Three rarely reported digeneans inhabiting amphibians from Vancouver Island, British Columbia, Canada

Three rarely reported species of digeneans were collected in amphibian hosts from Diversion Reservoir, Sooke, Vancouver Island, British Columbia, Canada. The endangered anuran Rana aurora hosted Glypthelmins californiensis, Gorgoderina multilobata, and Megalodiscus microphagus. In addition, the anuran Pseudacris regilla and the caudatan Taricha granulosa hosted Megalodiscus microphagus. Gorgoderina multilobata has been reported only once since 1936 and has never been reported outside of California. We note several characters not previously reported for Gorgoderina multilobata and G. aurora.     

Effects of nitrate on the interactions of the tadpoles of two ranids (Rana clamitans and R. catesbeiana)

Nitrogen pollution as a result of agricultural runoff and atmospheric deposition is a major challenge to aquatic ecosystems, and is likely to increase in the future. Nitrogenous pollutants are potential stressors of amphibian larvae through their toxicological impacts on individuals; however, they may also increase primary productivity. We examined how such an anthropogenic stressor could influence the interactions between two potentially competing species of tadpoles (Rana clamitans and R. catesbeiana). In a 42 d mesocosm experiment, R. catesbeiana survival, but not final mass, was reduced by nitrate addition. Rana catesbeiana survival was lower when in competition with R. clamitans than when only experiencing intraspecific competition. For R. clamitans, survival was not affected by nitrate addition or competition type. Rana clamitans in nitrate addition mesocosms were heavier than tadpoles from no nitrate mesocosms, and were heavier in intraspecific than in interspecific mesocosms. Our results suggest that nitrate can influence the performance of amphibian larvae, and that its effects could have complex effects on aquatic ecosystems.     

Survival of three species of anuran metamorphs exposed to UV-B radiation and the pathogenic fungus Batrachochytrium dendrobatidis

When exploring the possible factors contributing to population declines, it is necessary to consider multiple, interacting environmental stressors. Here, we investigate the impact of 2 factors, ultraviolet radiation and disease, on the survival of anuran amphibians. Exposure to ultraviolet-B (UV-B) radiation increases mortality and results in various sub-lethal effects for many amphibian species. Infectious diseases can also negatively impact amphibian populations. In this study, we exposed metamorphic individuals (metamorphs) to both UV-B and Batrachochytrium dendrobatidis (BD), a fungal pathogen and cause of the disease chytridiomycosis, and monitored survival for 3 wk. We tested for possible interactions between UV-B and BD in 3 species: the Cascades frog Rana cascadae; the Western toad Bufo boreas; and the Pacific treefrog Hyla regilla. We found strong interspecific differences in susceptibility to BD. For example, R. cascadae suffered a large increase in mortality when exposed to BD; B. boreas also experienced mortality, but this effect was small relative to the R. cascadae response. H. regilla did not show any decrease in survival when exposed to either factor. No synergistic interactions between UV-B and BD were found for any of the test species. A previous study investigating the impact of BD on larval amphibians showed different species responses (Blaustein et al. 2005a). Our results highlight the importance of studying multiple life history stages when determining the impact of environmental stressors. The contrast between these 2 studies emphasizes how vulnerability to a pathogen can vary between life history stages within a single species.     

Genetic comparison of water molds from embryos of amphibians Rana cascadae, Bufo boreas and Pseudacris regilla

Water molds that cause the disease saprolegniasis have been implicated in widespread mortality of amphibian embryos. However, because of the limitations of traditional identification methods, water mold species involved in die-offs or utilized in ecological studies often remain unidentified or identified only as Saprolegnia ferax. Furthermore, water mold taxonomy requires revision, so very distinct organisms may all be called S. ferax. Recent DNA-based studies indicate that the diversity of water molds infecting amphibian embryos is significantly higher than what was previously known, but these studies rely on culture methods, which may be biased towards taxa that grow best under laboratory conditions. In this study, total embryo-associated DNA was extracted from 3 amphibian species in a pond in central Washington, USA. The internal transcribed spacer (ITS) region of DNA was amplified with primers capable of amplifying a broad array of eukaryotic microorgansisms, and was used to construct clone libraries. Individual clones were sequenced and relationships among newly recovered sequences and previously studied taxa were analyzed using phylogenetics. These methods recovered several new taxa in association with amphibian embryos. Samples grouped into 11 distinct phylotypes with ITS sequence differences ranging from 4 to 28%. The water mold communities recovered differed among Rana cascadae, Bufo boreas, and Pseudacris regilla egg masses. Furthermore, the diversity of water molds increased as egg masses aged, and members comprising this diversity changed over time.     

Saprolegniaceae identified on amphibian eggs throughout the Pacific Northwest, USA, by internal transcribed spacer sequences and phylogenetic analysis

We assessed the diversity and phylogeny of Saprolegniaceae on amphibian eggs from the Pacific Northwest, with particular focus on Saprolegnia ferax, a species implicated in high egg mortality. We identified isolates from eggs of six amphibians with the internal transcribed spacer (ITS) and 5.8S gene regions and BLAST of the GenBank database. We identified 68 sequences as Saprolegniaceae and 43 sequences as true fungi from at least nine genera. Our phylogenetic analysis of the Saprolegniaceae included isolates within the genera Saprolegnia, Achlya and Leptolegnia. Our phylogeny grouped S. semihypogyna with Achlya rather than with the Saprolegnia reference sequences. We found only one isolate that grouped closely with S. ferax, and this came from a hatchery-raised salmon (Idaho) that we sampled opportunistically. We had representatives of 7-12 species and three genera of Saprolegniaceae on our amphibian eggs. Further work on the ecological roles of different species of Saprolegniaceae is needed to clarify their potential importance in amphibian egg mortality and potential links to population declines.     

An examination of multiple factors affecting community structure in an aquatic amphibian community

Summary The potential effects of multiple factors structuring certain larval amphibian communities were studied using a pen experiment in a natural pond. Potential factors (predation and competition from other species) were allowed to act in a stepwise fashion such that their relative importance could be evaluated. Based on a previous study, it was hypothesized that predation by Ambystoma salamander larvae on other larval amphibian species would be the most important factor. Survival of Ambystoma jeffersonianum salamander larvae and Rana sylvatica tadpoles was significantly depressed only by Ambystoma opacum predation. Survival of Ambystoma maculatum salamander larvae was significantly greater in the absence of both A. opacum and A. jeffersonianum predators. The virtual elimination of Hyla chrysoscelis larvae in all treatments also can be largely attributed to Ambystoma predation. Thus, Ambystoma predation was the dominant factor determining larval survival of four amphibian prey species in the experimental communities.     

环境因子对水霉菌生物膜形成的影响

【目的】体外构建水霉菌(Saprolegnia)生物膜(Biofilm,BF),研究环境因子对其生物膜形成的影响。【方法】采用改良的微孔板法研究静置培养条件下寄生水霉(Saprolegnia parasitica)ATCC200013在96孔酶标板上的成膜情况,CCK-8法(Cell Counting Kit-8)定量检测生物膜中水霉菌的活力。【结果】水霉菌的生物膜的OD450值在培养24 h达到峰值,48 h后趋于稳定。随着初始孢子浓度升高,水霉菌生物膜OD450值升高,差异显著(P0.05)。20-25°C生物膜形成量最多,OD450值显著高于其他温度组(P0.05)。在起始pH值为4-11的沙氏葡萄糖液体培养基中,水霉菌均能形成生物膜。在培养基中加入0.12 mmol/L以上CaCl2,能促进生物膜形成;添加0.03-2.00 mmol/L MgCl2,水霉菌生物膜形成量与未添加MgCl2对照组无显著性差异;Cu2+对水霉菌生物膜的形成有显著影响,0.5 mmol/L以上添加处理几乎不形成生物膜;NaCl能明显抑制水霉菌生物膜的形成,当NaCl质量分数低于0.12%时,对生物膜形成的影响较小(P0.05)。水霉菌在鲫皮和肌肉提取液包被后生物膜形成量与对照组相比无显著性差异,而鲫表皮黏液、鳃黏液包被后生物膜的形成量明显减少。【结论】研究首次采用微孔板法体外构建水霉菌生物膜,发现其生物膜的形成与多种环境因素有着密切的关系。这为了解水霉菌生物膜的形成规律提供了一定参考,为水霉菌生物膜的进一步研究奠定了基础。     

Synergistic effects of the invasive Chinese tallow (Triadica sebifera) and climate change on aquatic amphibian survival

Changes in climate and the introduction of invasive species are two major stressors to amphibians, although little is known about the interaction between these two factors with regard to impacts on amphibians. We focused our study on an invasive tree species, the Chinese tallow (Triadica sebifera), that annually sheds its leaves and produces leaf litter that is known to negatively impact aquatic amphibian survival. The purpose of our research was to determine whether the timing of leaf fall from Chinese tallow and the timing of amphibian breeding (determined by weather) influence survival of amphibian larvae. We simulated a range of winter weather scenarios, ranging from cold to warm, by altering the relative timing of when leaf litter and amphibian larvae were introduced into aquatic mesocosms. Our results indicate that amphibian larvae survival was greatly affected by the length of time Chinese tallow leaf litter decomposes in water prior to the introduction of the larvae. Larvae in treatments simulating warm winters (early amphibian breeding) were introduced to the mesocosms early in the aquatic decomposition process of the leaf litter and had significantly lower survival compared with cold winters (late amphibian breeding), likely due to significantly lower dissolved oxygen levels. Shifts to earlier breeding phenology, linked to warming climate, have already been observed in many amphibian taxa, and with most climate models predicting a significant warming trend over the next century, the trend toward earlier breeding should continue if not increase. Our results strongly suggest that a warming climate can interact with the effects of invasive plant species, in ways we have not previously considered, to reduce the survival of an already declining group of organisms.     

Experimental examination of the effects of ultraviolet-B radiation in combination with other stressors on frog larvae

Ultraviolet-B radiation (UVB) is a ubiquitous stressor with negative effects on many aquatic organisms. In amphibians, ambient levels of UVB can result in impaired growth, slowed development, malformations, altered behavior and mortality. UVB can also interact with other environmental stressors to amplify these negative effects on individuals. In outdoor mesocosm and laboratory experiments we studied potential synergistic effects of UVB, a pathogenic fungus, Batrachochytrium dendrobatidis (Bd), and varying temperatures on larval Cascades frogs (Rana cascadae). First, we compared survivorship, growth and development in two mesocosm experiments with UVB- and Bd-exposure treatments. We then investigated the effects of UVB on larvae in the laboratory under two temperature regimes, monitoring survival and behavior. We found reduced survival of R. cascadae larvae with exposure to UVB radiation in all experiments. In the mesocosm experiments, growth and development were not affected in either treatment, and no effect of Bd was found. In the laboratory experiment, larvae exposed to UVB demonstrated decreased activity levels. We also found a trend towards reduced survival when UVB and cold temperatures were combined. Our results show that amphibian larvae can suffer both lethal and sublethal effects when exposed to UVB radiation.     

Differences in susceptibility to Saprolegnia infections among embryonic stages of two anuran species

Many amphibians are known to suffer embryonic die-offs as a consequence of Saprolegnia infections; however, little is known about the action mechanisms of Saprolegnia and the host-pathogen relationships. In this study, we have isolated and characterized the species of Saprolegnia responsible for infections of embryos of natterjack toad (Bufo calamita) and Western spadefoot toad (Pelobates cultripes) in mountainous areas of Central Spain. We also assessed the influence of the developmental stage within the embryonic period on the susceptibility to the Saprolegnia species identified. Only one strain of Saprolegnia was isolated from B. calamita and identified as S. diclina. For P. cultripes, both S. diclina and S. ferax were identified. Healthy embryos of both amphibian species suffered increased mortality rates when exposed to the Saprolegnia strains isolated from individuals of the same population. Embryonic developmental stage was crucial in determining the sensitivity of embryos to Saprolegnia infection. The mortalities of P. cultripes and B. calamita embryos exposed at Gosner stages 15 (rotation) and 19 (heart beating) were almost total 72 h after challenge with Saprolegnia, while those exposed at stage 12 (late gastrula) showed no significant effects at that time. This is the first study to demonstrate the role of embryonic development on the sensitivity of amphibians to Saprolegnia.     

Predation risk and competition effects on the life-history characteristics of larval Oregon spotted frog and larval red-legged frog

We conducted an artificial pond experiment to test hypotheses about the effects of competition and non-lethal predator cues on metamorphic characteristics of sympatric Oregon spotted frogs (Rana pretiosa) and red-legged frogs (Rana aurora) in southwestern British Columbia. Tadpoles were exposed to the presence or absence of one another, two density levels and to the presence or absence of predacious odonate larvae (Aeshna palmata) isolated in enclosures. In the artificial pond study, R. aurora were significantly larger at metamorphosis (12%) and exhibited only slightly longer larval periods when exposed to Aeshna. In the presence of R. pretiosa, they significantly decreased time to metamorphosis, and were significantly larger at metamorphosis (12%) than those reared alone. Rana pretiosa in treatments with R. aurora were somewhat larger at metamorphosis when a non-lethal predator was present, and in treatments where R. pretiosa were alone with a predator tadpole mass at metamorphosis was smaller than those in the absence of Aeshna, but these results were not statistically significant. Both species reduced activity and moved away from the predator in the presence of an enclosed dragonfly larva in the laboratory. Most tadpole mesocosm experiments have found that the trade-off between size and timing of metamorphosis is extremely important to amphibians, but we suggest that the trade-off discussed in traditional amphibian models may not apply to species like R. pretiosa that are exposed to the same gape-limited predators upon reaching metamorphosis.     

Combined Effects of Atrazine and Chlorpyrifos on Susceptibility of the Tiger Salamander to Ambystoma tigrinum Virus

Several hypotheses have been examined as potential causes of global amphibian declines, including emerging infectious diseases and environmental contaminants. Although these factors are typically studied separately, animals are generally exposed to both stressors simultaneously. We examined the effects of the herbicide atrazine and the insecticide chlorpyrifos on the susceptibility of tiger salamander larvae, Ambystoma tigrinum, to a viral pathogen, Ambystoma tigrinum virus (ATV). Environmentally relevant concentrations of atrazine (0, 20, 200 μg/L) and chlorpyrifos (0, 2, 20, 200 μg/L) were used along with ATV in a fully factorial experimental design whereby individually housed, 4-week-old larvae were exposed for 2 weeks. Atrazine alone was not lethal to larvae, and chlorpyrifos alone was lethal only at the highest concentration. When combined with ATV, chlorpyrifos increased susceptibility to viral infection and resulted in increased larval mortality. A significant interactive effect between atrazine and ATV was detected. Atrazine treatments slightly decreased survival in virus-exposed treatments, yet slightly increased survival in the virus-free treatments. These findings corroborate earlier research on the impacts of atrazine, in particular, on disease susceptibility, but exhibit greater effects (i.e., reduced survival) when younger larvae were examined. This study is the first of its kind to demonstrate decreases in amphibian survival with the combination of pesticide and a viral disease. Further examination of these multiple stressors can provide key insights into potential significance of environmental cofactors, such as pesticides, in disease dynamics.     

FACULTATIVE PARASITISM OF SPIROGYRA SP. (CHLOROPHYTA) BY SAPROLEGNIA ASTEROPHORA AND PYTHIUM GRACILE (EUMYCOTA,OOMYCETES)1

Spirogyra sp. Link was found to be parasitized by filamentous fungi tentatively identified as Saprolegnia asterophora de Bary and Pythium gracile Schenk, in field samples and when maintained in unaltered pond water in an 18 h fluorescent light–6 h dark regime at 18 ± 2°C. Collections were made periodically between March 1978, and November 1979, from a pond in Mill Seek Sanctuary near Oyster Bay, Nassau Co., Long Island, New York. Initially, less than 1% of the total field population of Spirogyra sp. was infected by either fungal parasite with Saprolegnia asterophora being the dominant parasite present generally alone in most samples or present in 80–95% of the total number of infected algal filaments when occurring concurrently with P. gracile. However, in the laboratory, approximately 100% of the Spirogyra sp. filaments in any given sample became infected by Saprolegnia asterophora and/or P. gracile within a 1—2 wk and 3–4 wk period, respectively, with vegetative hyphae involved in the spread of infection to neighboring algal filaments. Infection of algal filaments occurred at random points with cell to cell hyphal extension within the filament causing disruption of host cells. Development of S. asterophora, and possibly P. gracile, sexual reproductive structures was common in relation with the host with asexual sporangial production not observed. Saprolegnia asterophora and P. gracile were cultured on glucose, yeast extract, malt extract (GYM) medium from infected Spirogyra sp. filaments, with infection of healthy algal filaments using these cultures by Saprolegnia asterophora, but not by P. gracile, induced in the laboratory. Growth responses and tropic responses of the fungi to the algal host could not be demonstrated.     

Effects of the mineral composition and water content of intact plants on the fitness of the African armyworm

The effects of organic nitrogen, nitrate, phosphorus, potassium and water content of leaves of intact maize plants, grown in a gravel culture system, on the fitness of the African armyworm, Spodoptera exempta (Walker)(Lepidoptera: Noctuidae) were studied. Organic nitrogen concentrations ranged from 1.3% to 3.7% over four treatments differing only in nitrate supply to the plants. Water content and other mineral levels were all positively correlated with the organic nitrogen level. Feeding damage by the caterpillars was most severe on the lowest nitrate treatments, where it could be least well compensated for by new leaf growth. Larval and pupal fitness variables were not affected by treatment, except for larval development on the lowest nitrate treatment which was delayed by just 1 day. The large compensatory capacity of the larvae was underlined by a similar mineral composition of the pupae in all treatments. Adult fitness variables hardly differed between the upper three nitrate treatments, but revealed a trend over all treatments: the higher the organic nitrogen content of the leaves, the shorter the pre-oviposition period and the higher the fecundity. This trend, however, might have been due to differences in available food quantity rather than in food quality. It is concluded that fitness of the African armyworm is only slightly affected over a wide range of nitrogen concentrations in its food. Though effects might be larger under field conditions, the large differences in outbreak development between years seem not to be attributable to observed differences in nitrogen levels in host plants between years in primary outbreak areas. Other environmental factors appear to be of greater importance.     

Amphibian survival,growth and development in response to mineral nitrogen exposure and predator cues in the field: an experimental approach

Mineral nitrogen (N) has been suggested as a potential factor causing declines in amphibian populations, especially in agricultural landscapes; however, there is a question as to whether it remains in the water column long enough to be toxic. We explored the hypothesis that mineral N can cause both lethal and sublethal toxic effects in amphibian embryos and larvae in a manipulative field experiment. We sampled 12 ponds, fertilizing half with ammonium nitrate fertilizer early in the spring, and measured hatching, survival, development, growth, and the incidence of deformities in native populations of wood frog (Rana sylvatica) and eastern tiger salamander (Ambystoma tigrinum tigrinum) embryos and larvae held in in situ enclosures. We found that higher ammonium concentrations negatively affect R. sylvatica more strongly than A. tigrinum. R. sylvatica tended to have lower survival as embryos and young tadpoles, slowed embryonic development, and an increased proportion of hatchlings with deformities at experimentally elevated ammonium. A. tigrinum did not experience significantly reduced survival, but their larval development was slowed in response to elevated ammonium and the abundance of large invertebrate predators. Variable species susceptibility, such as that shown by R sylvatica and A. tigrinum, could have large indirect effects on aquatic community structure through modification of competitive or predator-prey relationships. Ammonium and nitrate + nitrite concentrations were not correlated with other measures that might have affected amphibians, such as pH, pond area, depth, or vegetation. Our results highlight the potential importance of elevated ammonium on the growth, development and survival of amphibians, especially those that breed in surface waters receiving anthropogenic N inputs. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A reservoir species for the emerging Amphibian pathogen Batrachochytrium dendrobatidis thrives in a landscape decimated by disease

Chytridiomycosis, a disease caused by the fungal pathogen Batrachochytrium dendrobatidis (Bd), is driving amphibian declines and extinctions in protected areas globally. The introduction of invasive reservoir species has been implicated in the spread of Bd but does not explain the appearance of the pathogen in remote protected areas. In the high elevation (>1500 m) Sierra Nevada of California, the native Pacific chorus frog, Pseudacris regilla, appears unaffected by chytridiomycosis while sympatric species experience catastrophic declines. We investigated whether P. regilla is a reservoir of Bd by comparing habitat occupancy before and after a major Bd outbreak and measuring infection in P. regilla in the field, monitoring susceptibility of P. regilla to Bd in the laboratory, examining tissues with histology to determine patterns of infection, and using an innovative soak technique to determine individual output of Bd zoospores in water. Pseudacris regilla persists at 100% of sites where a sympatric species has been extirpated from 72% in synchrony with a wave of Bd. In the laboratory, P. regilla carried loads of Bd as much as an order of magnitude higher than loads found lethal to sympatric species. Histology shows heavy Bd infection in patchy areas next to normal skin, a possible mechanism for tolerance. The soak technique was 77.8% effective at detecting Bd in water and showed an average output of 68 zoospores per minute per individual. The results of this study suggest P. regilla should act as a Bd reservoir and provide evidence of a tolerance mechanism in a reservoir species.     

Effects of Ultraviolet Radiation on Locomotion and Orientation in Roughskin Newts (Taricha granulosa)

Environmental changes, including those associated with the atmosphere may significantly affect individual animals and ultimately populations. Ultraviolet (UV) radiation, perhaps increasing due to stratospheric ozone depletion, has been linked to mortality in a number of organisms, including amphibians. The eggs and larvae of certain amphibian species hatch at significantly lower rates when exposed to ambient ultraviolet light. Yet little is known about the sublethal effects of UV radiation. For example, UV radiation may affect specific behaviors of an animal that could alter its ability to survive. To examine if UV radiation affects amphibian behavior, we used roughskin newts ( Taricha granulosa ) as a model. Newts were exposed to low-level doses of UV in the laboratory and then tested in the field to examine if UV-exposed and control (no UV) newts differed in orientation towards water or in locomotor activity levels. UV-exposed and control newts both exhibited a significant orientation towards water in field tests but there was no significant difference in orientation between treatments. However, UV-exposed newts were significantly more active than control newts. Our results suggest that exposure to short-term low levels of UV radiation alters certain behaviors. Environmentally induced changes in behavior may have significant ecological and evolutionary consequences.     

Laboratory evaluation of entomopathogenic fungi against larvae and adults of onion maggot (Diptera: Anthomyiidae)

Laboratory experiments were done to measure the susceptibility of larvae and adults of the onion maggot, Delia antiqua (Meigen) (Diptera: Muscidae: Anthomyiidae) to 27 isolates of entomopathogenic fungi from four genera [Beauveria Vuillemin, Lecanicillium (Petch) Zare & W. Gams, Metarhizium Sorokin, and Paecilomyces Bainier]. A novel bioassay was developed for D. antiqua larvae by using a diet based on mixed vegetable powder. When evaluated in a virulence screen, the fungal isolates caused less mortality of D. antiqua larvae than adults. Only three isolates caused > 50% mortality of larvae, whereas 12 isolates caused > 50% mortality of adults. Fungal species was a statistically significant factor affecting the mortality of larvae but not of adults. The fungal isolates causing the most mortality of larvae tended to belong to Metarhizium anisopliae (Metschnikoff) Sorokin. Two M. anisopliae isolates (389.93 and 392.93) were evaluated in dose-response bioassays. The median lethal concentrations of the isolates against larvae were 6.1 x 10(7) conidia ml(-1) for isolate 389.93 and 7.6 x 10(7) conidia ml(-1) for isolate 392.93. The emergence of adult flies from pupae was reduced at high concentrations of conidia (3.0 x 10(8) and 1.0 x 10(8) conidia ml(-1)). The median lethal concentrations of the isolates against adults were 1.7 x 10(7) and 4.0 x 10(7) conidia ml(-1), respectively. Some of the fungal isolates examined may have potential as biological control agents of larvae of D. antiqua and related species.     

Rearing Costelytra zealandica (Coleoptera: Scarabaeidae)

Interaction between larvae can be a cause of mortality when scarabaeid larvae are concentrated in a confined volume of soil. Larvae of Costelytra zealandica (White), the New Zealand grass grub, were held for 30 days at population densities between 1 and 50 larvae per 200 ml of medium, and the effect of crowding on their survival and weight change was assessed. Larval density had no effect on survival in soil plus chopped sheep dung (3 :1), with up to 10 2nd‐instar larvae or 5 3rd‐instar larvae per 200 ml of medium. Larval survival and weight gain were increased by adding germinating ryegrass seed (Grasslands Ruanui) to the medium base, but the effect of density did not diminish. Weight gain of 3rd‐instar larvae decreased as their density increased. When the amount of germinating ryegrass seed in the medium was varied, there was no significant difference between the survival of larvae held in treatments with 5, 25, and 50 g of seed per litre of medium. Survival was significantly lower with 0 and 100 g of seed per litre of medium. It is concluded that, in rearing conditions, the final density of fully grown 3rd‐instar larvae should be a maximum of about 20 per litre of medium to prevent mortality caused by larval interaction—provided that adequate food is available.