Evaluation of culture, ELISA and PCR assays for the detection of Salmonella in seafood

Aims: The study evaluated the efficiency of culture, enzyme‐linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) assays for the detection of Salmonella in naturally contaminated seafood. Methods and Results: In this study, 215 seafood samples comprising fish, shrimp, crab, clam, mussel, oyster, squid, cuttlefish and octopus from fish market of Cochin (India), were compared by culture, ELISA and PCR methods. Bacteriological Analytical Manual (BAM), U.S. Food and Drug Administration (USFDA) method was followed for culture assay, and Salmonella Tek, a commercial sandwich ELISA kit, was used for ELISA assay. Salmonella‐specific PCR assay was developed for 284 bp Salmonella‐specific invA gene amplicon. PCR assay exhibited 31·6% seafood positive for Salmonella followed by ELISA (23·7%) and culture method (21·3%). There was fair to excellent agreement between culture, ELISA and PCR assays (kappa coefficient values ranging from 0·385 to 1·0) for different seafood samples. Conclusion: The investigation revealed the greater concordance between culture and ELISA methods for seafood. Among the three methods, PCR assay was most sensitive. Lower detection rate with culture and ELISA assays could be attributed to greater sensitivity of the PCR method in the detection of Salmonella in seafood. Significance and Impact of the Study: We propose the incorporation of dual tests based on different principle and procedure for the routine analysis of Salmonella in seafood.     

Comparison of culture and PCR for the detection of Brucella melitensis in blood and lymphoid tissues of serologically positive and negative slaughtered sheep

Aims: To compare the culture and PCR methods for detection of Brucella melitensis in blood and lymphoid tissue samples obtained from slaughtered sheep (n = 162) testing positive/negative in serological tests (Rose Bengal test and serum agglutination test). Methods and Results: Of 162 sheep examined, 45 were positive and 117 negative in serological tests. A PCR assay based on a pair of Br. melitensis‐specific primers was used to detect DNA in blood and lymphoid tissue. Brucella melitensis was isolated from 1·2% (2/162) and 17·2% (28/162) of the blood and lymphoid tissue samples respectively. Positive PCR products with a molecular size of 731 bp were obtained from 27·7% (45/162) of blood and 29·0% (47/162) of lymphoid tissue samples. Conclusions: The species‐specific PCR assay detected a higher number of Br. melitensis DNA both from serologically positive (P < 0·01 in blood PCR, P < 0·001 in tissue PCR) and serologically negative (P < 0·001 in both blood PCR and tissue PCR) sheep compared with classical bacteriological culture methods. Significance and Impact of the Study: The results emphasize the importance of using more than one type of diagnostic technique for the detection of animals positive for brucellosis, especially with epidemiological purposes.     

Molecular detection of enterotoxins E, G, H and I in Staphylococcus aureus and coagulase-negative staphylococci isolated from clinical samples of newborns in Brazil

Aims: The objective of this study was to investigate the detection of SEE, SEG, SEH and SEI in strains of Staphylococcus aureus and coagulase‐negative staphylococci (CNS) using RT‐PCR. Methods and Results: In this study, 90 Staph. aureus strains and 90 CNS strains were analysed by PCR for the detection of genes encoding staphylococcal enterotoxins (SE) E, G, H and I. One or more genes were detected in 54 (60%) Staph. aureus isolates and in 29 (32·2%) CNS isolates. Staphylococcus epidermidis was the most frequently isolated CNS species (n = 64, 71·1%), followed by Staphylococcus warneri (n = 8, 8·9%) and other species (Staphylococcus haemolyticus, Staphylococcus hominis, Staphylococcus lugdunensis, Staphylococcus simulans, Staphylococcus saprophyticus and Staphylococcus xylosus: n = 18, 20%). The genes studied were detected in Staph. epidermidis, Staph. warneri, Staph. haemolyticus, Staph. hominis, Staph. simulans and Staph. lugdunensis. The highest frequency of genes was observed in Staph. epidermidis and Staph. warneri, a finding indicating differences in the pathogenic potential between CNS species and highlighting the importance of the correct identification of these micro‐organisms. RT‐PCR used for the detection of mRNA revealed the expression of SEG, SEH and/or SEI in 32 (59·3%) of the 90 Staph. aureus isolates, whereas expression of some of these genes was observed in 10 (34·5%) of the 90 CNS isolates. Conclusions: Staphylococcus epidermidis was the most toxigenic CNS species. Among the other species, only Staph. warneri and Staph. lugdunensis presented a positive RT‐PCR result. PCR was efficient in confirming the toxigenic capacity of Staph. aureus and CNS. Significance and Impact of the Study: This study permitted to confirm the toxigenic capacity of CNS to better characterize the pathogenic potential of this group of micro‐organisms. In addition, it permitted the detection of SEG, SEH and SEI, enterotoxins that cannot be detected by commercially available immunological methods.     

Consistency in the host specificity and host sensitivity of the Bacteroides HF183 marker for sewage pollution tracking

Aims: The host specificity (H‐SPF) and host sensitivity (H‐SNV) values of the sewage‐associated HF183 Bacteroides marker in the current study were compared with the previously published studies in South East Queensland (SEQ), Australia, by testing a large number of wastewater and faecal DNA samples (n = 293) from 11 target and nontarget host groups. This was carried out to obtain information on the consistency in the H‐SPF and H‐SNV values of the HF183 marker for sewage pollution tracking in SEQ. Methods and Results: Polymerase chain reaction (PCR) analysis was used to determine the presence/absence of the HF183 marker in wastewater and faecal DNA samples. Among the human composite wastewater (n = 59) from sewage treatment plants and individual human (n = 20) faecal DNA samples tested, 75 (95%) were PCR positive for the HF183 marker. The overall H‐SNV of this marker in target host group was 0·95 (maximum of 1·00). Among the 214 nontarget animal faecal DNA samples tested, 201 (94%) samples were negative for the HF183 marker. Six chicken, five dog and two bird faecal DNA samples, however, were positive for the marker. The overall H‐SPF of the HF183 marker to differentiate between target and nontarget faecal DNA samples was 0·94 (maximum of 1·00). Conclusions: The H‐SNV (0·95) and H‐SPF (0·94) values obtained in this study was slightly lower than previous studies (H‐SNV value of 1·00 in 2007 and 1·00 in 2009; H‐SPF value of 1·00 in 2007 and 0·99 in 2009). Nonetheless, the overall high H‐SNV (0·98) and H‐SPF (0·97) values of the HF183 marker over the past 4 years (i.e. 2007–2011) suggest that the HF183 marker can be reliably used for the detection of sewage pollution in environmental waters in SEQ. Significance and Impact of the Study: In the current study, the HF183 marker was detected in small number nontarget animal faecal samples. Care should be taken to interpret results obtained from catchments or waterways that might be potentially contaminated with dog faecal matter or poultry litter.     

Multiplex PCR assay for simultaneous detection and differentiation of Mycobacterium tuberculosis, Mycobacterium avium complexes and other Mycobacterial species directly from clinical specimens

Aims: Polymerase chain reaction (PCR) is the most rapid and sensitive method for diagnosing mycobacterial infections and identifying the aetiological Mycobacterial species in order to administer the appropriate therapy and for better patient management. Methods and Results: Two hundred and thirty‐five samples from 145 clinically suspected cases of tuberculosis were processed for the detection of Mycobacterial infections by ZN (Ziehl Neelsen) smear examination, L‐J & BACTEC^TM MGIT‐960 culture and multiplex PCR tests. The multiplex PCR comprised of genus‐specific primers targeting hsp65 gene, Mycobacterium tuberculosis complex‐specific primer targeting cfp10 (Rv3875, esxB) region and Mycobacterium avium complex‐specific primer pairs targeting 16S–23S Internal Transcribed Spacer sequences. The multiplex PCR developed had an analytical sensitivity of 10 fg (3–4 cells) of mycobacterial DNA. The multiplex PCR test showed the highest (77·24%) detection rate, while ZN smear examination had the lowest (20%) detection rate, which was bettered by L‐J culture (34·4%) and BACTEC^TM MGIT‐960 culture (50·34%) methods. The mean isolation time for M. tuberculosis was 19·03 days in L‐J culture and 8·7 days in BACTEC^TM MGIT‐960 culture. Using the multiplex PCR, we could establish M. tuberculosis + M. avium co‐infection in 1·3% HIV‐negative and 2·9% HIV‐positive patients. The multiplex PCR was also highly useful in diagnosing mycobacteraemia in 38·09% HIV‐positive and 15·38% HIV‐negative cases. Conclusions: The developed in‐house multiplex PCR could identify and differentiate the M. tuberculosis and M. avium complexes from other Mycobacterial species directly from clinical specimens. Significance and Impact of the Study: The triplex PCR developed by us could be used to detect and differentiate M. tuberculosis, M. avium and other mycobacteria in a single reaction tube.     

Foraging behaviour and prey discrimination in the bluespotted maskray Dasyatis kuhlii

A study observing the foraging behaviours and prey discrimination of a common demersal stingray, the bluespotted maskray Dasyatis kuhlii was performed under controlled laboratory conditions. A selection of prey species and masses were offered at depths of 10 and 50 mm in sand. Foraging efficiency and prey selection at both burial depths were compared. Dasyatis kuhlii selected the ghost shrimps, Trypaea australiensis and T. australiensis >2·5 g, range ± 0·2 g though foraging errors represented by prey being excavated and not consumed suggested a limited discriminatory ability at the point of detection. Burial depth did not influence prey species, mass selection or discriminatory ability.     

Development and evaluation of a multiplex real-time PCR assay for the detection and differentiation of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in pure culture isolates and lacrimal swabs collected from conventionally raised cattle

Aim: To develop a multiplex real‐time PCR assay for the detection and differentiation of Moraxella bovis (M. bovis), M. bovoculi and M. ovis. Methods and Results: The multiplex real‐time PCR assay was validated on three reference strains, 57 pure culture isolates and 45 lacrimal swab samples. All reference strains were identified correctly with no cross‐reactions between species. Sequencing of 53 of the 57 culture isolates confirmed the results obtained with the multiplex real‐time PCR, and the assay had 96·5% (55/57) concordance with a Moraxella spp. multiplex conventional PCR assay on the isolates. Among the lacrimal swab samples, the concordance between the multiplex real‐time PCR and culture was 86·7% (39/45) for M. bovoculi and 75·6% (34/45) for M. bovis. Conclusions: The multiplex real‐time PCR assay is specific and sensitive and can be used directly on lacrimal swab samples. Significance and Impact of Study: The lack of a rapid, specific and sensitive detection method is a barrier for determining the roles of M. bovis, M. bovoculi and M. ovis in infectious bovine keratoconjunctivitis cases, and the developed PCR assay will contribute to improved understanding of the epidemiology and pathogenesis of these three Moraxella species.     

Stem water storage capacity and efficiency of water transport: their functional significance in a Hawaiian dry forest

We investigated the contribution of internal water storage and efficiency of water transport to the maintenance of water balance in six evergreen tree species in a Hawaiian dry forest. Wood‐saturated water content, a surrogate for relative water storage capacity, ranged from 70 to 105%, and was inversely related to its morphological correlate, wood density, which ranged between 0·51 and 0·65 g cm^?3. Leaf‐specific conductivity (k_L) measured in stem segments from terminal branches ranged from 3 to 18 mmol m^?1 s^?1 MPa^?1, and whole‐plant hydraulic efficiency calculated as stomatal conductance (g) divided by the difference between predawn and midday leaf water potential (Ψ_L), ranged from 70 to 150 mmol m^?2 s^?1 MPa^?1. Hydraulic efficiency was positively correlated with k_L (r² = 0·86). Minimum annual Ψ_L ranged from ? 1·5 to ? 4·1 MPa among the six species. Seasonal and diurnal variation in Ψ_L were associated with differences among species in wood‐saturated water content, wood density and k_L. The species with higher wood‐saturated water content were more efficient in terms of long‐distance water transport, exhibited smaller diurnal variation in Ψ_L and higher maximum photosynthetic rates. Smaller diurnal variation in Ψ_L in species with higher wood‐saturated water content, k_L and hydraulic efficiency was not associated with stomatal restriction of transpiration when soil water deficit was moderate, but avoidance of low minimum seasonal Ψ_L in these species was associated with a substantial seasonal decline in g. Low seasonal minimum Ψ_L in species with low k_L, hydraulic efficiency, and wood‐saturated water content was associated with higher leaf solute content and corresponding lower leaf turgor loss point. Despite the species‐specific differences in leaf water relations characteristics, all six evergreen tree species shared a common functional relationship defined primarily by k_L and stem water storage capacity.     

A review of the genus Dysalotus (Percomorphacea: Chiasmodontidae), with the description of Dysalotus pouliulii sp. nov.

A new species of Dysalotus (family Chiasmodontidae) known only from off the Hawaiian archipelago is described here as Dysalotus pouliulii sp. nov. It differs from all other species of Dysalotus in having a greater number of teeth on the premaxilla (151–198 v. 60–138) and dentary (136–199 v. 76–132) and in a shorter upper jaw [51·9–54·9% of head length (L_H) v. 62·4–74·4% L_H] and lower jaw (64·8–67·4% in L_H v. 75·3–88·1% in L_H). A key for the species of Dysalotus and an updated distribution map are provided. http://zoobank.org/urn:lsid:zoobank.org:pub:E9B5F1DC‐52E0‐4F53‐9109‐2A8E252AE8CE .     

Characterization of enterococci populations collected from a subsurface flow constructed wetland

Aims: The aim of this study was to identify and characterize the population of Enterococcus sp. in domestic wastewater as it flows through a constructed wetland. Methods and Results: Four hundred and eighty‐four Enterococcus isolates were collected from the inlet, various sites within and from the outlet of a plastic lined constructed wetland in College Station, TX. The wetland treated septic tank effluent that passed sequentially through two 1·89 m³ septic tanks and a 1·89 m³ pump tank allowing 48 l doses at a 24 l min^?1 rate. The Enterococcus isolates were identified to species using the commercial Biolog system. The 484 Enterococcus isolates were comprised of ten different species, including Enterococcus faecalis (30·6%), Enterococcus pseudoavium (24·0%), Enterococcus casseliflavus (12·8%), Enterococcus faecium (11·2%), Enterococcus mundtii (7·9%), Enterococcus gallinarum (6·2%), Enterococcus dispar (3·7%), Enterococcus hirae (2·1%), Enterococcus durans and Enterococcus flavescens both 0·8%. Of the 88 isolates collected from the inlet, only 9·1% of the isolates were identified as Ent. faecalis and Ent. pseudoavium (36·4%) was identified as the predominant species. Whereas of the 74 isolates collected from the outlet, the predominant species were identified as Ent. faecalis (29·7%). Species identification varied among sites within the wetland, but often Ent. faecalis was the predominant species. Conclusions: Our data suggest that while Ent. faecalis is the predominant species of Enterococcus found in domestic wastewater, the populations may shift during treatment as the wastewater flows through the constructed wetland. Significance and Impact of the Study: We found that shifts in Enterococcus species composition occurred during domestic wastewater treatment. This has implications for the identification of faecal pollution based on the presence of specific bacterial types associated with domestic wastewater.     

Redescription of Chlorophthalmus corniger,a senior synonym of Chlorophthalmus bicornis (Family: Chlorophthalmidae)

Chlorophthalmus corniger is redescribed on the basis of recently collected specimens. The species is redefined as a species of Chlorophthalmus with the lower jaw terminating in a distinctly projecting horizontal plate with strong, spine‐like processes directed forward from the plate's corners; body silvery grey, with numerous minute black spots and traces of broad darker crossbars; base of anterior dorsal fin spines and distal parts of dorsal fins black; adipose fin tiny with numerous black spots; caudal fin black; 3·5 scales above lateral line; three rows of cheek scales; head very large, 34·3–40·1% standard length (L_S); eye large, 29·8–40·8% head length (L_H); pectoral fin long, extending to beyond dorsal fin base, 21·7–26·2% L_S. Chlorophthalmus bicornis is a junior synonym of C. corniger based on the examination of the type series of both species. It is confined to the northern half of the Indian Ocean, reliably recorded from Somalia and the Gulf of Aden to southern Java, Indonesia, at depths between 200 and 500 m. A lectotype and three paralectotypes were designated for C. corniger. DNA barcodes for Indian species of Chlorophthalmus were generated.     

Occurrence of Gobiidae larvae in a tropical Brazilian estuary,with particular emphasis on the use of size classes to categorize species guilds

The structure and seasonal dynamics of larvae of the Gobiidae family in the Mucuri Estuary (Bahia, Brazil) were studied for nine consecutive years. Sampling was conducted at three stations in the lower estuary channel, between 2002 and 2010, in relation to season, day and night and tidal variations. A total of 5802 Gobiidae larvae, representing 15 taxa (12 species and three morphotypes), were collected in the Mucuri Estuary during this time. The highest mean ± s.d. density of fish larvae, 54·7 ± 79·8 larvae 100 m^?3, was recorded during the flood tide and night sampling. Ctenogobius boleosoma was the most abundant species (68%), being dominant in the rainy and dry seasons and had a long reproductive period. This species was classed as a marine estuarine‐opportunist because it was observed at high frequencies and active larvae entering the estuary between 6·1 and 12·0 mm standard length (L_S). Gobionellus oceanicus, second in abundance (12%), occurred only in later larval stages but did not use the estuary as a preferred location for spawning, being classed a marine estuarine dependent. Microgobius carri (11%) was represented in all L_S classes and was resident in the estuary for spawning, remaining there throughout their life cycle. The other species were considered rare due to their low densities and could not be classified in any guild.     

An energy budget for juvenile thick-lipped mullet, Crenimugil labrosus (Risso)

A series of experiments were carried out to construct an energy budget for juvenile thick lipped mullet, Crenimugil labrosus Risso. A partial factorial experimental design was used to examine the effects of temperature, fish size and meal size on growth. The maximum ration that the fish were able to ingest completely per day was found to be 0·8, 1·4 and 2·3% wet body weight (b.w.) at 13,18 and 23°C, respectively. Ingested maintenance requirements (M.R.) were estimated to be 137, 205 and 288 cal fish^-1 day^-1 at 13, 18 and 23°C, respectively. At 18deg; C, M.R. varied as 25 W^1.04 cal day^-1, where W= fish weight (g). Growth rate increased with increasing temperature. Maximal conversion efficiency was 21–24% and was achieved closer to the maximum ingested ration with increasing temperature. The relationship between respiration rate and W at 18deg; C for 3-20 g fish is described by: respiration rate (ml O₂ h^-1) = 0·128 W^0.976 The energy cost of apparent specific dynamic action at 18deg; C was found to vary between 5·1% and 23·6% of the calorific value of the ingested meal (1% wet b.w.) , mean (± S.E.)=10·2 ± 2·0%. Post mortem analyses of groups of fish fed 0·2, 0·8 and 1·5% wet b.w. meals showed a significant increase in total lipid and a significant decrease in water content with increasing ratio size. A negative correlation was found between body water content and total lipid (and calories). The mean assimilation efficiency (±s.e.) for 5–10 g mullet at 18deg; C was 73·9 ± 3·6%. The observations reported in this study were brought together to construct an energy budget for juvenile C. labrosus which was found to give a reliable prediction (within 10%) of energy demand and growth under the prevailing experimental conditions. Both gross (K₁) and net (K₂) growth efficiencies, based on energy values, increased with increasing ratio size up to satiation and were independent of temperature. The maximum values of K₁ and K₂ observed were 0·33 and 0·46, respectively. The third order efficiency (K₃) appeared to be independent of temperature and ration size; mean values ranged between 0·66 and 0·84.     

The assimilation efficiency of Asellus aquaticus L. (Crustacea, Isopoda)

(1) Comparative studies of the ash-ratio and gravimetric methods of determining assimilation efficiency in Asellus aquaticus showed that the differential use of minerals by this species rendered the ash-ratio method unreliable. Results obtained by the gravimetric method were therefore employed in the analysis of further experiments. (2) The assimilation efficiencies estimated for A. aquaticus ranged between 26 and 44%, and varied according to population density and reproductive condition. (3) Individual winter males had a significantly lower assimilation efficiency (26%) than grouped animals (35%), but the assimilation efficiency of summer males (33%) did not differ significantly from that of winter males at the same density. It is concluded that density affects assimilation efficiency in A. aquaticus. (4) The assimilation efficiency of summer males (33 %) is significantly different from that of summer females (non-ovigerous, 41 %; ovigerous, 44%). A mean assimilation efficiency of 40% is proposed for the summer period whereas an overall annual mean of 30% is suggested. (5) Despite the various assimilation efficiencies reported within any one season consumption rate per unit weight is fairly constant (winter, 0·04–0·05 cal/24 h; summer, 0·36–0·38 cal/24 h) and it is suggested that the different assimilation rates are a mechanism whereby the additional energy and material requirements of females for breeding can be met without increasing food intake.     

Some observations on the biology of two rarely seen deep‐sea chimaerids,Chimaera carophila and Hydrolagus homonycteris

Chimaera carophila (n = 45) and Hydrolagus homonycteris (n = 11), two deep‐sea chimaerids rarely caught in the waters off New Zealand, were collected from research trawl catches and commercial fishery catches around New Zealand at depths between 400 and 1300 m, between 2014 and 2016. Additional preserved specimens of both species (n = 58) from museum collections were analysed for size, sex and maturity. External assessment of male claspers and a combination of internal assessments of female gonad mass and oviducal gland width, were used to determine maturity. For both species, length at first maturity was 0·70–0·82 of their maximum observed chimaera length (L_C), with females maturing at a larger size. Length at maturity for C. carophila (L_C range: 28·7–103·9 cm) was estimated at 72·5 cm L_C for males (n = 163) and 82·5 L_C for females (n = 58). In H. homonycteris, length at maturity (length range: 78·6–99·8 cm L_C) was estimated at 79·1 cm L_C for males (n = 51) and 80·1 cm L_C for females (n = 17). Ovarian fecundity was up to 31 for C. carophila and sperm storage was confirmed in the oviducal gland of this species. Both species preyed on benthic invertebrates. Some C. carophila and H. homonycteris inhabit depths beyond most current fisheries, but both species appear to be relatively rare and have reproductive parameters characteristic of low productivity, which may make these species vulnerable to population decline if mortality was to increase in the future.     

Assessing the faecal source sensitivity and specificity of ruminant and human genetic microbial source tracking markers in the central Ethiopian highlands

This study tested genetic microbial source tracking (MST) methods for identifying ruminant- (BacR) and human-associated (HF183/BacR287, BacHum) bacterial faecal contaminants in Ethiopia in a newly created regional faecal sample bank (n = 173). BacR performed well, and its marker abundance was high (100% sensitivity (Sens), 95% specificity (Spec), median log₁₀ 8·1 marker equivalents (ME) g⁻¹ ruminant faeces). Human-associated markers tested were less abundant in individual human samples (median: log₁₀ 5·4 and 4·2 (ME + 1) g⁻¹) and were not continuously detected (81% Sens, 91% Spec for BacHum; 77% Sens, 91% Spec for HF183/BacR287). Furthermore, the pig-associated Pig2Bac assay was included and performed excellent (100% Sens, 100% Spec). To evaluate the presence of MST targets in the soil microbiome, representative soil samples were tested during a whole seasonal cycle (n = 60). Only BacR could be detected, but was limited to the dry season and to sites of higher anthropogenic influence (log₁₀ 3·0 to 4·9 (ME + 1) g⁻¹ soil). In conclusion, the large differences in marker abundances between target and non-target faecal samples (median distances between distributions ≥log₁₀ 3 to ≥log₁₀ 7) and their absence in pristine soil indicate that all tested assays are suitable candidates for diverse MST applications in the Ethiopian area.     

Catch composition,reproductive biology and diet of the bramble shark Echinorhinus brucus (Squaliformes: Echinorhinidae) from the south‐eastern Arabian Sea

Fishery and biological data are presented for the poorly known bramble shark Echinorhinus brucus (Squaliformes: Echinorhinidae), from the deep waters of the south‐eastern Arabian Sea. A total of 5318 individuals from by‐catch landings of deep‐water bottom set longlines, gillnets and shrimp trawl fisheries operating at depths of 200–1200 m were recorded between January 2008 and December 2011 at the Kochi Fisheries Harbour (Kerala). A total of 431 individuals, from 46 to 318 cm total length (L_T) and 0·8 to 132 kg total mass (M_T), were examined to determine biological data for this species. The L_T at which 50% were mature (L_T50) for females and males was estimated at 189 and 187 cm L_T. Litter size ranged from 10 to 36 and size at birth was between 42 and 46 cm L_T. Dietary analysis of stomach contents revealed E. brucus feeds on a variety of prey including crustaceans (69% index of relative importance, I_RI), teleosts (25·8% I_RI), cephalopods (1·7% I_RI) and elasmobranchs (0·7% I_RI). This study provides the first detailed biological data for this species and also highlights the extent of the by‐catch fishery for this species in Indian waters.     

Early growth and development of reciprocal hybrids of the starry flounder Platichthys stellatus and stone flounder Kareius bicoloratus

Larval growth and development of hybrid flounder were observed and compared with those of their parent species. The reciprocal hybrids of female starry flounder Platichthys stellatus and male stone flounder Kareius bicoloratus (hybrid Sb) and of female K. bicoloratus and male P. stellatus (hybrid Bs) both survived and grew to juveniles. Development was divided into nine stages (A–I). Many of the hybrids' traits were identical and intermediate to those of their parents. The position of the eye, however, was primarily sinistral in both hybrids (80% in Sb and 76% in Bs), a trait possessed by P. stellatus (80%) in the western Pacific Ocean. The daily growth rates of the larvae were similar. In the parent species, development was more rapid in P. stellatus than in K. bicoloratus whereas rate of development was intermediate in both Sb and Bs hybrids. The size at settlement [standard length (L_S) at stage H (mean ± s.d. )] was 9·82 ± 1·47 mm for the hybrid Sb and 9·99 ± 0·90 mm for the hybrid Bs, while the minimum age at metamorphosis (initial age at stage H) was 29 days after hatching (DAH) in both hybrids. In comparison, L_S at settlement in parent species was 6·43 ± 0·25 mm for P. stellatus and 12·87 ± 1·29 mm for K. bicoloratus. Minimum age at metamorphosis for the parents was 23 DAH at stage G in P. stellatus and 34 DAH at stage H in K. bicoloratus. Thus, the timing of settlement of hybrids was different from that of their parent species. These traits may occur with high frequency in a natural habitat.     

A new species of Anchoviella (Clupeiformes: Engraulidae) from the western Amazon River in Peru,with comments on congeners in the Peruvian Amazon River

Anchoviella hernanni sp. nov. is described from the upper Amazon River basin, in tributaries of the Marañon, Ucayali and Madre de Dios river drainages that drain the Peruvian Andes. The new taxon can be distinguished from all congeners except Anchoviella jamesi, Anchoviella manamensis and Anchoviella perezi, by having 12–15 gill rakers in the lower branch of the first gill arch (v·16–35) and from those species by the distance between verticals through the posterior margin of the orbit to the posterior margin of the upper jaw 9·5–14·8% head length; L_H (v. up to 6·0% L_H). An updated identification key of all freshwater species of Anchoviella and morphological comparisons between all species of the genus occurring in Peru are provided.     

The photoreceptor system in the retinae of two dogfishes, Scyliorhinus canicula and Galeus melastomus: possible relationship with depth distribution and predatory lifestyle

The small spotted dogfish Scyliorhinus canicula and the blackmouth dogfish Galeus melastomus, whose depth distributions overlap in the upper part of the slope (c. 500 m depth), where they have access to the same prey community, have well‐developed eyes and a pure‐rod retina with a single layer of photoreceptors. Interspecific differences in rod outer segment length (L_ROS) within retinal regions were found. In the periphery and the retinal centre G. melastomus showed a L_ROS 24 and 30% longer, respectively, than S. canicula and, therefore, a potential for increased sensitivity. In both species longer L_ROS were always found in correspondence with the retinal centre where the ganglion cell topography formed a horizontal meridian that allowed for better discrimination of the horizon in the visual field. In this area L_ROS reached 53·4±4·1μm in S. canicula and 77·1±10·5μm in G. melastomus against 46·3±4·2μm and 61·1±10·1μm in the retinal periphery. No significant differences were recorded in L_ROS and rod density during growth. In both species, a rapid increase of theoretical visual acuity was found to be related to an increase in fish L_T and lens size. Visual acuity ranged between 1·7 and 3 cycles degree^‐1 in S. canicula and 2·4 and 4·2 in G. melastomus. The G. melastomus rod visual pigment showed the characteristic spectral adaptation to vision in deep‐water (λ_max of 481 nm), but was also well placed to detect the bioluminescence of some of its main prey species. In S. canicula the visual pigment absorption (λ_max of 496 nm) was more typical of shallow water living fishes. The opsin sequences of the two visual pigments are discussed and key amino acid sites were identified where sequence changes could be responsible for the spectral absorption differences between the two species. The possible relationship between L^ROS, visual acuity, visual pigment absorption, depth distribution and feeding behaviour are discussed.