The occurrence of Lepeophtheirus salmonis and Caligus clemensi (Copepoda: Caligidae) on three-spine stickleback Gasterosteus aculeatus in coastal British Columbia

Infections with sea lice species belonging to Lepeophtheirus and Caligus are reported from examinations of 1,309 three-spine sticklebacks collected in coastal British Columbia. Over 97% of the 19,960 Lepeophtheirus specimens and nearly 96% of the 2,340 Caligus specimens were in the copepodid and chalimus developmental stages. The parasites were identified as Lepeophtheirus salmonis and Caligus clemensi based on morphology of adult stages. Between 1,763 and 1,766 base pairs (bp) of 18S rDNA from adult specimens collected from sticklebacks and salmon differed from the GenBank L. salmonis reference sequence by a single bp and were distinct from those of 2 other Lepeophtheirus species. A 530-bp region of 18S rDNA from chalimus stages of Lepeophtheirus obtained from sticklebacks and salmon was identical to that of the L. salmonis reference sequence. The three-spine stickleback is a new host record for L. salmonis. The prevalence of L. salmonis was 83.6% and that of C. clemensi was 42.8%. The intensities of these infections were 18.3 and 4.2, respectively. There was no significant relationship between sea lice abundance and stickleback condition factor. Significant spatial and temporal variations both in abundance of sea lice and surface seawater salinities were measured. The abundance of both sea lice species was lowest in zones in which surface seawater salinity was also lowest. Sticklebacks appear to serve as temporary hosts, suggesting a role of this host in the epizootiology of L. salmonis. The stickleback may be a useful sentinel species with which to monitor spatial and temporal changes in the abundance of L. salmonis and C. clemensi.     

Comparison of small subunit ribosomal RNA gene and internal transcribed spacer sequences among isolates of the intranuclear microsporidian Nucleospora salmonis

Nucleospora salmonis is an intranuclear microsporidian associated with a proliferative disorder of the lymphoid cells of captive salmonid fish in the northwestern and northeastern regions of North America, in France, and in Chile. Newer diagnostic approaches have used the polymerase chain reaction (PCR) to detect the parasite in fish tissues. The target sequences for these assays lie in the small subunit ribosomal RNA (ssu rRNA) gene or internal transcribed spacer (ITS) as determined from N. salmonis from chinook salmon (Oncorhynchus tshawytscha) from the Pacific Northwest of North America. The lack of sequence data on parasites from diverse geographic origins and hosts led us to compare several isolates of N. salmonis. There was a high degree of similarity in the ssu rDNA sequences (> 98%) among all the isolates of N. salmonis examined, regardless of host or geographic origin. The greatest sequence differences were found between isolates from the Pacific regions of America. Isolates from Chile shared sequences with one or both geographic groups from North America. A similar distribution of sequence types was observed when ITS-1 sequences of selected isolates were analyzed. Sequence data from two N. salmonis-like isolates from marine non-salmonid fish showed one closely related and the second less closely related to N. salmonis isolates from salmonid fish. These results provide evidence for a homogeneous group of aquatic members of the genus Nucleospora found among salmonid fish (N. salmonis) that can be detected using diagnostic PCR assays with ssu rDNA target sequences. The presence of parasites related to N. salmonis among marine fish suggests a potentially broad host and geographic distribution of members of the family Enterocytozoonidae.     

tRNA genes were found in Piscirickettsia salmonis 16S-23S rDNA spacer region (ITS)

Piscirickettsia salmonis is the etiological agent of Salmonid Rickettsial Septicemia, a disease affecting salmon aquaculture industry. We analyzed the 16S-23S rDNA spacer region (internal transcribed spacer, ITS) of Chilean P. salmonis isolates LF-89 and EM-90. Two main ITS amplification products were obtained by PCR using L1 and G1 primers, differing from that described where only one ITS region was found. By Southern blot, it was established that these two amplification products contained sequences related to P. salmonis ITS. Sequence analysis confirmed that P. salmonis had two ITS regions: ITS A and ITS B. In both isolates, the smaller (ITS B) corresponded to ITS sequences previously described for each one, and the larger (ITS A) were almost the same as their respective ITS B sequences interrupted by an insert which contained two tRNAs genes: tRNA-Ile and tRNA-Ala.     

Isolates of Piscirickettsia salmonis from Scotland and Ireland show evidence of clonal diversity

Salmonid rickettsial septicemia, caused by Piscirickettsia salmonis, causes major mortalities in Chilean salmonid aquaculture and is an increasing problem in Atlantic salmon in Ireland and Scotland. Analysis of 16S-to-23S internal transcribed sequences and 16S ribosomal DNA (rDNA) shows that Irish isolates of P. salmonis form two new groups of the organism while Scottish isolates cluster together with Norwegian and Canadian isolates from Atlantic salmon.     

Confirmation of Piscirickettsia salmonis as a pathogen in European sea bass Dicentrarchus labrax and phylogenetic comparison with salmonid strains

European sea bass Dicentrarchus labrax from the Mediterranean were diagnosed with a severe encephalitis. Rickettsia-like organisms (RLOs) were associated with brain lesions in routine paraffin sections. These were found to share common antigens with the Piscirickettsia salmonis type-strain, LF-89, by indirect fluorescent antibody test (IFAT) and by immunohistochemistry (IHC). In addition, we compared the DNA sequences of the 16S rDNA and 16S-23S internal transcribed spacer region (ITS) with those published for P. salmonis strains and found that the sea bass piscirickettsia-like organism (SBPLO) was another strain of P. salmonis, closely related to the salmonid pathogens. Furthermore, we showed that the SBPLO possessed at least 2 ITS regions, 1 of which contained tRNA genes.     

The diversity of microsporidia in parasitic copepods (Caligidae: Siphonostomatoida) in the Northeast Pacific Ocean with description of Facilispora margolisi n. g., n. sp. and a new Family Facilisporidae n. fam

Three distinct microsporidia were identified from parasitic copepods in the northeast Pacific Ocean. Sequencing and phylogenetic analysis of a partial small subunit ribosomal RNA gene (SSU rDNA) sequence identified a genetically distinct variety of Desmozoon lepeophtherii from Lepeophtheirus salmonis on cultured Atlantic salmon Salmo salar, and this was confirmed by transmission electron microscopy. Phylogenetic analysis resolved the SSU rDNA sequence of the second organism in a unique lineage that was most similar to microsporidia from marine and brackish water crustaceans. The second occurred in L. salmonis on Atlantic, sockeye Oncorhynchus nerka, chum O. keta and coho O. kisutch salmon, in Lepeophtheirus cuneifer on Atlantic salmon, and in Lepeophtheirus parviventris on Irish Lord Hemilepidotus hemilepidotus. Replication occurred by binary fission during merogony and sporogony, diplokarya were not present, and all stages were in contact with host cell cytoplasm. This parasite was identified as Facilispora margolisi n. g., n. sp. and accommodated within a new family, the Facilisporidae n. fam. The third, from Lepeophtheirus hospitalis on starry flounder Platichthys stellatus, was recognized only from its unique, but clearly microsporidian SSU rDNA sequence. Phylogenetic analysis placed this organism within the clade of microsporidia from crustaceans.     

Glacial refugia and reticulate evolution: the case of the Tasmanian eucalypts

Tasmania is a natural laboratory for investigating the evolutionary processes of the Quaternary. It is a large island lying 40-44 degrees S, which was repeatedly glaciated and linked to southeastern continental Australia during the Quaternary. Climate change promoted both the isolation of species in glacial refugia, and an exchange between Tasmanian and mainland floras. Eucalyptus is a complex and diverse genus, which has increased in abundance in Australia over the past 100 kyr, probably in response to higher fire frequency. Morphological evidence suggests that gene flow may have occurred between many eucalypt species after changes in their distribution during the Quaternary. This paper summarizes recent genetic evidence for migration and introgressive hybridization in Tasmanian Eucalyptus. Maternally inherited chloroplast DNA reveals a long-term persistence of eucalypts in southeastern Tasmanian refugia, coupled with introgressive hybridization involving many species. Detailed analysis of the widespread species Eucalyptus globulus suggests that migration from mainland Australia was followed by introgression involving a rare Tasmanian endemic. The data support the hypothesis that changes in distribution of interfertile species during the Quaternary have promoted reticulate evolution in Eucalyptus.     

Phylogenetic analysis of Piscirickettsia salmonis by 16S, internal transcribed spacer (ITS) and 23S ribosomal DNA sequencing

Piscirickettsia salmonis, the etiologic agent of piscirickettsiosis, is a systemic disease of salmonid fish. Variations in virulence and mortality have been observed during epizootics at different geographical regions and in laboratory experiments with isolates from these different locations. This raises the possibility that biogeographical patterns of genetic variation might be a significant factor with this disease. To assess the genetic variability the 16S ribosomal DNA, the internal transcribed spacer (ITS) and the 23S ribosomal DNA of isolates from 3 different hosts and 3 geographic origins were amplified using the polymerase chain reaction (PCR). Results of this analysis confirm that P. salmonis is a member of the gamma subgroup of the Proteobacteria and show that the isolates form a tight monophyletic cluster with 16S rDNA similarities ranging from 99.7 to 98.5%. The ITS regions were 309 base pairs (bp), did not contain tRNA genes, and varied between isolates (95.2 to 99.7% similarity). Two-thirds of the 23S rRNA gene was sequenced from 5 of the isolates, yielding similarities ranging from 97.9 to 99.8%. Phylogenetic trees were constructed based on the 16S rDNA, ITS and 23S rDNA sequence data and compared. The trees were topologically similar, suggesting that the 3 types of molecules provided similar phylogenetic information. Five of the isolates are closely related (> 99.4% 16S rDNA similarity, 99.1% to 99.7% ITS and 99.3 to 99.8% 23S rDNA similarities). The sequence of one Chilean isolate, EM-90, was unique, with 16S rDNA similarities to the other isolates ranging from 98.5 to 98.9%, the ITS from 95.2 to 96.9% and the 23S rDNA from 97.6 to 98.5%.     

Tasmanian rotifera: Affinities with the Australian fauna

Plankton was collected from more than 100 lentic and lotic habitats in Tasmania. Of the 131 rotifer taxa identified, 126 are first records for the island, and 9 are first records for Australia. Three new species, Aspelta tilba, sp. nov., Lepadella tyleri sp. nov., and Testudinella unicornuta sp. nov. are described and figured. Horaëlla brehmi, which has a disjunct distribution (equatorial regions and southeastern Australia), also occurs in Tasmanian waters. The morphology and development of this rare species are discussed. Geographical affinities of the Tasmanian assemblage are considered. There appears to be a significant tropical component in the Tasmanian Rotifera.     

A review of parasites in the Tasmanian devil (<Emphasis Type="Italic">Sarcophilus harrisii</Emphasis>)

Threatened by devil facial tumour disease, the Tasmanian devil (Sarcophilus harrisii), a carnivorous marsupial confined to Tasmania, Australia, is the subject of conservation management under the Save the Tasmanian Devil Program. Conservation actions such as captive breeding and translocation may impact upon parasite ecology, presenting risk of increased disease through stress and impaired immunity, and by exposing hosts to parasites to which they are immunologically naïve. Given the importance of parasites to ecosystem function, it has been argued from a biodiversity perspective that parasites should be conserved in their own right. In this review we describe current knowledge, and limitations in our knowledge, of Tasmanian devil parasites. We then discuss the potential for changes in host–parasite interactions as a result of host-population decline and conservation management, both generally and with examples from the Tasmanian devil. The review closes with a recommendation for a systematic evaluation of parasites in captive and wild devils to aid conservation of this host–parasite system in its entirety.     

GENETIC VARIATION AMONG STRAINS OF THE TOXIC DINOFLAGELLATE GYMNODINIUM CATENATUM (DINOPHYCEAE)

The toxic dinoflagellate Gymnodinium catenatum Graham has formed recurrent toxic blooms in southeastern Tasmanian waters since its discovery in the area in 1986. Current evidence suggests that this species might have been introduced to Tasmania prior to 1973, possibly in cargo vessel ballast water carried from populations in Japan or Spain, followed by recent dispersal to mainland Australia. To examine this hypothesis, cultured strains from G. catenatum populations in Australia, Spain, Portugal, and Japan were examined using allozymes and randomly amplified polymorphic DNA (RAPD). Allozyme screening detected very limited polymorphism and was not useful for population comparisons; however, Australian, Spanish, Portuguese, and Japanese strains showed considerable RAPD diversity, and all strains examined represented unique genotypes. Multidimensional scaling analysis (MDS) of RAPD genetic distances between strains showed clear separation of strains into three nonoverlapping regional clusters: Australia, Japan, and Spain/Portugal. Analysis of genetic distances between strains from the three regional populations indicated that Australian strains were almost equally related to both the Spanish/Portuguese population and the Japanese population. Analysis of molecular variance (AMOVA) found that genetic variation was partitioned mainly within populations (87%) compared to the variation between the regions (8%) and between populations within regions (5%). The potential source population for Tasmania’s introduced G. catenatum remains equivocal; however, strains from the recently discovered mainland Australian population (Port Lincoln, South Australia, 1996) clustered with Tasmanian strains, supporting the notion of a secondary relocation of Tasmanian G. catenatum populations to the mainland via a shipping vector. Geographic and temporal clustering of strains was evident among the Tasmanian strains, indicating that genetic exchange between neighboring estuaries is limited and that Tasmanian G. catenatum blooms are composed of localized, estuary-bound subpopulations.     

Molecular detection and phylogenetic placement of a microsporidian from English sole (Pleuronectes vetulus) affected by X-cell pseudotumors

Flatfish tissue samples exhibiting X-cell pseudotumors were tested with a number of ribosomal DNA (rDNA) general primers in polymerase chain reactions (PCRs). Microsporidian primers resulted in the amplification of an rDNA fragment and molecular phylogenetic analysis indicated that although the organism did not relate closely with any current microsporidian genera, it was most similar to Nucleospora salmonis and branched within the Enterocytozoonidae. Re-examination of the original tissues used for DNA extractions revealed the presence of putative microsporidian spores in PCR-positive samples. These observations reiterate the highly sensitive diagnostic feature of PCR, allowing detection of organisms overlooked by conventional methods and demonstrate the occurrence of rare, coinfecting organisms.     

HMGB in Mollusk Crassostrea ariakensis Gould: Structure,Pro-Inflammatory Cytokine Function Characterization and Anti-Infection Role of Its Antibody

Background

Crassostrea ariakensis Gould is a representative bivalve species and an economically important oyster in China, but suffers severe mortalities in recent years that are caused by rickettsia-like organism (RLO). Prevention and control of this disease is a priority for the development of oyster aquaculture. It has been proven that mammalian HMGB (high mobility group box) can be released extracellularly and acts as an important pro-inflammatory cytokine and late mediator of inflammatory reactions. In vertebrates, HMGB’s antibody (anti-HMGB) has been shown to confer significant protection against certain local and systemic inflammatory diseases. Therefore, we investigated the functions of Ca-HMGB (oyster HMGB) and anti-CaHMGB (Ca-HMGB’s antibody) in oyster RLO/LPS (RLO or LPS)-induced disease or inflammation.

Methodology/Principal Findings

Sequencing analysis revealed Ca-HMGB shares conserved structures with mammalians. Tissue-specific expression indicates that Ca-HMGB has higher relative expression in hemocytes. Significant continuous up-regulation of Ca-HMGB was detected when the hemocytes were stimulated with RLO/LPS. Recombinant Ca-HMGB protein significantly up-regulated the expression levels of some cytokines. Indirect immunofluorescence study revealed that Ca-HMGB localized both in the hemocyte nucleus and cytoplasm before RLO challenge, but mainly in the cytoplasm 12 h after challenge. Western blot analysis demonstrated Ca-HMGB was released extracellularly 4–12 h after RLO challenge. Anti-CaHMGB was added to the RLO/LPS-challenged hemocyte monolayer and real-time RT-PCR showed that administration of anti-CaHMGB dramatically reduced the rate of RLO/LPS-induced up-regulation of LITAF at 4–12 h after treatment. Flow cytometry analysis indicated that administration of anti-CaHMGB reduced RLO/LPS-induced hemocyte apoptosis and necrosis rates.

Conclusions/Significance

Ca-HMGB can be released extracellularly and its subcellular localization varies when stimulated with RLO. Ca-HMGB is involved in oyster immune reactions and functions as a pro-inflammatory cytokine. Anti-CaHMGB can significantly suppress RLO/LPS-induced inflammatory responses and hemocyte necrosis and apoptosis, suggesting that Ca-HMGB is a potential target to prevent and control RLO/LPS-induced disease or inflammation.     

Allograft Inflammatory Factor 1 Functions as a Pro-Inflammatory Cytokine in the Oyster,Crassostrea ariakensis

The oyster Crassostrea ariakensis is an economically important bivalve species in China, unfortunately it has suffered severe mortalities in recent years caused by rickettsia-like organism (RLO) infection. Prevention and control of this disease is a priority for the development of oyster aquaculture. Allograft inflammatory factor-1 (AIF-1) was identified as a modulator of the immune response during macrophage activation and a key gene in host immune defense reaction and inflammatory response. Therefore we investigated the functions of C. ariakensis AIF-1 (Ca-AIF1) and its antibody (anti-CaAIF1) in oyster RLO/LPS-induced disease and inflammation. Ca-AIF1 encodes a 149 amino acid protein containing two typical Ca²⁺ binding EF-hand motifs and shares a 48–95% amino acid sequence identity with other animal AIF-1s. Tissue-specific expression analysis indicates that Ca-AIF1 is highly expressed in hemocytes. Significant and continuous up-regulation of Ca-AIF1 is detected when hemocytes are stimulated with RLO/LPS (RLO or LPS). Treatment with recombinant Ca-AIF1 protein significantly up-regulates the expression levels of LITAF, MyD88 and TGFβ. When anti-CaAIF1 antibody is added to RLO/LPS-challenged hemocyte monolayers, a significant reduction of RLO/LPS-induced LITAF is observed at 1.5–12 h after treatment, suggesting that interference with Ca-AIF1 can suppress the inflammatory response. Furthermore, flow cytometric analysis indicated that anti-CaAIF1 administration reduces RLO/LPS-induced apoptosis and necrosis rates of hemocytes. Collectively these findings suggest that Ca-AIF1 functions as a pro-inflammatory cytokine in the oyster immune response and is a potential target for controlling RLO infection and LPS-induced inflammation.     

Genetic characterization and experimental pathogenesis of Piscirickettsia salmonis isolated from white seabass Atractoscion nobilis

An intracellular bacterium originally isolated from hatchery-reared juvenile white seabass Atractoscion nobilis in southern California, USA, was identified by sequences of the small and large subunit ribosomal (16S and 23S) DNA and the internal transcribed spacer (ITS) as Piscirickettsia salmonis. Considering all rDNA sequences compared, the white seabass isolate (WSB-98) had a 96.3 to 98.7% homology with 4 previously described strains of P. salmonis isolated from salmon in Chile, Norway, and British Columbia, Canada. Experimental infections induced by intraperitoneal injections of juvenile white seabass with WSB-98 resulted in disease and mortality similar to that observed in P. salmonis infections in salmon. After 60 d, the cumulative mortality among P. salmonis-injected white seabass was 82 and 40%, respectively, following a high (1.99 x 10(4) TCID50) or low (3.98 x 10(2) TCID50) dose-challenge with WSB-98. The bacterium was recovered by isolation in cell culture or was observed in stains from tissues of injected white seabass but not from control fish. There were no external signs of infection. Internally, the most common gross lesion was a mottled appearance of the liver, sometimes with distinct nodules. Microscopic lesions were evident in both the capsule and parenchyma of the liver and were characterized by multifocal necrosis, often with infiltration of mononuclear leukocytes. Macrophages filled with bacteria were present at tissue sites exhibiting focal necrosis. Foreign body-type granulomas were prevalent in livers of experimentally infected white seabass, but not in control fish. Similar granulomatous lesions were observed in the spleen, kidney, intestine and gills, but these organs were considered secondary sites of infection, with significantly fewer and less severe histologic lesions compared to the liver. The results from this study clearly indicate that infections with P. salmonis are not restricted to salmonid fishes and that the bacterium can cause a disease similar to piscirickettsiosis in nonsalmonid hosts.     

Endemism in Tasmanian cool temperate rainforest: alternative hypotheses

Endemism in Tasmanian cool temperate rainforest: alternative hypotheseS. Evidence is presented which suggests that hypotheses presented by Kirkpatrick & Brown relating to endemic species in Tasmania are either invalid or of limited importance for woody rainforest species.
In their place three hypotheses are presented to account for the presence of endemic species in Tasmanian cool temperate rainforest on the basis of the fossil record and the distribution of species which are closely related to the endemics. The first two hypotheses relate to the presence of the endemic species in the general Tasmanian region. They are:

• 1. 

  Some species evolved in southeastern Australia during the Tertiary in response to the changing climate. Some of the ancestral species still occur in temperate rainforest at lower latitudes in Australia.
• 2. 

  Some species have remained essentially unchanged in Tasmania during the Tertiary and Quaternary climatic changes.

The third hypothesis relates to the restriction of these cool temperate rainforest species to Tasmania:

• 1. 

  Post-glacial climatic changes (especially a decrease in rainfall) and the human influence (especially land clearing and fire) may have combined to eliminate some cool temperate rainforest species from mainland Australia.

     

Characterization of proteases in the skin mucus of Atlantic salmon (Salmo salar) infected with the salmon louse (Lepeophtheirus salmonis) and in whole-body louse homogenate

As part of an investigation of the biochemical interactions between the salmon louse Lepeophtheirus salmonis and Atlantic salmon Salmo salar, we characterized protease activity in the skin mucus of noninfected Atlantic salmon and Atlantic salmon infected with L. salmonis and in an L. salmonis whole-body homogenate. Zymography revealed that mucus from infected salmon contained a series of low-molecular-mass (17-22 kDa) serine proteases that were not present in the mucus of noninfected salmon. Based on molecular mass, inhibition studies, and affinity chromatography, the series of proteases was identified as being trypsin-like. Similar proteases were observed in the L. salmonis homogenate and in mucus from noninfected Atlantic salmon following a 1-hr incubation with live L. salmonis. An antibody raised against Atlantic salmon trypsin failed to recognize any proteases in the mucus of noninfected salmon or infected salmon or in the L. salmonis homogenate. Collectively, these findings suggest that the trypsin-like proteases present in the mucus of infected Atlantic salmon were produced by L. salmonis, possibly to aid in feeding and evasion of host immune responses.     

Low major histocompatibility complex diversity in the Tasmanian devil predates European settlement and may explain susceptibility to disease epidemics

The Tasmanian devil (Sarcophilus harrisii) is at risk of extinction owing to the emergence of a contagious cancer known as devil facial tumour disease (DFTD). The emergence and spread of DFTD has been linked to low genetic diversity in the major histocompatibility complex (MHC). We examined MHC diversity in historical and ancient devils to determine whether loss of diversity is recent or predates European settlement in Australia. Our results reveal no additional diversity in historical Tasmanian samples. Mainland devils had common modern variants plus six new variants that are highly similar to existing alleles. We conclude that low MHC diversity has been a feature of devil populations since at least the Mid-Holocene and could explain their tumultuous history of population crashes.     

Lepeophtheirus salmonis: characterization of prostaglandin E(2) in secretory products of the salmon louse by RP-HPLC and mass spectrometry

Lepeophtheirus salmonis is an ectoparasitic copepod that causes serious disease outbreaks in both wild and farmed salmonids. As the relationship between L. salmonis and its hosts is not well understood, the current investigation was undertaken to investigate whether any immunomodulatory compounds could be identified from secretions of L. salmonis. By incubating live L. salmonis adults with the neurotransmitter dopamine in seawater, we were able to obtain secretions from the parasite. These were analyzed by RP-HPLC column, as well as LC-MS. L. salmonis secretions contained a compound with the same retention time and mass of PGE(2). The identity of this compound as PGE(2) was confirmed by MS-in source dissociation. The concentrations of PGE(2) in L. salmonis secretions ranged from 0.2 to 12.3 ng/individual and varied with incubation temperature and time kept off the host. Prostaglandin E(2) is a potent vasodilator and thought to aid in parasite evasion from host immune responses. This is the first reported evidence of prostaglandin production in parasitic copepod secretions and its implications for the host-parasite relationship are discussed.     

Geographic variation in competitive ability in Drosophila melanogaster

The aim of this study was to examine the latitudinal variation in preadult competitive ability of Drosophila melanogaster. Two pairs of populations from Queensland and Tasmania, Australia, were examined. Queensland flies are genetically smaller and develop more slowly than the Tasmanian flies. Survival and body size of flies raised at different temperatures and densities were compared when larvae were challenged with a common competitor. No latitudinal variation in larval survival was detected. Body size (measured as wing length) decreased with increasing temperature and larval density. Flies from the Tasmanian populations were more sensitive to the effects of temperature and density and to the joint effect of increased temperature and density. This could explain the evolution of greater growth efficiency and larger body size at lower temperatures.