Single-step nested multiplex PCR to differentiate between various bivalve larvae

A nested multiplex polymerase chain reaction (PCR) assay has been developed that allows the discrimination between six bivalve larvae common to Danish coastal waters (Cerastoderma edule, Macoma balthica, Mytilus edulis, Spisula subtruncata, Ensis americanus and members of the order Myoida). This assay involves the simultaneous use of a pair of general universally targeted 18S rRNA gene primers, five specific 18S rRNA gene targeted oligonucleotide primers internal (nested) to the universal primer pair and one species-specific primer that is not nested (Mya). The specificity of each primer was evaluated in silico, empirically, and verified further by sequencing of amplification products from single larvae collected from plankton samples. Identification of individually isolated bivalve larvae from plankton samples was based on the size of the PCR product produced by the specific primers after visualisation by agarose gel electrophoresis. Preliminary studies indicated that this method was suitable for use with freshly collected and preserved larvae, and is therefore suitable for field application.Communicated by M. Kühl, Helsingør     

Nearshore distributional gradients of larval fish (15 taxa) and planktonic crustaceans (6 Taxa) in Hawaii

Fifteen taxa of fish larvae (most identified to species) and 6 taxa of crustaceans (most identified to species) were studied in zooplankton samples collected 0.2, 0.5, 1.0 and 3.0 km off the leeward coast of Oahu, Hawaii in May and July 1975. The following distributional patterns were elucidated: inshore (2 spp); inshore-neritic (2 spp); neritic (7 spp); and offshore-neritic (7 spp). Three of these 18 species had age-related differences in behaviour that led to age-dependent distributional patterns, and 4 further species had a random distribution. All abundant types of fish larvae were either oceanic as adults or were hatches from the demersal or brooded eggs of reef species. The larvae of reef fishes with pelagic eggs were not abundant in the nearshore region sampled and were probably to be found more than 3 km offshore. The offshore-neritic distributional pattern evidently resulted from relatively passive movement with currents. In contrast, maintenance of inshore and neritic patterns probably required relatively active swimming by the animals. The current regime, including a tidal eddy and possible nearshore upwelling, probably helped maintain the inshore and neritic patterns of such animals. The limited area occupied by these inshore coastal plankton communities could make them particularly vulnerable to environmental changes, including anthrogenic ones.     

Evaluation of combined morphological and molecular techniques for marine nematode (<Emphasis Type="Italic">Terschellingia</Emphasis> spp.) identification

Marine nematodes, which play an important role in many ecosystems, include a number of apparently cosmopolitan taxa that exhibit broad biogeographic ranges even though there is no obvious dispersal phase in their lifecycle. In this study, standard taxonomic approaches to marine nematode identification in conjunction with multivariate statistical analysis of morphometric data were compared with molecular techniques. Specimens of the marine nematode Terschellingia longicaudata that had been identified by their morphological features were investigated from a range of localities (East and West Atlantic, Bahrain, Malaysia) and habitats (estuarine, intertidal, subtidal) using molecular approaches based on the amplification and sequencing of the small subunit ribosomal RNA (18S rRNA). The study revealed that the majority of the morphologically defined T. longicaudata specimens share a single 18S rRNA sequence and apparently belong to a single taxon distributed from the British Isles to Malaysia. In addition, 18S rRNA analysis also revealed two additional sequences. One of these sequences was found in both the British Isles and Mexico, the other was recorded only from British waters. Individuals collected in Bahrain and identified from their morphology as T. longicaudata had two highly divergent 18S rRNA sequences. Separate morphological and morphometric approaches to identification of specimens from the same sites that had been formalin-preserved did not support evidence of multiple genotypes revealed previously by molecular analysis. Current taxonomy based on morphological characters detected using light-microscopy may be unable to discriminate possible species complexes. Biodiversity of marine nematodes may often be underestimated due to the presence of morphologically cryptic species complexes. High-throughput techniques such as DNA barcoding would aid in species identification but may require thorough analysis of multiple nuclear and mitochondrial molecular markers.     

Characterization in the archaeological excavation site of heterotrophic bacteria and fungi of deteriorated wall painting of Herculaneum in Italy

Microbiological characterization of frescos in four different locations (Collegio degli Augustali, Casa del Colonnato Tuscanico, Casa dello Scheletro and Casa del Gran Portale) of excavation sites of Herculaneum was carried out. The use of infrared thermography allowed detecting sample points on frescos with greatest moisture not visible to the naked eye, resulting in structural damage. The microclimatic conditions provided perfect habitat for bacteria and fungi, particularly of spore forming and mould. In fact, heterotrophic bacteria were prevalent in all wall paintings monitored (ranging from 18 +/- 2 CFU 100 cm(-2) to 68 +/- 4 CFU 100 cm(-2)), whereas fungi were also detected but at lower levels (ranging from 9 +/- 2 CFU 100 cm(-2) to 45 +/- 3 CFU 100 cm(-2)). Cultural-based method allow us to identify by 16S and 26S rRNA partial sequence analysis heterotrophic microorganisms belonging to different genera of Bacillus and Aspergillus, Penicillium and Fusarium together with the unusual genera as Microascus and Coprinus. By using this approach, Bacillus-related species (B. cereus/B. thuringiensis group, B. simplex/B. muralis group, B. megaterium and B. subtilis) were isolated in all sample points analysed with the exception of the Casa dello Scheletro in which Micrococcus luteus/Arthrobactersp. group and Streptomyces fragilis were found. DGGE analysis of PCR amplified V3 region of rDNA from DNA directly recovered from frescos samples, enabled identification of bacterial species not identified using culturable technology asthose closest related to Microbacterium group, often associated with Brevibacterium, Streptomyces and Stenotrophomonas. Combination of culture-dependent and independent methods provided better microbiology characterization of heterotrophic microbiota present on the surface of ancient frescos of this important archaeological site.     

RNA/DNA ratio and expression of 18S ribosomal RNA, actin and myosin heavy chain messenger RNAs in starved and fed larval Atlantic cod (Gadus morhua)

Levels of total RNA, total DNA, 18S ribosomal RNA (rRNA), poly(A) messenger RNA (mRNA), and two mRNAs coding for abundant myofibrillar proteins were estimated in laboratory-reared Atlantic cod larvae (Gadusmorhua Linnaeus) under conditions of feeding and starvation. DNA probes specific for cod 18S rRNA, β-actin mRNA and myosin heavy chain mRNA were developed. In two experiments on newly hatched larvae in fed and starved treatments, changes in 18S rRNA and mRNA were similar to changes in total RNA during the first weeks after hatching. RNA levels in fed and starved larvae in both experiments were stable, or increased, over the first 3 d after hatching, and then decreased to minima at 9 d. RNA levels increased after 9 d, with the degree and timing of the increase varying among the individual classes of RNA. Complete mortality of starved larvae in both experiments was observed shortly after 11 d, corresponding to exhaustion of endogenous yolk reserves. Total RNA content, RNA/DNA ratio, 18S rRNA levels, total mRNA pool, and actin and myosin heavy chain mRNA levels showed significant differences in fed and starved first-feeding larvae after yolk exhaustion. In another experiment with 3- to 4-week-old cod larvae, 18S rRNA levels were significantly lower in starved versus fed larvae after 3 d. Total RNA responded to feeding and starvation within a similar time as 18S rRNA and the mRNAs examined. Analysis of bulk nucleic acids using fluorometric dyes was simpler and faster than analysis of individual RNAs using hybridization probes, and provides valuable information on recent growth and condition of individual larvae. However, analysis of specific RNAs can provide information on expression of the corresponding genes and reveal the changes underlying trends seen in bulk RNA. Received: 9 February 1996 / Accepted: 7 June 1999     

The use of polymerase chain reaction for the identification of sciaenid eggs

Traditionally, sciaenid eggs have been identified based on morphological characteristics such as size, number of oil globules and/or pigmentation patterns. Identification of sciaenid eggs by these procedures is time consuming and often inaccurate due to considerable egg size overlap among species. The utilization of molecular techniques for the identification of economically important species has become a fundamental component in ecological studies involving fish eggs and larvae. This study reports the development of a series of both multiplex and individual polymerase chain reactions to identify the eggs of 11 sciaenid species commonly found in the Gulf of Mexico and estuaries near Port Aransas and Corpus Christi, TX, USA. Following method development, the discriminatory power of the assay was first determined with samples from adult fish collected from Aransas and Corpus Christi Bays, Galveston Bay and the lower Laguna Madre in northern Mexico. Most (97%) of these fishes were correctly identified to the level of species. To demonstrate the applicability of the assay, wild fish eggs were collected and analyzed from the Aransas Pass tidal inlet from September through December 2005. During this period, the eggs of four target species were positively identified which was in keeping with current knowledge regarding the spawning areas and seasons of these sciaenids based on the presence of mature females, eggs and/or larvae. Future use of this method can provide valuable information to better discriminate spawning sites and seasons of these species.     

Determining the diet of larvae of the red rock lobster (Jasus edwardsii) using high-throughput DNA sequencing techniques

The diet of Jasus edwardsii during its ~2-year larval (phyllosoma) phase is largely unknown. High mortalities experienced during larviculture might be reduced if their diet were nutritionally modelled on the natural diet. Here, prey species were identified from phyllosoma midgut glands using 454 pyrosequencing of 18S rDNA. We found that gelatinous zooplankton, particularly Siphonophora and Ctenophora, occurred frequently in the midgut glands of phyllosomas, resolving previous conjecture that these animals are in the diet of J. edwardsii phyllosomas. A high occurrence of sequencing reads from unicellular microbes may also reflect a reliance on scavenging detritus.     

A molecular approach for the identification of meiofaunal turbellarians (Platyhelminthes,Turbellaria)

In the absence of reliable morphological characters, or in conjunction with morphology-based identifications, meiofaunal turbellarians may also be identified using the nucleotide sequence of a portion of the large subunit of the ribosomal RNA (26/28S rRNA). A 284 base pair-long region of the 26/28S rRNA has been identified by isolating genomic DNA from ten species of turbellarians belonging to four orders, namely, the Proseriata, Macrostomida, Prolecithophora and Acoela. The proseriates had been collected from localities in Europe and Israel and were preserved in ethanol. The remaining turbellarians were isolated from intertidal sediment samples collected from two sites on the Maine and New Hampshire coast, USA in 1992. Amplification of the genomic DNA was carried out using two primers designed to match the nucleotide sequence of a portion of the 26/28S rDNA gene of the terrestrial nematode,Caenorhabditis elegans (Maupas 1900). This area consists of a highly variable, about 150 base pair-long region, called the D3 expansion segment, followed by a very conserved stretch of sequence. When folded into its secondary structure, the conserved region will form stem structures that correspond to helices 15 to 18 of theC. elegans structural model. The sequence alignment program PILEUP was used to perform a cluster analysis (unweighted pair group method using arthmetic averages, UPGMA) on the sequences. This analysis revealed that the helices allow for the classification of the turbellarians at the level of families and above, whereas if the D3 expansion segment itself was included in the analysis, intrageneric and intraspecific groupings could be established.     

Dispersal of phytoplanktotrophic shipworm larvae (Bivalvia: Teredinidae) over long distances by ocean currents

Shipworms or Teredinidae may be dispersed either as adults in floating wooden objects or as pelagic larvae drifting near the sea surface. Five shipworm species, i.e., half of those having an amphi-Atlantic geographical distribution, are known also to have pelagic phytoplanktotrophic larvae which can be carried by ocean currents. From a series of 742 plankton samples taken from throughout the temperate and tropical North Atlantic Ocean, it can be shown that shipworm larvae are not uncommon in the open sea. Teredinid veligers were found in 19% of all samples taken. One species of larvae, identical in all details to that described by Rancurel (1965), is particularly prevalent and is tentatively identified as Teredora malleolus (Turton). A definitive identification will be possible only after the pelagic larvae of the other Atlantic species are known. The larvae of Teredora malleolus are found throughout the North Atlantic Gyre and the adjacent temperate and tropical seas, and from scattered records in the South Equatorial Current. Larvae of other unidentified Teredinidae species were also found. The distance that larvae may be transported depends upon the length of pelagic larval development and the velocity of the currents. From the known current velocities it can be shown that, even in a few weeks, larvae may be dispersed many hundreds of kilometers. The geographical distribution of shipworm larvae suggests that they are carried along the coasts of continents and even across ocean basins, and that this dispersal must be an important factor in the geographical distribution of the adult forms and in the maintenance of genetic continuity between populations otherwise isolated from one another.Contribution No. 2555 from the Woods Hole Oceanographic Institution, Woods Hole, Massachusetts, USA     

Genetic population structure of the southern oyster drillStramonita (=Thais)haemostoma

Nine collections in the southeastern USA of the thaidid molluscStramonita haemastoma, comprising 963 individuals, were made between June 1988 and November 1989, and examined for allozyme variation at 18 electrophoretically-detected loci. Two genetically-differentiated groups were identified and are referred to ascanaliculata-like andfloridana-like snails, based on previously-described subspecies ofS. haemastoma. The two groups were differentiated genetically at a level that is characteristic of congeneric species in other molluscan taxa. The two groups maintained their genetic differences in areas of sympatry, although rare hybrid individuals occurred (about 1.2% of the total set). Shell and radular characters showed little variation within or between the two groups. Within each group, allozyme allele frequencies showed little geographic variation across distances as great as 1 500 km. This result is consistent with the suggestion that species with planktonic larvae (such asS. haemastoma) should have higher rates of gene flow than related brooding species.     

The transport of oyster larvae in an estuary

Vertical and horizontal distributions of 3 larval stages of the oyster Crassostrea virginica were measured concurrently with phytoplankton species compositions, phytoplankton size distributions and physical hydrographic parameters in tributaries of the Chesapeake Bay (USA) during the oyster spawning seasons of 1980 and 1981. The superposition of the biological distributions upon the physical hydrographic data provide instantaneous distributions of the entire system which are consistent with the upstream transport of oyster larvae. Oyster larvae distributions in the Choptank River and its Broad Creek and Tred Avon River tributaries can be described in terms of three contiguous regions: (1) a common spawning region, (2) an intermediate, upstream transport region and (3) a seed bed region where major spat set occurs. The phytoplankton species compositions and abundances in the size fraction less than 10 m in the tributary system during the transport were sufficient to supply optimum growth requirements of developing larvae. The transport proposed can explain the 30 yr record of consistently higher spat set success in one tributary, Broad Creek, relative to an adjoining tributary, the Tred Avon River. This may be a general mechanism whereby oysters maintain reproductive success and emigrate to seed bed regions in the Chesapeake Bay.Contribution No. 1144 of the McCollum-Pratt Institute and Department of Biology     

Larval ecology of the great barracuda, <Emphasis Type="Italic">Sphyraena barracuda</Emphasis>, and other sphyraenids in the Straits of Florida

The great barracuda (Sphyraena barracuda) is a widespread, ecologically and socioeconomically important coastal fish, yet very little is known about its larvae. We examined spawning and larval ecology of Western Atlantic sphyraenids using monthly ichthyoplankton samples collected over 2 years along a transect spanning the east–west axis of the Straits of Florida (SOF). Samples were dominated by the great barracuda (92.8%) and sennets (Sphyraena borealis and Sphyraena picudilla; 6.6%). While larval sennets and S. barracuda displayed similar vertical distributions (majority in upper 25 m), horizontal and temporal patterns of abundance suggested a spatial and temporal species replacement between larval S. barracuda and sennets that tracks adult ecology. The diet of both taxa consisted largely of copepods, with inclusion of fish larvae at 8 mm SL, and in S. barracuda alone, a switch in the wet season to exclusive piscivory by 12 mm SL (18 days post-hatch). A lack of piscivory in S. barracuda larvae captured in the dry season corresponded to slower larval growth than in the wet season. Larval growth was also related to size-at-hatch and larval age such that larvae that were larger at hatch or larger (older) at capture grew faster at earlier ages, suggesting faster larval growth, and indirectly larger hatch size, conveys a survival advantage. Unlike larval growth, instantaneous mortality rate did not differ with season, and no lunar cyclic patterns in spawning output were identified. Our results provide insight into the pelagic phase of sphyraenids and highlight the importance of both diet and hatch size to the growth and survival of fish larvae in low latitude oceanic environments.     

Correlating gene expression with larval competence,and the effect of age and parentage on metamorphosis in the tropical abalone<Emphasis Type="Italic"> Haliotis asinina</Emphasis>

The non-geniculate crustose coralline alga (CCA) Mastophora pacifica can induce the metamorphosis of competent Haliotis asinina (Vetigastropoda) larvae. The ability to respond to this natural cue varies considerably with larval age, with a higher proportion of older larvae (e.g. 90 h) able to metamorphose in response to M. pacifica than younger larvae (e.g. 66 h). Here we document the variation in time to acquisition of competence within a larval age class. For example, after 18 h of exposure to M. pacifica, approximately 15 and 36% of 84 and 90-h-old H. asinina larvae had initiated metamorphosis, respectively. This age-dependent response to M. pacifica is also observed when different aged larvae are exposed to CCA for varying periods. A higher proportion of older larvae require shorter periods of exposure to CCA than younger larvae in order to initiate metamorphosis. In this experiment, as in the previous, a small proportion of young larvae were able to respond to brief periods of CCA exposure, suggesting that they had developed the same state of competency as the majority of their older counterparts. Comparisons of the proportions of larvae undergoing metamorphosis between families reveals that parentage also has a significant (P<0.05) affect on whether an individual will initiate metamorphosis at a given age. These familial differences are more pronounced when younger, largely pre-competent larvae (i.e. 66 h old) are exposed to M. pacifica, with proportions of larvae undergoing metamorphosis differing by as much as 10 fold between families. As these data suggest that variation in the rate of development of the competent state has a genetic basis, and as a first step towards identifying the molecular basis to this variation, we have identified numerous genes that are differentially expressed later in larval development using a differential display approach. Spatial expression analysis of these genes suggests that they may be directly involved in the acquisition of competence, or may play a functional role in the postlarva following metamorphosis.Communicated by M.S. Johnson, Crawley     

Cuticular hydrocarbon profiles indicate reproductive status in the termite <Emphasis Type="Italic">Zootermopsis nevadensis</Emphasis>

Reproductive division of labor in social insects is accompanied by the reliable communication of individual fertility status. A central question is whether there exists a general mechanism underlying this communication system across species. The best way to produce reliable information is through physiological markers tightly associated with reproductive status. Cuticular hydrocarbons exhibit this link to individual fertility in several species of ants, bees, and wasps, and we present the first evidence for such a system in a non-Hymenopteran eusocial species. In the termite Zootermopsis nevadensis, we identified four polyunsaturated alkenes, which only occur in significant amounts on reproductives that are actively producing gametes. These compounds are either absent or only occur in small amounts in soldiers, worker-like larvae, and secondary reproductives with inactive gonads. In contrast to Hymenopteran social insects, both sexes express the reproductive peaks. The reproductive-specific hydrocarbons may promote tending behavior by worker-like larvae or act as a primer pheromone, inhibiting the reproductive development of immature conspecifics.     

Phylogenetic relationship among several genera of Dictyotaceae (Dictyotales, Phaeophyceae) based on 18S rRNA and partial rbcL gene sequences

The partial sequences of the 18S rRNA gene and the rbcL gene, RuBisCo spacer region of representatives of the Dictyotaceae were determined and compared to resolve their phylogenetic relationships. A total of 1,861 base pairs of 18S rDNA sequences were aligned and examined. There was a high similarity of nucleotide sequences within the tribe Dictyoteae (99.4-99.6%) and the tribe Zonarieae (92.8-99.8%). The largest intergeneric divergence within the Dictyotaceae was 7.31%, between Dilophus and Zonaria. The aligned 422 bases of partial rbcL gene sequences gave a similar phylogenetic relationship among taxa to those of the 18S rDNA sequences, although our alignment covered less than 30% of the whole gene. All genera of Dictyotales consisted of several clades forming a polychotomy in MP trees based on partial rbcL gene sequences and on 18S rDNA sequences. The genera within the tribe Dictyoteae formed a clade, a Dictyota-Phacydictyon-Dilophus clade, while genera of the tribe Zonarieae formed four clades: Padina; Distromium-Lobophora-Zonaria; Spatoglossum-Lobospira; and Dictyopteris-Spatoglossum. All genera of the Dictyotaceae included in these sequence analyses are classified into five groups, which have little phylogenetic resolution among them. The genera classified in the tribe Zonarieae according to Womersley's criteria do not appear to be monophyletic, based on our analyses. Although not all genera of Dictyotaceae were analysed, our data suggest that the status of the meristem cells should not be a criterion for separating tribes within the family Dictyotaceae and that other hierarchical systems should be adopted to properly reflect phylogenetic relationships.     

Spatio-temporal modeling of striped-bass egg, larval movement, and fate in the San Francisco Bay-Delta

Most models developed for the movement and fate of eggs and larvae of aquatic species are based on a particle tracking approach. Although this method has many advantages due to its high flexibility, particle tracking may become computationally intensive for complex geometries and when large numbers of particles are needed to simulate the population properly. In continuous models based on advection and dispersion mechanisms, the computational burden is independent of the size of the population. We developed a continuous fate and transport model for striped bass eggs and larvae in the San Francisco Bay-Delta. The model predicts the concentration of eggs and larvae at any location over time. The method of moments was used to account for the effect of temperature and age on the transition of eggs to larvae and larvae to juveniles. Egg and larval mortality were represented as functions of temperature, and eggs also experienced settling mortality. The fate and transport model used the same one-dimensional spatial grid as the existing Delta Simulation Model II (DSM2) hydrodynamics model. DSM2 output of flow rates, water depths, and cross-sectional areas were inputted into the fate and transport model to determine transport. The model was applied to striped bass eggs and larvae data collected during years 1990-1994; agreement between the modeled and the measured data was acceptable in most cases. Exploratory simulations were performed to demonstrate how the model could be used to evaluate the effects on egg and larval survival and total juvenile production of water diversions for supply and agricultural use and changes in the long-term mean water temperature. The model can be further used to examine the impact of various operation strategies in the San Francisco Bay-Delta, where diversion losses of early life stages of fishes remain a major management issue.     

DNA barcoding of Pacific Canada’s fishes

DNA barcoding—sequencing a standard region of the mitochondrial cytochrome c oxidase 1 gene (COI)—promises a rapid, accurate means of identifying animals to a species level. This study establishes that sequence variability in the barcode region permits discrimination of 98% of 201 fish species from the Canadian Pacific. The average sequence variation within species was 0.25%, while the average distance separating species within genera was 3.75%. The latter value was considerably lower than values reported in other studies, reflecting the dominance of the Canadian fauna by members of the young and highly diverse genus Sebastes. Although most sebastids possessed distinctive COI sequences, four species did not. As a partial offset to these cases, the barcode records indicated the presence of a new, broadly distributed species of Paraliparis and the possibility that Paraliparis pectoralis is actually a species pair. The present study shows that most fish species in Pacific Canadian waters correspond to a single, tightly cohesive array of barcode sequences that are distinct from those of any other species, but also highlights some taxonomic issues that need further investigation.     

Implications of life history for genetic structure and migration rates of southern African coastal invertebrates: planktonic,abbreviated and direct development

The amount of genetic structure in marine invertebrates is often thought to be negatively correlated with larval duration. However, larval retention may increase genetic structure in species with long-lived planktonic larvae, and rafting provides a means of dispersal for species that lack a larval dispersal phase. We compared genetic structure, demographic histories and levels of gene flow of regional lineages (in most cases defined by biogeographic region) of five southern African coastal invertebrates with three main types of larval development: (1) dispersal by long-lived planktonic larvae (mudprawn Upogebia africana and brown mussel Perna perna), (2) abbreviated larval development (crown crab Hymenosoma orbiculare) and (3) direct development (estuarine isopod Exosphaeroma hylecoetes and estuarine cumacean Iphinoe truncata). We hypothesized that H. orbiculare, having abbreviated larval development, would employ a strategy of larval retention, resulting in genetic structure comparable to that of the direct developers rather than the planktonic dispersers. However, regional population structure was significantly lower in all species with planktonic larvae, including H. orbiculare, than in the direct developers. Moreover, nested clade analysis identified demographic histories resulting from low levels of gene flow (isolation by distance and allopatric fragmentation) in the direct developers only, and migration rates were significantly higher in all three species having planktonic larvae than in the direct developers. We conclude that the amount of genetic structure within marine biogeographic regions strongly depends on the presence or absence of free-swimming larvae. Whether such larvae are primarily exported or retained, whether they have long or short larval duration, and whether or not they are capable of active dispersal seems to have little effect on connectivity among populations.     

Permanence of Euscorpius carpathicus (L.) larvae on the mother's back (Scorpiones,Chatidae)

Summary The tendency of Euscorpius carpathicus larvae to remain on immobilized live or dried scorpions of the same or different species (Euscorpius flavicaudis) has been tested. Dried scorpions are less attractive than live ones, which are equally attractive regardless of species, sex or reproductive phase. The larvae find old (one year) dried specimens less attractive than freshly or recently (15 days) killed females, which are equally as attractive as live mothers (Table 1). A substratum treated with a chloroform extract of the mother's cuticle is consistently preferred by the larvae. The maternal behaviour (tolerating the larvae on the back) usually disappears about a week after the moult of the larvae, but it can be protracted if they are continually replaced by younger larvae (Table 2). The survival rate of larvae on live mothers is higher than on dried specimens.