Effect of commercial grade endosulfan on growth and reproduction of the fighting fish Betta splendens.

To study the effects of endosulfan on survival, growth and reproduction of the obligate air-breathing male heterogametic fighting fish Betta splendens, posthatchlings of the fighting fish were discretely immersed for 3 h/day during the labile period on the 2nd, 5th, and 8th day posthatching (dph) at selected concentrations of commercial grade endosulfan ranging from 175 to 1400 ng/L. The immersions at 1,400 ng/L led to 21% mortality, among the 79% of surviving fry, 80% developed into females. The endosulfan reduced the air-breathing frequency of 5- and 8-day old hatchlings, and the reduction in the frequency persisted even after a depuration period of 172 days. In the ovary of the treated females, reduced number of vitellogenic oocytes with increased vacuolar cavities was observed. In the testis of the treated males, the reduced number of spermatogonia with increased vacuolar cavities was observed. The treated male induced the female to spawn a fewer eggs, which were subsequently incubated in his smaller bubble nest. The control females attained puberty on the 138th dph and spawned 120 eggs once in every 15 days, the females, which were previously treated at 1400 ng/L, postponed puberty to the 179th dph and spawned 70 eggs once in every 32 days. During the 240-day experiment, endosulfan is found to reduce significantly the cumulative progeny production from 760 to 144, reducing significantly to 19% of the control.

Interspecific androgenetic restoration of rosy barb using cadaveric sperm.

Interspecific androgenetic rosy barb (Puntius conchonius) was generated using its cadaveric (-20 degrees C) or fresh sperm to activate nuclear genome inactivated oocytes of gray tiger barb (Puntius tetrazona). UV irradiation was used to inactivate nuclear genome of tiger barb oocytes. Thermal shock restored diploidy of rosy barb in the oocytes of tiger barb. Survival of androgenotes was 14% or 7% when fresh or cadaveric sperm was used. The diploid or haploid nuclear genome of rosy barb, individually or jointly with that of tiger barb, regulated the time sequence of embryonic development in an alien cytoplasm of tiger barb oocytes. Androgenetic males (Y2Y2) attained sexual maturity earlier and had significantly higher gonadosomatic index and sperm concentration, albeit suffering a slight decrease in fertilizing ability. Conversely, androgenetic females (X2X2) suffered extended interspawning period, reduced fecundity, and poor hatchability of their progenies. These results are discussed with respect to their significance for conservation biology.

Hormonal induction of supermale golden rosy barb and isolation of Y-chromosome specific markers.

Viable supermales of the golden rosy barb Puntius conchonius are generated through hormonal sex reversal and progeny testing. Discrete immersion (3 h/day on the second, fourth, and sixth day after hatching) of the fry in Estradiol-17beta (E2) at doses of 400-600 microg/L ensures greater than 40% survival and production of more than 98% F1 females; of these 50% are heterogametic females, that when crossed with normal males, sire 25% YY supermales. Supermales sire female progenies at the frequencies between 0 and 8%. Reproductive performance of hormonally sex-reversed females and androgenetic females is inferior to the normal ones. Conversely, the performance of androgenetic males is superior but suffers from low fertilizability. The relative performance of supermale produced by breeding sex-reversed parents is superior to those produced by androgenesis. Using the SRY-specific primers, the PCR analysis of the genomic DNA of the male golden rosy barb produces three products of 588, 333, and 200 bp length. However only the 200 bp product is amplified in the female genome. Hence it is possible to use the first two products as molecular markers to rapidly identify fish possessing a Y chromosome. The presence of 333 and 588 bp fragments in normal (X1Y2), hormonally induced (Y1Y2) and androgenetic (Y2Y2) males and the absence of relation between the 200 bp fragment and the X-chromosome indicates that the male specific markers are specific to Y-chromosome. For the first time, a Y-chromosome specific molecular marker for a cyprinid has been identified, isolated, and characterized.

Enhanced expression of beta-thymosin mRNA in the ovary of GnRH analog or estradiol-17beta-treated paradise fish, Macropodus opercularis.

cDNA clones were isolated as expressed sequence tags (ESTs) from the ovarian cDNA library of Macropodus opercularis. The EST sequences showed similarity with many housekeeping genes and ribosomal proteins. One of the ESTs showed similarity to beta-thymosin, a 5-kDa polypeptide expressed under different physiological conditions. The cDNA corresponding to beta-thymosin of M. opercularis is 368 bp in length and codes for a putative polypeptide of 42 amino acids. Multiple alignment of the deduced amino acid sequence showed 61% similarity with piscine beta-thymosins and 56% similarity with mammalian beta-thymosins. Administration of a gonadotropin releasing hormone analog or estradiol-17beta induced an increase in the gonadosomatic index, oocyte diameter and also enhanced expression of beta-thymosin m-RNA in the recrudizing ovary. This report indicates that both GnRH analog and E(2) might induce similar pathways for the differentiation of ovarian cells for the maturation of oocytes.

Molecular cloning of growth hormone encoding cDNA of Indian major carps by a modified rapid amplification of cDNA ends strategy.

A modified rapid amplification of cDNA ends (RACE) strategy has been developed for cloning highly conserved cDNA sequences. Using this modified method, the growth hormone (GH) encoding cDNA sequences of Labeo rohita, Cirrhina mrigala and Catla catla have been cloned, characterized and overexpressed in Escherichia coli. These sequences show 96-98% homology to each other and are about 85% homologous to that of common carp. Besides, an attempt has been made for the first time to describe a 3-D model of the fish GH protein.

Molecular cloning of growth hormone-encoding cDNA of an Indian major carp, Labeo rohita, and its expression in Escherichia coli and zebrafish.

The cDNA clones encoding for growth hormone (GH) of an Indian major carp rohu Labeo rohita were isolated from a cDNA library constructed from the poly(A)(+) RNA extracted from the pituitary glands of rohu. Partial GH cDNA of the rohu (3'-end) was amplified by RT-PCR and used as probe to screen the cDNA library. Full-length GH-specific cDNA clones (1180 bp) were isolated and sequenced. The sequence contains 48-bp 5'-noncoding region followed by an ORF of 621 bp and a 3'-noncoding region of 521 bp. The peptide shares about 90% identity with the GH of Cyprinus carpio (Linn.) and >84% identity with GH sequences of other cyprinids. The GH-encoding cDNA of rohu has been cloned into expression vectors and GH protein has been over expressed in Escherichia coli and purified as a soluble protein. The GH cDNA was cloned into a bicistronic vector with EGFP; injection of in vitro transcribed GH-EGFP mRNA into zebrafish embryo has resulted in EGFP expression confirming the cloned GH cDNA is functional in fish and the IRES element could be effectively used in fish for bicistronic expression of foreign genes.

Cloning, sequencing and expression of cDNA encoding growth hormone from Indian catfish (Heteropneustes fossilis).

A tissue-specific cDNA library was constructed using polyA+ RNA from pituitary glands of the Indian catfish Heteropneustes fossilis (Bloch) and a cDNA clone encoding growth hormone (GH) was isolated. Using polymerase chain reaction (PCR) primers representing the conserved regions of fish GH sequences the 3' region of catfish GH cDNA (540 bp) was cloned by random amplification of cDNA ends and the clone was used as a probe to isolate recombinant phages carrying the full-length cDNA sequence. The full-length cDNA clone is 1132 bp in length, coding for an open reading frame (ORF) of 603 bp; the reading frame encodes a putative polypeptide of 200 amino acids including the signal sequence of 22 amino acids. The 5' and 3' untranslated regions of the cDNA are 58 bp and 456 bp long, respectively. The predicted amino acid sequence of H. fossils GH shared 98% homology with other catfishes. Mature GH protein was efficiently expressed in bacterial and zebrafish systems using appropriate expression vectors. The successful expression of the cloned GH cDNA of catfish confirms the functional viability of the clone.

Cloning of partial putative gonadotropin hormone receptor sequence from fish.

A search for the presence of mariner-like elements in the Labeo rohita genome by polymerase chain reaction led to the amplification of a partial DNA sequence coding for a putative transmembrane domain of gonadotropin hormone receptor. The amplified DNA sequence shows a high degree of homology to the available turkey and human luteinizing and follicle stimulating hormone receptor coding sequences. This is the first report on cloning such sequences of piscine origin.

Interspecific hybridization in poeciliids.

Seventy per cent attempts to ensure interspecific hybridization between Poecilia velifera and P. sphenops were successful and led to the production of true hybrids, but not gynogens or triploids, as evidenced by the mottled or striped colour, chromosome number (2n = 46) and response from scale transplantation. Most hyrbids were infertile as they failed to cross among themselves or with their respective parents; however they were more closely related to P. sphenops as indicated by mating responses and scale transplantation studies. Heterospecific impregnation resulted in 40% reduction in fecundity but retention of interparturition period characteristic of the female species. A skewed ratio of 3 Female Female:2 Male Male, observed in the laboratory populations of both P. velifera and P. sphenops, was also sustained among the progenies sired from heterospecifically inseminated females. The colour patterns of hybrids were of two types: mottled and striped, the latter one being reported for the first time.

Sublethal effects of textile dye stuff effluent on selected oxidative enzymes and tissue respiration of Cyprinus carpio (Linn.).

Toxic effects of sublethal concentration of dye stuff effluent on succinic dehydrogenase (SDH) and lactic dehydrogenase (LDH) activities and tissue respiration were studied in C. carpio. While the sublethal exposure significantly reduced SDH activity and tissue respiration, LDH activity increased in gill, brain, liver, muscle and kidney. The maximum inhibition of SDH activity (74%) was recorded in gill and the minimum (38%) in liver. The percentage reduction of oxygen consumption in the tested tissues was in the order of gill greater than brain greater than liver greater than muscle greater than kidney. The muscle showed the highest level (96%) of increase in LDH activity whereas the kidney cells showed the minimum increase. Exposure to sublethal concentration suppressed the aerobic respiration and triggered the anaerobic respiration.

Physiological correlates of surfacing behaviour--effect of aquarium depth on surfacing, growth and metamorphosis in Rana tigrina.

Rana tigrina were reared from hatching to metamorphosis in aquaria containing different depths but equal volume of water. To exchange atmospheric air, tadpoles in the shallow aquarium surfaced less frequently, travelled shorter distance at less metabolic energy cost and hence grew larger (3.1 g) at a higher (25%) efficiency than those (2.5 g; 19% efficiency) reared in the deepest aquarium. Aquarium depth is a more potent factor than pO2 in the regulation of surfacing behaviour. The significant and positive correlations observed between feeding, metabolism and growth, on one hand, and surfacing behaviour, on the other, can serve as an index of metabolism of the tadpole.

Effects of size hierarchy on surfacing behaviour and conversion rate in an air-breathing fish Channa striatus.

Effects of size hierachy on surfacing behaviour and food conversion rate in relation to density and feeding frequency were studied in Channa striatus. In general, crowding increased surfacing frequency in all the weight classes, but affected the food conversion rate of small individuals alone. At any tested density, conversion rate of smaller individual was depressed by the presence of larger individuals; increased feeding frequency did not compensate the negative effects of density. The possible reasons for growth inhibition are: (1) The smaller individuals spend much of their time and energy to escape from the aggressive larger ones than on feeding, and (2) the smaller individuals spend a considerable amount of assimilated food energy on increased surface activity. Unlike density, frequency of feeding improved the conversion rate of both the weight classes and it suggests that the larger individuals could compensate the negative effects of crowding by increased feeding.

Effects of interaction of ration and temperature on growth and bioenergetics of Achaea janata Linnaeus (Lepidoptera: Noctuidae).

Growth and bioenergetics of the last instar larvae of Achaea janata fed on a wide range of rations of leaves of Ricinus communis (ad libitum to 50 or 100 mg leaf per larva per day) at 22, 27, 32 and 35° C were studied. Increase in larval mortality, extension of larval duration and decrease in final body weight were some of the adverse effects of restricted rations. Whereas larval duration was influenced by ration level, pupal period was dependent on temperature. The larvae partially compensated for restricted rations by enhancing feeding rate over the limit expected in proportion to the ration offered. The level of compensation was higher at 27° C. Influence of temperature or its interaction with ration on assimilation efficiency was more significant than the independent influence of ration; the efficiency ranged from 57 to 67%. Restriction of ration below a critical level (300 mg/larva/day at 35° C and 200 mg/larva/day at other temperatures) significantly influenced the net conversion efficiency; the lowest efficiency of 1.3% was recorded for the larvae fed 100 mg leaf/day. Energy content of the terminal larva determined the percentage of energy transferred to the imago, which ranged from 37 to 55% of the terminal larval energy.

Energy balance in Speliphron violaceum (Hymenoptera) and use of meconium weight as an index of bioenergetics components.

Speliphron violaceum deposits its larva and a few paralysed spiders inside the holes of electrical sockets. The larva feeds on the spiders, completes development in 6 days and emerges after 18 days of pupation. When provided with spiders to the maximum capacity of the hole, a larva consumed 1.746 KJ and converted 1.360 KJ. Restriction of the quantity of spiders provided, resulted in decreased consumption and extension of larval duration to 7 or 8 days. Consumption of 0.710·718 KJ was the minimum requirement for the completion of larval development. Despite variations in food consumption, assimilation efficiency of the larva remained constant around 97%. Gross production efficiency of the larvae averaged 77%. Larvae ingesting different amounts of host energy emerged with a wide range of body weight (16-62 mg). The efficiency with which pupal energy is transferred to adult was around 30%. Dry weight of meconium egested by the test individuals varied from 1.3 to 4.6 mg. Using simple regression equations, it is possible to predict the bioenergetics components of S. violaceum from the dry weight of meconium.

Energy flow from spider eggs through dipteran parasite and hymenopteran hyperparasite populations.

The incidence of the parasites in the egg sacs of the spider Argiope pulchella was 100% for Sarcophaga banksi and 25% for Tachinobia repanda. In 1976, 237 spider eggs equivalent to 131 gcal/m2 were present. Of these, 212 eggs/m2 (=117 gcal) were consumed by S. banksi larvae, leaving 25 spiderlings/m2 (=14 gcal) to emerge. The density of S. banksi larvae was l/m2 (=87 gcal), of which 0.7/m2 S. banksi (=41 gcal) successfully emerged; as few as 0.3 S. banksi/m 2 (=25 gcal) were infected by T. repanda; only 14 T. repanda/m2 (=8 gcal) successfully emerged. Exploitation efficiency was 89% for S. banksi and 29% for T. repanda. Ecological efficiency was 66% for S. banksi and only 9% for T. repanda. The egg sac area of A. pulchella holds a straight line relationship to the energy content of the eggs; the sacs were grouped into 8 different sizes and each one further into groups containing 1, 2, and 3 S. banksi larvae per sac. Analysis of the sacs at the appropriate time revealed that an S. banksi larva consumed a minimum of 114 eggs (=70 gcal), when present as one of a pair in the smallest sac (0.6 cm2 area), and a maximum of 476 eggs (=234 gcal), when present alone in the largest sac (1.3 cm2). Despite this wide difference in food intake, all S. banksi (barring those infected by T. repanda) successfully emerged. The energy content of a pharate pupa, which was 125, 92, and 68 gcal in a sac with 1 cm2 area containing 1, 2, or 3 S. banksi, depended on the size of the sac and the number of S. banksi per sac. The corresponding values for the imago were 82, 61, and 45 gcal. The efficiency of S. banksi ranged between 60 and 80% for food conversion and between 35 and 56% for pupation.

Use of feeding rate as an indicator of caloric value in some lepidopterous larvae.

A significant, positive correlation between feeding rate and caloric value becomes apparent in 6 species of lepidopterous larvae exposed to different rations, food plants and temperatures. There is a possibility of using feeding rate of the final instar larvae as an indicator of its caloric value. Larvae feeding at the rates of 100, 200, 400 and 600 mg dry weight/g live insect/day contained around 5,320, 5,670, 5,800 and 5,900 g cal/g dry weight, respectively.

Use of faecal weight as an indicator of food consumption in some lepidopterans.

Correlation coefficients obtained between food consumed and feces defecated are highly significant in the larva of Achaea junta and other lepidopteran larvae tested. Different factors like species, stage of life, temperature, and food quality do not significantly affect the value of the correlation coefficient in the larvae tested. The reference value (i.e. the ratio between dry food consumed and dry feces weight) of 1.5 for the moths and 1.9 for the butterflies is suggested for use in the estimation of total food consumption in phytophagous lepidopterans, and its applicbility is discussed. In all tested cases, the correlation coefficients between food consumed and number of pellets defecated showed no significance, indicating the unreliablity of fecal count as an index of food consumption.