes australislarvae

Abstract A study was undertaken to elucidate the mode of entry of Coelomomyces opifexi into Aedes australis larvae and the copepod intermediate host, Tigriopus sp. cf. angulatus, using the fluorescent antibody technique coupled with direct examination of whole mounted specimens under the light microscope. Zygotes and zoospores of C. opifexi attached and encysted on the cuticle of mosquito larvae and copepods respectively; their overall distribution on the cuticle is described. Penetration by zygotes and zoospores appears to be mainly confined to the intersegmental membranes. The zygote protoplasm is injected through the cuticle, and forms a hyphal body which may either be released to circulate in the haemolymph or remain attached to the epidermal layer. The development of the hyphal body in the mosquito coelom is discussed.

Coelomomyces opifexiPillai &amp; smith (Coelomomycetaceae: Blastocladiales) V. Development of the fungus in pupae and adults ofAedes australis

Abstract Late-instar Aedes australis larvae were experimentally infected with Coelomomyces opifexi, and the subsequent development of the fungal parasite was studied. Five separate experiments involving 1190 infected larvae were conducted. Of these larvae, 114 continued development and pupated; 38 of them contained sporangia. Histological studies indicated that parasite development slowed down, if not ceased, following pupation of infected larvae, and the presence of sporangia in adults reflected the degree of advancement of infection in the larvae at pupation.

Coelomomyces opifexiPillai &amp; Smith (Coelomomycetaceae: Blastocladiales) IV. Host range and relative susceptibility ofAedes australisandOpifex fuscuslarvae

Abstract The host range of Coelomomyces opifexi was investigated. Of the three species of mosquito larvae tested, Aedes australis and Opifex fuscus were susceptible whereas Culex pervigilans was refractory to infection. The results of laboratory infection experiments using inoculum derived from larvae of both species clearly indicated that Ae. australis larvae are more susceptible to infection than O. fuscus; this conclusion is supported by observations of natural infection pools. Of three species of copepods also tested, only Tigriopus sp. cf. angulatus became infected; Cyclops vernalis and C. bicuspidatus proved refractory to infection.