Embryo development and maternal–embryo nutritional relationships of piked spurdog (<Emphasis Type="Italic">Squalus</Emphasis><Emphasis Type="Italic"> megalops</Emphasis>)

The maternal–embryo relationship was determined for the piked spurdog (Squalus megalops). In addition, the increase in offspring size with maternal size was studied and the embryonic development was described. Wet weight of in utero eggs and offspring size was correlated with maternal size; larger females produced larger embryos which would have higher survival rate and reproductive value. All embryos present in a female were at a similar stage of development. The external yolk sac is reabsorbed late in gestation, suggesting that embryos are mostly nourished by yolk sac reserves. Embryo size-at-birth varied considerably (180–244 mm total length) as a result of the significant variability in ova size at ovulation. The amounts of water, organic and inorganic matter of embryos at different stages of development were measured to determine possible maternal contributions during embryonic development. Total wet weight from smallest and largest in utero eggs to smallest and largest term embryos changed by +46 and +58%, respectively. This pattern was due to a change in water content by +137 and +154%, and inorganic matter by +100 and +156%. Organic matter of smallest and largest in utero eggs changed by −23 and −17%, respectively. The uterus of pregnant females became specialised for water and mineral transport, not nutrient provision. These results indicate that S. megalops is a strict yolk-sac viviparous species with no maternal contribution of organic matter during development.     

Changes in yolk total proteins and lipid components and embryonic growth rates during lobster (<Emphasis Type="Italic">Homarus americanus</Emphasis>) egg development under a simulated seasonal temperature cycle

From August 2000 to June 2001, seven egg-carrying female lobster (Homarus americanus) from the Îles de la Madeleine population (Gulf of St. Lawrence, Canada) were held under a simulated seasonal temperature cycle to monitor egg development from extrusion to hatching. For the first time, changes in the yolk components (total lipids and major lipid classes, total proteins) and embryo growth of single eggs were monitored separately over the entire development period. Under the controlled temperature conditions, egg development proceeded in three phases. (1) Autumn, from extrusion to early December, was marked by a rapid increase in the Perkinss eye index and rapid declines in yolk total proteins and triacylglycerols (TAG). Embryo daily growth rate was estimated between 1 and 2 µg proteins day^–1. (2) Winter, from late December to early April (temperature stable at ca. 1°C) was characterized by a stationary phase in the evolution of the eye index and yolk lipid use, and embryo growth slowed significantly. (3) Spring, from late April to hatching in June was the period with the most rapid changes in yolk TAG and embryo growth rates >6 µg proteins day^–1 were recorded. Almost 65% of the live biomass (total proteins) of the hatching larvae was accumulated during the last few weeks of development. An index of embryo growth efficiency was estimated as the slope of the relationship between embryo total proteins and yolk TAG during egg development. A relationship was found between the initial mean egg dry weight and the embryo growth efficiency index suggesting that under the same experimental conditions bigger eggs used yolk lipids more efficiently and sustained faster embryonic growth than smaller eggs. The relationship may also explain why larger larvae originate from larger eggs.Communicated by R.J.Thompson, St Johns     

Ecophysiological studies on the developing eggs and embryos of the European lobster Homarus gammarus

During the course of its embryonic development, the European lobster Homarus gammarus Linnaeus exhibits progressive increases in content of water (54.0 to 83.1%), ash (2.7 to 16.7%), protein (47.4 to 50.9%) and non-protein nitrogen (1.0 to 2.4%), and steady decreases in content of fat (43.8 to 25.4%) and energy (6343 to 5431 cal/g dry organic substance). Cumulative yolk utilization efficiency during the total development is 81.8% for dry weight; the corresponding value for energy is 60.1, for protein 75.6 and for fat 47.4%. Energy content of a single egg is 10.49 cal. Of 4.20 cal expended for metabolic processes of the embryo, only 13.3% energy is drawn from protein oxidation; fat oxidation supplies as much as 87.7% energy, that of carbohydrate only 2.3%. Embryonic development results in a remarkable decrease in net yolk utilization efficiency, which falls from 85.5% in the early developmental stages, to less than 70% in later developmental stages. The mean dry weight of a single egg membrane increases from 38 g (2.2% of egg weight) in a freshly laid and attached egg, to 81 g in an egg with an almost completely developed embryo. This result supports the earlier observation of Cheung (1966) that the formation of the inner chitinous egg membrane occurs after the egg is laid and attached to the setum. Protein seems to be the major constituent of the egg membrane (4049 cal/g dry weight), which has the following composition: protein 70.4%, non-protein nitrogen 0.13%, ash 2.83%. Initial permeability of the egg membrane to water (about 6% of the total water requirement is let in) is followed by a period during which the egg membrane is almost impermeable to water (stages I to III); the egg membrane becomes permeable to water again and lets in 85% of the total water requirement (the rest, i.e. 9%, is metabolic water) at a relatively advanced stage of development. These assumed changes in egg membrane permeability appear to be indicative of variations in the egg's osmoconcentration leading to shiftings in net transport of water. Rates of water and salt uptake during embryonic development are essentially parallel (Fig. 1). The egg membrane remains permeable to salts throughout development; salt intake almost doubles after the egg passes through stage III. A single egg, weighing 3.7 mg requires 4.9 mg water for successful completion of embryonic development. The imbibition of water by the developing marine demersal egg seems to (1) serve in osmotic hatching; (2) float the hatched larva by means of specific gravity reduction; (3) aid the larva to quickly adjust its body temperature. The simple osmotic hatching mechanism, proposed by previous workers, seems to be inadequate to account for the events and timing of the hatching process in the lobster. It is suggested that hatching time is determined not solely by increased internal pressure caused by inflow of water and salts, but also by some unknown internal factor. In the lobster egg, as well as in many other marine demersal eggs, protein metabolism is suppressed to a considerable extent, and fat metabolism is geared up Thus, the non-cleidoic lobster eggs exhibit metablic properties which are typical of cleidoic eggs. This finding is discussed in the light of Needham's (1950) concept of cleidoicterrestrial and non cleidoic-aquatic eggs.Dedicated to Professor P. Kochukutta Menon, Montreal, on his 62nd birthday, June 3, 1970.     

Sex ratio manipulation in response to maternal condition in pigeons: evidence for pre-ovulatory follicle selection

A number of recent reports have documented offspring sex ratio biases in birds. However, to date the potential mechanisms that have been put forward to explain the proximate basis for these deviations are entirely speculative. Using a captive population of domestic pigeons (Columba livia domestica), I tested the hypothesis that mothers in relatively poor physical condition should overproduce daughters by manipulating maternal body condition around the time of egg laying by continuous egg removal and differing feeding regimes. During treatment, females were fed a controlled quantity of food. This, combined with the high energetic costs of repeated egg production caused a significant reduction in maternal body weight. In contrast, during control when food was available ad libitum, maternal body weight did not decline, despite repeated egg production. No significant deviation from parity was evident in the sex ratio of either the first or second eggs during control, whereas during treatment a significant female bias was evident in not only the first egg, but also in the second egg. The absence of single-egg clutches, the rarity of infertile eggs and the lack of laying delays between eggs strongly suggests that the mechanism of sex ratio adjustment in pigeons occurs prior to ovulation. The highly skewed sex-distribution within the two-egg clutches and the unexpectedly large amount of variation in the yolk weight of eggs produced during treatment (but not control) are consistent with the expectations of pre-ovulatory selective resorption of wrong sex ovarian follicles.     

Yolk utilization in the gastropod Crepidula fornicata

The increases in constituents per unit weight of eggs during embryonic development of the gastropod Crepidula fornicata amounted to 14.6% for ash, 1.0% for protein, and 0.3% for non-protein nitrogen. During the same stages, fat content decreased from 33.7 to 20.3%, carbohydrate from 10.2 to 7.7% and energy content from 6209 to 5298 cal/g dry organic substance. The cumulative efficiencies for yolk utilization were 83.8% for dry weight, 61.0% for energy, 85.1% for protein, 50.7% for fat, and 63.6% for carbohydrate. A single egg contained 0.0269 cal, a single veliger 0.0164 cal. Of the 0.0105 cal expended on metabolic processes of the embryo, oxidation of fat contributed as much as 65.3%, while that of protein and carbohydrate amounted only to 18.8 and 6.3%, respectively. On the basis of ecophysiological considerations, a new classification of eggs is proposed.This paper is based on a lecture presented during the first Convention of the Indian Association of Biological Sciences in Madras, December, 1968.     

Determination of copper in embryos and very young specimens of Sepia officinalis

The total amount of copper in embryos and newly hatched young individuals of Sepia officinalis L. has been determined by microtechnique, using bathocuproine-sulfonate as complexing reagent. During embryonic life, the total amount of copper does not change; it remains at a level close to 3.8 g. The copper is found in the yolk sac of very early embryos; it is subsequently transferred into the embryo proper. After hatching, the copper content diminishes quickly in starved individuals. Fed S. officinalis also usually lose copper. The reason for this may be that the inner yolk sac of newly hatched individuals contains a great deal of the total copper, which is excreted with the yolk after the latter has become superfluous. Later on, copper must be taken up from the food. The mobilization of protein and copper from the yolk into the blood may account for the early appearance of embryonic hemocyanin in the blood.     

Sexual reproduction in the compound ascidian Diplosoma listerianum (Tunicata). II. Sperm penetration through ovary wall and evidence of internal fertilization

Diplosoma listerianum differs from most ascidians in that, at ovulation, eggs are emitted at the bottom of the ovary and segregated into the tunic, so that fertilization occurs far from seawater. A fertilization canal, a hollow extension of the ovary, conducts sperm towards the egg. In the present paper, ultrastructural evidence is reported on the morphological relationship between the ovary, egg envelopes and oocyte and on the mechanism by which sperm-egg interaction is established. In the ovary, the very complex sperm, equipped with a spiral dense groove, undergo metamorphosis as the first step in a sperm reaction and then pass through the ovary epithelium insinuating themselves between the intercellular junctions which appear to be mouldable, although able to maintain the egg-ovary barrier. Sperm then reach the vitelline coat, where a further step in the sperm reaction occurs. Before the egg abandons the ovary, the sperm head is incorporated into the oocyte by a process recalling phagocytosis, with the formation of an engulfing pocket. Sperm-egg contact and incorporation in D. listerianum occur in a way, never previously reported for other ascidians, in which fusion of plasma membranes takes place immediately after sperm-egg contact. Unlike other cytoplasmic components, the dense groove persists until the sperm enters the egg. It gives a corkscrew-like configuration to the sperm head and allows close adhesion to cell membranes, facilitating sperm movement. Expulsion of numerous cortical granules and features of a cortical reaction were observed in the egg penetrated by the sperm. The mode of internal fertilization of this species in comparison with that of other tunicates and phylogenetic aspects are discussed. Ripe colonies of D. listerianum collected in the Lagoon of Venice, Italy in 1986 and 1991 and colonies reared on glass in aquaria were used for our investigations.     

Digestive carbohydrases from the hepatopancreas of two Antarctic euphausiid species (Euphausia superba and E. crystallorophias)

The hepatopancreatic extracts of Euphausia superba Dana and E. crystallorophias Holt and Tattersall collected from the Antarctic. Ocean during January 1985, are most effective in hydrolysing substrates containing (13)--glucosidic linkages. Three enzymes appear to be involved in the depolymerization of (13)--D-glucans in the euphausiid diet: (1) an exo-(13)--D-glucanase, (2) an endo-(13)--D-glucanase and (3) a -D-glucosidase. The glucanases have a pH optimum of 5.4, a temperature optimum of 50°C and are optimally extracted in bistripropane buffer, pH 7.2. Levels of (13)-D-glucanase in laboratory-cultured E. superba are inversely affected by food availability, and activities double after starvation for 12 d. The increase is due mainly to higher activities of exo-acting glucanases. -Amylase and endo-(14)--D-glucanase (cellulase) activity are also present in the extracts in addition to glycosidase activity against a range of p-nitrophenyl substrates (-and -D-glocose, - and -D-galactose, -D-xylose, - and -D-mannose). Digestive activity against several acidic polysaccharides, including the acidic mucilage polysaccharide of the ice diatom Stauroneis amphioxys, is minimal and is not induced when the polysaccharide is present in the diet of E. superba. These results indicate that some, but not all, components of the algal material in the euphausiid diet can be hydrolyzed and assimilated.     

Comparison of larval development and growth of the sea cucumberActinopyga echinites: Ovary-induced ova and DTT-induced ova

AdultActinopyga echinites (Jaeger) were collected from northern Taiwan in September 1989. Oocytes were induced to mature by bathing them in ovary juice (ovary-induced ova) or in 10^–2 M dithiothreitol (DTT-induced ova). The percentage of germinal vesicle breakdown (GVBD) increased from 0.4 to 6.4% in the former treatment and to 84% in the latter. After artificial fertilization, the embryos were cultured in seawater (35 S) at 25 to 28°C. Larvae were fed with the algaIsochysis aff.galbana at 10^4–5 cells/ml. Larvae from ovary-induced ova developed faster (18 d to the doliolaria stage) and grew to a larger size (1.13 mm length) than those from DTT-induced ova (20 d to the doliolaria stage and 0.62 mm in length). On the twelfth day, larvae from ovary-induced ova bear lipid spheres. The number of spheres is positively correlated with larval size. Lipid spheres may provide nutrient reserves for larvae during metamorphosis.     

Water and salt balance of halibut eggs and larvae (Hippoglossus hippoglossus)

Eggs of halibut [Hippoglossus hippoglossus (L.)] have a negative buoyancy in sea water of 35 S, in contrast to eggs of most flatfish species. The cause of this was investigated. The osmolality of the yolk is 350–420 mOsm during embryonic development. This is within the range for marine teleost serum and for yolk of pelagic eggs. Concentrations of major inorganic ions are comparable with those of pelagic plaice eggs [Pleuronectes platessa (L.)]. The values for Na⁺, K⁺ and Cl^- are 6, 85, and 64 mmol · (1 H₂O)^-1 after fertilization, and at the time of hatching the corresponding values are 17, 11, and 80. Large amounts of other inorganic constitutents are excluded for osmotic reasons. Malfunction in the regulation of osmolality or of inorganic constituents is thus unlikely to be the cause of negative buoyancy. The relative dry weight of the chorion (egg shell) in halibut eggs is less than in several pelagic egg types, excluding the chorion as the main contributor of negative buoyancy. It is concluded that a high content of organic matter in the rest of the egg is the cause of the negative buoyancy.     

Aspects of the reproduction and life cycle of Rhabdopleura compacta (Hemichordata)

Rhabdopleura compacta Hincks is shown to be monoecious. It is suggested that the ovum is ovulated and extruded into the lumen of the coenecium via a temporary opening at the base of the metasome, and that the oviduct itself may serve only as a way for sperm to fertilise the ova internally. The zooids degenerate when reproducing sexually and when conditions are unfavourable. The larva and initial stages of astogeny are described. Some observations on seasonal variation in the colonies indicate that the dormant buds serve for storage of nutrients. Reproduction seems to occur throughout the year, but most actively in spring. Relationships with Cephalodiscus and the Bryozoa are briefly discussed.     

Fecundity of the Argentinean mackerel Scomber japonicus marplatensis

Research on the spawning and fecundity of the Argentinean mackerel Scomber japonicus marplatensis was carried out during the spawning season 1966/67. The purpose of the present study was to obtain data for the estimation of the number of eggs which can be produced and spawned by one female of a known size, during one season of reproduction: 152 females were studied. All yolked ova of the biggest size group were taken into consideration. On the basis of the relatively short spawning season, the great homogeneity in meristic characteristics, and histological examination, it is assumed that the Argentinean mackerel spawns once a year. The fecundity of the smallest female of 315 mm in total length was calculated to be 101,859 eggs, and the total number of yolked ova 181,000; for the biggest female of 470 mm in length, 522,370 and 853,500, respectively. The relation fecundity/length and fecundity/weight is almost rectilineal. The equations: F=8.17 ^0.008L for fecundity/length relationship, and F=0.42 W ^1.043 for fecundity/weight relationship, were calculated. Prediction curves were established in each case. It would appear that the fecundity of smaller fish is associated more with length, and the fecundity of bigger fish more with weight. Large differences in fecundity of specimens of the same size were observed.Communicated by M. Vannucoi, So Paulo     

Oogenesis and spawn-formation in the pigmy lion fish Dendrochirus brachypterus (Pteroidae)

Dendrochirus brachypterus possesses a special type of ovary in which the germinative epithelium surrounds a spongy stroma hanging in the lumen. The inner side of the ovarian wall is covered by a special type of epithelium that produces a gelatinous envelope around the gametes (spawn) during reproduction. In general, oogenesis in the lion fish resembles that of other teleosts, the characteristic difference being in the formation of ovigerous peduncles-protrusions of the spongy stroma, each of which bears a single oocyte. During oogenesis the oocytes rise above the germinal epithelium, enveloped in their follicular epithelia. The larger the oocyte the greater the length of its peduncles and the further its position from the germinal layer (Fig. 3). During this phase of development, a marked multiplication of yolk granules occurs; they increase their diameter and finally homogenize at ovulation. Under favorable thermal conditions, oogenesis continues all year round. The peduncular growth of oocytes enables the growing egg cells to move away from the egg-producing surface, thus making space available for the ovary to produce more eggs. This ability should have a survival value for these fish if we take into consideration that the most vulnerable stage in their life history is that of planktonic larvae (Fishelson, 1975).     

Spawning behaviour,egg and larval development of the myctophid fish Benthosema pterotum

The distribution of eggs and early larval stages of the myctophid fish Benthosema pterotum was studied during three cruises to the Gulf of Oman during January to February 1981 and February 1983. The eggs and yolk sac larvae were identified and described after artificial fertilization on board the Research Vessel Dr. Fridtjof Nansen. The mature fish spawn in the Gulf at 300 to 100 m depths during early night. The eggs and smallest larvae were found at 300 to 200 m depths. The eggs hatch within 12 h at 21°C. Hatching occurs before the slightly bouyant eggs reach the upper 50 m of the water column.     

Marine Gammaridea (Crustacea: Amphipoda) from the Indian region. Family Ampithoidae

This paper deals with 6 species of marine gammaridean amphipods belonging to the family Ampithoidae. The specimens described were collected from Mandapam (Gulf of Mannar), India and Thankasserry (Quilon, Kerala), India. The collection includes 3 new species, namely Ampithoe (Ampithoe) serraticauda, Ampithoe (Pleonexes) auriculata and Cymadusa imbroglio. Ampithoe (Ampithoe) kergueleni Stebbing (1888) is a new record for this region, while Ampithoe (Ampithoe) ramondi Audouin (1826) and Cymadusa filosa Savigny (1816), the two well-known tropicopolitan members, are briefly redescribed. The study supports Barnard's (1970, p. 23) contention that the genus Pleonexes Bate (1857) (since reduced to subgeneric status under the genus Ampithoe Leach, 1814) represents only a grade of evolution, but suggests that the structure of the 1st maxillary and mandibular palps and the outer lobes of the lower lip should also be taken into consideration, in combination with the telsonic processes, for distinguishing Pleonexes from Ampithoe. Based on this concept, a reappraisal of some of the species of the genera is suggested. The subgenus Ampithoe is created to hold those species of Ampithoe showing typical characteristics of the genus.     

Reproductive biology of the common antarctic crinoid Promachocrinus kerguelensis (Echinodermata: Crinoidea)

Histological examinations of the gonads of Promachocrinus kerguelensis Carpenter, 1888 collected in November 1984 from McMurdo Sound, Antarctica, revealed ripe ovaries containing three distinct size classes of oocytes: 20 to 90, 91 to 150 and 151 to 180 m diameter as well as ova. Testes were ripe, and filled with mature sperms. There was marked inter-pinnule and inter-individual synchrony of gametogenesis and spawning is probably synchronous, at least within the sub-population sampled. The number of eggs per pinnule is allometrically related to body size. Body weight and arm length are positively correlated with total egg number. Annual fecundity was estimated to be 29 000 eggs (full-grown oocytes and ova) for a crinoid weighing 53.6 g wet weight with an arm length of 16.3 cm. The eggs float. Female genital pinnules contain more lipid and energy than male pinnules. Reproductive output and effort (23.4 to 65.2 kJ and 26 to 38%, respectively) increase with body size and are similar to values reported for other echinoderms with lecithotrophic larvae. The greatest overall energetic contribution is represented by the arms and genital pinnules. Spawning in P. kerguelensis probably occurs in November and December within McMurdo Sound. If development is slow, as in all other antarctic echinoderms studied to date, then settlement most likely occurs 2 to 3 mo later, after the short summer period of high phytoplankton productivity. High fecundity, a pelagic mode of reproduction, and the high probability of a slow rate of development may contribute to the wide geographic distribution of this common antarctic crinoid.     

Yolk utilization and hatching time in the Canadian lobster Homarus americanus

During the course of its embryonic development, the Canadian lobster Homarus americanus Milne-Edwards exhibits steady increases in water content (56.2 to 86.8%) and ash (5.8 to 21.2%), and a progressive decrease in energy content from 6636 to 4292 cal/g dry weight. Mean dry weight of a single egg is 965 g, equivalent to 6.4 cal; a freshly hatched egg. The lobster hatches about 1,500 larvae per night over a period of 4 to 5 days. Dry weight, ash and calorific contents of larvae hatched on different days show considerable variations. After larvae hatch on the first day, continuous salt absorption by eggs to hatch on subsequent days leads to a steady increase in ash content from 143 g/larva hatched on the first day to 255 g/larva hatched on the fourth day, and consequently, to an increase in dry weight from 854 to 956 g/larva. Metabolism of embryos (0.1 cal/day), which are yet to be hatched on subsequent days, depletes the calorific content per unit weight (from 4637 to 3837 cal/g dry weight) as well as per larva (from 3.98 to 3.67 cal).Dedicated to my colleague K.-H. Schumann (Biologische Anstalt Helgoland) who died on December 12, 1969 due to a diving accident.     

No evidence for selective follicle abortion underlying primary sex ratio adjustment in pigeons

Primary sex ratio adjustment in birds has been extensively studied, yet the underlying physiological mechanisms are far from understood. Avian females are the heterogametic sex (ZW), and the future sex of the offspring is determined at chromosome segregation during meiosis I, shortly before the oocyte is ovulated. Assuming that the mother can detect the sex of the developing oocyte before ovulation, it has been suggested that a follicle of the un-preferred sex could selectively be induced to become atretic and regress instead of being ovulated (selective follicle abortion). This potential mechanism has been proposed to underlie biased primary sex ratios in birds, including the homing pigeon (Columba livia domestica), which produces a modal clutch size of two eggs. However, without replacement by an additional, already mature follicle, abortion of a preovulatory follicle would most likely result in either reduced clutch sizes or laying gaps, since a not-yet-recruited follicle still needed to undergo the whole maturation phase. In the current study we killed female pigeons, which were adjusting embryo sex of first eggs according to change in body mass. We examined ovaries for signs of follicle abortion but did not find any supporting evidence. All females produced one or two mature follicles but only two out of the 56 experimental birds produced an additional third mature follicle. Therefore, our results do not corroborate the hypothesis that pigeon mothers manipulate primary offspring sex by selectively aborting follicles of the un-preferred sex.     

Egg development in a littoral pycnogonidNymphon gracile

Aspects of the reproductive biology ofNymphon gracile (Leach) are examined. The eggs develop between August and February, when they are passed to the ovigerous legs of the male. The process of vitellogenesis closely resembles that of annelids andLimulus polyphemus, in which the majority of the yolk is synthesised intra-oöcytecally, with only a small extra-oöcytic contribution.     

Effect of hatching time on larval mortality and survival of the prawn Macrobrachium idae

The prawn Macrobrachium idae Heller carries 40 to 160 eggs depending upon the body size of the mother animal. After incubation, 63, 35 and 2% eggs are hatched on the first, second and third hatching nights, respectively. Mean dry weight of a single larva relased on the first night is 420 g (equivalent to 2.86 cal). Larvae released on the second or third hatching nights weighed 380 g (=2.56 cal) or 308 g (=2.02 cal). The total reserve yolk-energy available in a larva hatched on the first night is 1 cal. There is a definite shift from protein to fat metabolism as hatching is delayed. Oxygen consumption of developing eggs awaiting release on the first or second hatching night is 1.9 l/mg dry weight/h. In comparison to larvae released on the first night, those released on the second night exhibit 2% increase in total body-length and 2.4 or 14.3% decrease in the lengths of the 6th abdominal segment or the orbit. Larvae released on the first, second or third hatching nights, on exposure to starvation stress, survive for 3.8, 2.3 or 1.5 days. The swimming speed of larvae released on the first or second night is 1.0 or 0.6 cm/sec. Larvae released on the subsequent hatching nights committed nearly 2 times greater number of mistakes per cm distance in the horizontal plane. Since hatching of all developing eggs simultaneously on the first hatching night is possible by means of artificial technique(s), it is possible to improve survival rate of decapod larvae.