Isolation and partial characterization of three isoamylases of Rhyzopertha dominica F. (Coleoptera: Bostrichidae)

Three isoamylases of Rhyzopertha dominica (termed RdA70, RdA79, and RdA90 according to their relative mobility in gel electrophoresis) were isolated by ammonium sulfate fractionation and hydrophobic interaction chromatography. RdA70 and RdA79 showed an optimal pH of 7.0, whereas for RdA90 the optimal pH was 6.5. The three isoamylases remained stable at 50 °C for 1 h, but at 60 °C, all lost 50% of their activity in 20 min and were completely inactivated in 1 h. RdA70 and RdA79 were inhibited by albumin extracts from wheat samples varying widely in amylase inhibitory activity; however, RdA90 was highly resistant to inhibition. β-Mercaptoethanol up to 30 mM increased the activity of the three isoamylases by 2.5-fold. The action pattern of the three isoamylases was typical of endoamylases; however, differences were observed on the hydrolytic efficiency rates measured as V_max/K_m ratio on starch, amylopectin, and amylose. The hydrolyzing action of RdA90 on starch and amylopectin (V_max/K_m = 90.4 ± 2.3 and 78.9 ± 6.6, respectively) was less efficient than that on amylose (V_max/K_m = 214 ± 23.2). RdA79 efficiently hydrolyzed both amylopectin and amylose (V_max/K_m = 260.6 ± 12.9 and 326.5 ± 9.4, respectively). RdA70 hydrolyzed starch and amylose at similar rates (V_max/K_m = 202.9 ± 5.5 and 215.9 ± 6.2, respectively), but amylopectin was a poor substrate (V_max/K_m = 124.2 ± 7.4). The overall results suggest that RdA70 and RdA79 appear to belong to a group of saccharifying isoamylases that breaks down long fragments of oligosaccharide chains produced by the hydrolytic action of RdA90. The simultaneous action of the three isoamylases on starch, aside from the high resistance of RdA90 to wheat amylase inhibitors, might allow R. dominica to feed and reproduce successfully on the wheat kernel.     

Effects of temperature on the activity and kinetics of the granulovirus infecting the potato tuber moth Phthorimaea operculella Zeller (Lepidoptera: Gelechiidae)

The granulovirus infecting the potato tuber moth (PoGV) is an important biocontrol agent, especially for managing the pest in rustic potato storerooms. For efficient propagation and use of baculoviruses in pest control strategies, information on the effects of temperature on virus multiplication and activity is crucial. The interaction between PoGV infection and incubation temperature on P. operculella was studied in laboratory bioassays by determining the survival, yield of virus-infected larvae, and the kinetics of virus in vivo increase. Bioassays for LC₅₀ determination by using the egg-dip method were repeated over a period of six years in controlled incubation chambers at six constant temperatures ranging from 16 to 28 °C. Additionally, at temperatures of 17 and 24 °C the kinetics of virus development and increase in larva were assessed in destructive time-series experiments. Three different virus concentrations were used for inoculation. Control mortality was significantly temperature-dependent and was well described by a second-order polynomial function, with lowest mortality at 25 °C (20%) and highest at 16 °C (>60%). LC₅₀ values and slopes of probit-mortality curves were not significantly different between temperatures. Numbers of virus-infected larvae increased exponentially with increasing log-concentration of virus inoculum; an effect of temperature was not evident. Virus granules per larva correlated highly with larval age and larval weight. Multiple regression revealed minor direct effects of temperature on virus numbers; however, with decreasing temperature, larval weight and hence virus numbers increased. As a result, temperature is an important factor to be considered in virus-production facilities. Rearing temperature in virus-production facilities should be maintained at temperatures around 24 °C.     

Hibernation and daily torpor in an armadillo, the pichi (Zaedyus pichiy)

Hibernation and daily torpor are physiological strategies to cope with energetic challenges that occur in many mammalian and avian taxa, but no reliable information exists about daily torpor or hibernation for any xenarthran. Our objective was to determine whether the pichi (Zaedyus pichiy), a small armadillo (Xenarthra, Dasypodidae) that inhabits arid and semi-arid habitats in central and southern Argentina and Chile, enters shallow daily torpor or prolonged deep hibernation during winter when environmental temperature and food availability are low. We studied body temperature changes during winter in semi-captive pichis by means of temperature dataloggers implanted subcutaneously. All individuals entered hibernation, characterized by torpor events of 75 ± 20 h during which the subcutaneous temperature (T_sc) decreased to 14.6 ± 2.1 °C. These events were interrupted by periods of euthermia of 44 ± 38 h with a T_sc of 29.1 ± 0.7 °C. After the hibernation season, daily torpor bouts of 4 to 6 h occurred irregularly, with T_sc dropping to as low as 24.5 °C. We conclude that the pichi is a true hibernator and can enter daily torpor outside of the hibernation season.     

Reduced insecticide susceptibility among populations of Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) in strawberry production

Chilli thrips, Scirtothrips dorsalis Hood (Thysanoptera: Thripidae) is a major, economically important, and recent invasive pest of strawberries and other horticultural crops in United States. Several conventional insecticides are used for S. dorsalis management, and resistance development threatens loss of few available tools. Hence, our objectives were to: (1) determine the susceptibility of S. dorsalis to commonly used conventional insecticides: spinetoram, acetamiprid, cyantraniliprole and bifenthrin, and (2) establish LC₅₀ and LC₉₀ dosages for spinetoram against S. dorsalis. Sampling of S. dorsalis populations was conducted twice in seven strawberry fields in Florida during the strawberry field season between 2019 and 2020. Leaf-disc bioassays were performed with field collected populations along with a susceptible 2-year-old laboratory culture of S. dorsalis. Overall, at highest recommended rate the percent mortality of late season S. dorsalis populations from five out of seven collection sites was lower (~41%) than average mortality observed with early season populations (~72%). Populations from at least four out of seven sampling sites exhibited significantly lower mortality than the laboratory susceptible culture in late season. The LC₅₀ and LC₉₀ values for spinetoram for the susceptible laboratory population were 0.026 and 8.64 ppm, respectively. On the other hand, LC₅₀ values of field collected populations to spinetoram varied with resistance ratios ranging from 6 to 269 fold as compared against the laboratory strain. Our results suggest that susceptibility of S. dorsalis to commonly used insecticides in strawberries varies significantly between early and late season populations within the same crop season. The efficacy of bifenthrin against S. dorsalis was particularly low (~ reduced to half in late season), especially among field collected populations. Our results indicate an urgent need to incorporate other pest management strategies, as well as effective rotation programs to reduce selection for resistance among populations of S. dorsalis in strawberry production.     

Acaricidal effects of <Emphasis Type="Italic">Corymbia citriodora</Emphasis> oil containing <Emphasis Type="Italic">para</Emphasis>-menthane-3,8-diol against nymphs of <Emphasis Type="Italic">Ixodes ricinus</Emphasis> (Acari: Ixodidae)

The toxicity of para-menthane-3,8-diol (PMD), the main arthropod-repellent compound in the oil of the lemon eucalyptus, Corymbia citriodora, was evaluated against nymphs of Ixodes ricinus using five methods (A–E) of a contact toxicity bioassay. Mortality rates were estimated by recording numbers of dead nymphs at 30 min intervals during the first 5 h after the start of exposure and at longer intervals thereafter. The mortality rate increased with increasing concentration of PMD and duration of exposure with a distinct effect after 3.5 h. From the results obtained by methods A, C and E, the LC₅₀ range was 0.035–0.037 mg PMD/cm² and the LC₉₅ range was 0.095–0.097 mg PMD/cm² at 4 h of exposure; the LT₅₀ range was 2.1–2.8 h and the LT₉₅ range was 3.9–4.2 h at 0.1 mg PMD/cm². To determine the duration of toxic activity of PMD, different concentrations (0.002, 0.01, 0.1 mg PMD/cm²) were tested and mortality was recorded at each concentration after 1 h; thereafter new ticks were tested. This test revealed that the lethal activity of PMD remained for 24 h but appeared absent after 48 h. The overall results show that PMD is toxic to nymphs of I. ricinus and may be useful for tick control.     

Characterization of phenoloxidase activity in Sydney rock oysters (Saccostrea glomerata)

Phenoloxidase (PO) activity was studied in Sydney rock oysters (Saccostrea glomerata). As in other molluscs, PO was found to exist as a pro-enzyme (proPO) in hemocytes. ProPO could be activated to PO by exogenous proteases (trypsin and chymotrypsin), exposure of hemocytes to pathogen-associated molecular patterns (PAMPs) and by the detergents, Triton X-100 and sodium dodecyl sulphate (SDS). Inhibition studies confirmed the proPO activating system of Sydney rock oysters is a proteinase cascade in which Ca²⁺ dependent serine proteinases proteolytically convert proPO into active PO. Activated PO was found to be a tyrosinase-like enzyme that is responsible for both monophenolase and diphenolase activity. The bifunctional PO had higher affinity for the monophenol, hydroquinine monomethyl ether (4HA) (K_m = 4.45 ± 1.46 mM) than for the diphenol, l-DOPA (K_m = 10.27 ± 1.33 mM). Maximum enzyme activity was evident at 37 °C, pH 8 and at salinities of between 30 and 37 ppt. Melanogenesis catalysed by the active enzyme is a composite of eumelanin and the product of a sclerotin pathway combining DOPA decarboxylase with PO activity.     

The development and multiple uses of a standardised bioassay method to select hypocrealean fungi for biological control of aphids

A technically standardised bioassay method was designed, evaluated and used to assess virulence and host range of hypocrealean fungi against aphids. A track mounted sprayer was used to apply conidia because hand held versions of the same sprayer can be used for field applications, thereby allowing the outcome from laboratory experiments to predict activity in the field accurately. Eighteen fungal isolates were assessed in single concentration bioassays against the black bean aphid Aphis fabae Scopoli. Isolates comprised commercially available mycoinsecticides (based on Beauveria bassiana and Lecanicillium longisporum) and isolates of B. bassiana, Lecanicillium spp., Paecilomyces fumosoroseus and Metarhizium anisopliae. Aphid mortality was in excess of 80% for 15 isolates, and HRI 1.72 (L. longipsorum), Z11 (P. fumosoroseus), Mycotech strain GHA (B. bassiana) and ARSEF 2879 (B. bassiana) were studied further. Multiple concentration bioassays identified HRI 1.72 as the most virulent isolate against A. fabae with significantly smaller LC₅₀ and LT₅₀ values compared to other isolates. A precise LC₅₀ value (2.95 × 10² conidia ml⁻¹) was calculated for HRI 1.72 using a second multiple concentration assay with smaller concentrations of conidia. The four isolates were applied at a single concentration (1 × 10⁸ conidia ml⁻¹) against Myzus persicae, A. fabae, Acyrthosiphon pisum, Metopolophium dirhodum, Sitobion avenae and Rhopalosiphum padi. A ranking of aphid susceptibility was obtained, such that S. avenae > M. persicae, A. pisum, A. fabae > R. padi. Results indicate the importance of standardising bioassay methods to reduce bioassay variability without compromising the ability to use the bioassay to investigate fungus–host interactions under varying abiotic and biotic conditions.     

Hymenopteran specificity of Bacillus thuringiensis strain PS86Q3

The biological activity of Bacillus thuringiensis (Bt) strain PS86Q3 against five Hymenopteran species was determined by means of bioassays adapted to each species. Four species of sawfly that are important pests of conifers (Diprion pini, Gilpinia hercyniae and Pristiphora abietina) or ornamental plants (Arge rosae), as well as the non-target honeybee, Apis mellifera, were studied. Two out of the four sawfly species tested were found to be sensitive to PS86Q3 crystals or spore/crystal suspensions. A sporulated culture of this strain was moderately active on D. pini, and a complete bioassay with solubilized crystals was performed to estimate the LC₅₀ of 4.9 mg/ml. Pristiphora abietina was also found to be sensitive to PS86Q3, with an LC₅₀ of 1.6 mg/ml. By contrast, at the concentrations tested, PS86Q3 did not prove active on the remaining sawflies, G. hercyniae and A. rosae. The strain was administered orally to check its effects on honeybees which were fed sucrose solutions supplemented with a PS86Q3 sporulated suspension, in a field assay using commercial beehives. No significant differences in larval mortality (as deduced by comparing the number of larvae, pupae and empty cells) were found between the Bt and control treatments. On the basis of the results presented here, the suitability of PS86Q3 for the control of Hymenopteran pests, particularly sawflies, in terms of both potency and environmental safety, is discussed.     

The relationship between tribuyltin (TBT) accumulation and toxicity to Hyalella azteca for use in identifying TBT toxicity in the field

The relationship between bioaccumulation and toxicity of tributyltin in Hyalella azteca was determined for future use in identifying freshwater sites of TBT induced toxicity in the field. Hyalella accumulated waterborne TBT rapidly, reaching equilibrium within 1 week. Short exposure times are, therefore, sufficient to measure TBT availability in laboratory or field exposures. Accumulation was not affected significantly by body size for animals between 0.1 and 0.6 mg dry weight, eliminating the need for a body size correction factor. Young amphipods were more sensitive to TBT than adults when both were exposed for 1 week. The 4-wk LC50 initiated with 0–1-week-old young was 4.8 nM. TBT accumulation was approximately proportional to TBT in water, and the 4-week LC50 expressed on a body concentration basis was 110 nmole/g dry weight. Accumulation of TBT by adult Hyalella to concentrations of about 100 nmole/g or more during short term (1-wk) exposures to environmental samples would suggest chronic TBT-induced toxicity is present at those sites.     

Febrile response to infection in the American alligator (Alligator mississippiensis)

Temperature probes were inserted into the stomachs of juvenile American alligators (Alligator mississippiensis) maintained outdoors at ambient fluctuating temperatures. Internal body temperatures (T_b) were measured every 15 min for two days, and then the alligators were injected with bacterial lipopolysaccharide (LPS), pyrogen-free saline, or left untreated. Alligators injected intraperitoneally with LPS exhibited maximum T_bs 2.6 ± 1.1 °C and 3.5 ± 1.2 °C higher than untreated control animals on days one and two after treatment, respectively. T_bs for these animals fell to within control ranges by day three postinjection. Similarly, mean preferred body temperatures (MPBTs) were significantly higher for LPS-injected alligators on days one (4.2 ± 1.8 °C) and two (3.5 ± 1.6 °C) after treatment. Intraperitoneal injection of heat-killed Aeromonas hydrophila, a gram-negative bacterium known to infect crocodilians, resulted in a fever while injection of Staphylococcus aureus (gram positive) did not elicit a febrile response. Injection of LPS in alligators maintained indoors in a constant temperature environment resulted in no increase in internal T_b. These results indicate that alligators did not exhibit a febrile response in the absence of a thermal gradient, and suggest that febrile responses observed are probably behavioral in nature.     

Histopathology in Pacific oyster (Crassostrea gigas) spat caused by the dinoflagellate Prorocentrum rhathymum

The recognition of an apparent association between seasonal oyster spat mortalities (up to 40%) and high Prorocentrum rhathymum density in the Little Swanport Estuary, Tasmania, prompted further experimental investigation into the toxicity by this dinoflagellate. Standard brine shrimp, haemolysis assays and intraperitoneal mouse bioassays revealed fast acting toxins in methanol but not aqueous extracts of P. rhathymum, with mice dying in less than 20 min. Oyster bioassays involved feeding spat (4 mm shell width) for 21 consecutive days on a diet of cultured P. rhathymum at simulated bloom densities (10⁴ cells ml⁻¹). No oyster mortality was observed, however, histopathological signs of thin, dilated gut tubules and sloughing of gut cells resembled those seen in affected field samples. In contrast to field samples, gill pathology was also observed in experimental exposure oysters.     

Ontogenetic differentiation of swimming performance in Gilthead seabream (Sparus aurata, Linnaeus 1758) during metamorphosis

The critical swimming speed (U_crit) of gilthead seabream (Sparus aurata, Linnaeus 1875) was studied in two ontogenetic phases, early (13.7-18.7 mm total length, TL) and late metamorphosis (20.4-34.3 mm TL, after the full development of fin meristics and during squamation ontogeny), under four exercise temperatures (15, 20, 25 and 28 °C). Both the exercise temperature and the ontogenetic stage had a significant effect on the relative U_crit (RU_crit) of S. aurata, with the fish of early metamorphosis phase (E group) presenting significantly higher RU_crit than those of the late metamorphosis stage (L group). This ontogenetic shift in swimming performance was accompanied by significant ontogenetic shifts of body shape and of muscle anatomy. Compared to the L group, S. aurata of the E group were characterized by a streamline body shape and significantly higher relative contribution of the slow-red muscle to the cross-sectional area of the body (31.0 ± 1.3% vs 12.0 ± 1.2% in the L group).     

An autecological study of the potentially toxic dinoflagellate Alexandrium affine isolated from Vietnamese waters

The potentially toxic dinoflagellate species Alexandrium affine isolated from Ha Long Bay (Tonkin Gulf), Vietnam was cultured and maintained for morphological, physiological and toxicological studies. Classical morphological examinations including plate pattern were in good agreement with the international nomenclature of the species. The fine structure of A. affine, including morphology of its developmental stages during vegetative and sexual reproduction was found to be typical of other species in the genus. Two general trends in growth of A. Affine from Vietnamese waters were apparent: (1) growth rates were low at low salinities (10 and 15 psu) in all experimental temperatures (21–27 °C); (2) growth rates were high at salinities 25, 30, and 35 psu in all temperatures. There were no significant differences in growth rates at different salinities at low temperature (21 °C), and the most significant difference in growth rate was between high temperature–high salinity and high temperature–low salinity. The optimum temperature and salinity for growth were 24 °C and 30 psu. Maximum division rates per day (0.5–0.7) were at salinities 30 and 35 psu and at temperatures 24 and 27 °C. But the best conditions for division rate were 21 and 24 °C at salinities 30 and 35 psu. Toxicity analyses indicated A. affine to be both toxic and non-toxic at certain times. In the former case, toxicity was very low, 2.28 fmol  per cell; the toxicity component of A. affine was compared with that of A. leei and the mussel Perna viridis including neoSTX, STX, and GTX₁–GTX₄.     

Comparative ultrastructure of spermatozoa from two regular and two irregular New Zealand echinoids

Spermatozoa from four species of echinoids found in New Zealand had morphological characteristics typical of other echinoids, including a conical sperm head with an acrosome‐capped nucleus, a midpiece, and a single long flagellum. The spermatozoa of Fellaster zelandiae, Echinocardium cordatum, Evechinus chloroticus, and Centrostephanus rodgersii also showed statistically significant differences in species‐specific morphological characteristics. Evechinus chloroticus showed the most variable sperm morphology. The irregular urchins (F. zelandiae and E. cordatum) had short, wide sperm heads (head length:width ratios 2.93:1 & 2.97:1, respectively) with a long acrosome complex, while the regular urchins (E. chloroticus and C. rodgersii) had longer, narrower heads with a short acrosome complex (ratios 5.29:1 & 3.37:1). Spermatozoa of E. cordatum from the New Zealand population shared more characteristics with those of conspecifics from the Sea of Japan than those of conspecifics from the Baltic, reflecting the membership of the former two populations in a distinct Pacific clade. Volumetric calculations showed no evidence of phylogenetic grouping. Mitochondria of E. chloroticus spermatozoa were less than half the volume of those of C. rodgersii and E. cordatum, and those of F. zelandiae were intermediate in volume. These volume measurements will be useful in physiological studies of sperm performance and quality.     

Diet and physiological responses of Spondyliosoma cantharus (Linnaeus, 1758) to the Caulerpa racemosa var. cylindracea invasion

Marine invasions are a worldwide problem that involves changes in communities and the acclimation of organisms to them. The invasive Chlorophyte Caulerpa racemosa var. cylindracea is widespread in the Mediterranean and colonises large areas from 0 to 70 m in depth. The omnivorous fish Spondyliosoma cantharus presents a high frequency of occurrence of C. racemosa in the stomach contents at invaded areas (76.3%) while no presence of C. racemosa was detected in control areas. The isotopic composition of muscle differed significantly between invaded and non-invaded sites for δ¹³C (− 16.67‰ ± 0.09 and − 17.67‰ ± 0.08, respectively), δ¹⁵N (10.22‰ ± 0.22 and 9.32‰ ± 0.18, respectively) and the C:N ratio (2.01 ± 0.0002 and 1.96 ± 0.009, respectively). Despite the high frequency of occurrence of C. racemosa in the stomach contents of S. cantharus and its important contribution to the δ¹³C source (20.7% ± 16.2), the contribution of C. racemosa to the δ¹⁵N in S. cantharus food sources was very low (6.6% ± 5.8). Other invertebrate prey such as decapods and polychaetes were more important contributors to the δ¹⁵N source at both invaded and non-invaded sites. Activation of enzymatic pathways (catalase, superoxide dismutase, glutathione-s-tranferase, 7-ethoxy resorufin O-de-ethylase) but not a significant increase in lipid peroxidation MDA (0.49 ± 0.01 nmol/mg prot at non-invaded and 0.53 ± 0.01 nmol/mg prot at invaded sites) was observed in S. cantharus individuals living in C. racemosa-invaded sites compared with control specimens. The low δ¹⁵N contribution values of C. racemosa by S. cantharus together with the toxicity demonstrated by the activation of the antioxidant defences and the important contribution of invertebrate prey to the δ¹⁵N could mean that the ingestion of C. racemosa by S. cantharus might be unintentional during the predation of invertebrate preys living underneath the entanglement of the C. racemosa fronds and stolons mats.     

Neuronal membrane potential is mildly depolarized in the anoxic turtle cortex

Neuronal membrane potential (E_m) regulates the activity of excitatory voltage-sensitive channels. Anoxic insults lead to a severe loss of E_m and excitotoxic cell death (ECD) in mammalian neurons. Conversely, anoxia-tolerant freshwater turtle neurons depress energy usage during anoxia by altering ionic conductance to reduce neuronal excitability and ECD is avoided. This wholesale alteration of ion channel and pump activity likely has a significant effect on E_m. Using the whole-cell patch clamp technique we recorded changes in E_m from turtle cortical neurons during a normoxic to anoxic transition in the presence of various ion channel/pump modulators. E_m did not change with normoxic perfusion but underwent a reversible, mild depolarization of 8.1 ± 0.2 mV following anoxic perfusion. This mild anoxic depolarization (MAD) was not prevented by the manipulation of any single ionic conductance, but was partially reduced by pre-treatment with antagonists of GABA_A receptors (5.7 ± 0.5 mV), cellular bicarbonate production (5.3 ± 0.2 mV) or K⁺ channels (6.0 ± 0.2 mV), or by perfusion of reactive oxygen species scavengers (5.2 ± 0.3 mV). Furthermore, all of these treatments induced depolarization in normoxic neurons. Together these data suggest that the MAD may be due to the summation of numerous altered ion conductance states during anoxia.     

Growth and protein synthesis of barramundi, Lates calcarifer, fed lupin as a partial protein replacement

Protein synthesis is an essential growth process in all animals. Little information is available on post-prandial protein synthesis and even less where different protein sources are compared. Protein synthesis was measured at 4 and 24 h after feeding juvenile barramundi in order to determine the effect of using lupin as a partial protein replacement for fish meal on the post-prandial protein metabolism. Juvenile barramundi (4.3 ±0.6 g) were held in a recirculation system (27 °C, salinity 10‰ and 24 h light) for 15 days. Fish were fed one of two isonitrogenous isoenergetic diets (40% crude protein, 16% lipid and 18.5 GE MJ kg^− 1). One diet was formulated with 100% fish meal as the protein source while the other had 45% of the protein replaced with lupin ingredients (lupin kernel meal (Lupinus angustifolius) and lupin protein concentrate). All fish were fed a ration of 6%·d^− 1 and feed intake was not significantly different between the two diets. Specific growth rate (SGR) and growth efficiency (in relation to protein (PPV) and energy (PEV)) were 6.5 ± 0.14%·d^− 1, 43.8 ± 2.72% and 38.31 ± 1.56%, respectively, and were not significantly different between the two diets. There was no significant difference in protein synthesis between the two diets at 4 and 24 h after feeding, however protein synthesis was significantly higher 4 h after feeding than at 24 h (p = 0.02). Neither growth performance nor protein metabolism was altered by replacing 45% of the protein with lupin protein and indicated this to be a suitable protein source for barramundi feeds.     

Virulence of a South African isolate of the <Emphasis Type="Italic">Cryptophlebia leucotreta</Emphasis> granulovirus to <Emphasis Type="Italic">Thaumatotibia leucotreta</Emphasis> neonate larvae

Surface inoculation dose–response and time–response bioassays and detached fruit bioassays were conducted with a novel South African isolate of the Cryptophlebia leucotreta granulovirus (CrleGV-SA) against Thaumatotibia leucotreta (Meyrick) (Lepidoptera: Noctuidae) neonate larvae. LC₅₀ and LC₉₀ values were estimated to be 4.095 × 10³ and 1.185 × 10⁵ OBs ml⁻¹, respectively. LT₅₀ and LT₉₀ values were estimated to be 4 days 22 h and 7 days 8 h, respectively, categorising the virus as a fast or type 2 granulovirus. There was a conspicuous difference in behaviour between larvae on inoculated diet and untreated diet, resulting in a significant reduction in penetration of diet. Bioassays on detached Navel oranges revealed LC₅₀ and LC₉₀ values of 9.310 × 10⁷ and 1.515 × 10⁹ OBs ml⁻¹, when using data on numbers of larvae per fruit rather than on numbers of infested fruit. Field trials will be conducted.     

Benefits to host sea anemones from ammonia contributions of resident anemonefish

Large ectosymbionts (especially fishes and crustaceans) may have major impacts on the physiology of host cnidarians (sea anemones and corals), but these effects have not been well quantified. Here we describe impacts on giant sea anemone hosts (Entacmaea quadricolor) and their endosymbiotic zooxanthellae (Symbiodinium spp.) from the excretion products of anemonefish guests (Amphiprion bicinctus) under laboratory conditions. Starved host anemones were maintained with anemonefish, ammonia supplements (= NH₃ gas and NH₄⁺ ion), or neither for 2 mo. In the presence of external ammonia supplements or resident anemonefish, the zooxanthellae within host anemones increased in abundance (173% and 139% respectively), and provided the hosts with energy that minimized host body size loss. In contrast, anemones cultured with neither ammonia nor anemonefish harbored significantly lower abundances of zooxanthellae (84% of initial abundance) and decreased > 60% in body size. Although they maintained higher zooxanthella abundances, anemones cultured with either ammonia supplements or resident anemonefish exhibited significantly lower ammonia uptake rates (0.065 ± 0.005 µmol g^- 1 h^- 1, and 0.052 ± 0.018 µmol g^- 1 h^- 1 respectively) than did control anemones (0.119 ± 0.009 µmol g^- 1 h^- 1), indicating that their zooxanthellae were more nitrogen sufficient. We conclude that, in this multi-level mutualism, ammonia supplements provide essentially the same level of physiological contribution to host anemones and zooxanthellae as do live resident fish. This nutrient supplement reduces the dependence of the zooxanthellae on host feeding, and allows them to provide abundant photosynthetically-produced energy to the host.