Note: Descriptions are shown in the official language in which they were submitted.
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CONTROL OF CRUSTACEAN INFESTATION OF AQUATIC ANIMALS
FIELD OF INVENTION
The present invention relates in its broadest aspect to the production of
aquatic animals
such as fish. In particular there is provided novel and improved means of
controlling para-
sitic crustacean infestations of fish, including farmed fish, using juvenile
hormone ana-
logue compounds.
TECHNICAL BACKGROUND AND PRIOR ART
Ectoparasitic infestations constitute considerable problems in the fish
farming industry as
well as in wild fish. However, the problems are particularly serious in farmed
fish in both
fresh water and sea water environments. Damages due to ectoparasitic
infestations of fish
result in considerable losses and increased workloads for fish farmers. Among
ectopara-
sites in fish and other aquatic animals, parasitic species of crustacean
ectoparasites, also
generally referred to as sea lice, are particularly harmful. Thus, infestation
with sea lice in-
cluding Lepeophtheirus salmonis and Caligus elongatus is considered to be one
of the
most important disease problems in farming of salmonids, especially in
Atlantic salmon
(Salmo salary and rainbow trout (Oncorhynchus mykiss). In addition to the
costs that are
associated with treatment, lower classification ratings of slaughtered fish
and reduced
growth rate due to reduced feed intake contribute to the economic losses
caused by sea
lice.
The life cycle of Lepeophtheirus salmonis is direct, meaning that no
intermediate host
is required for the development of the adult lice. The two nauplius stages of
this species
are pelagic. The infective stage, the copepodites, attach to fish, normally on
their ventral
surfaces and on the fins. It moults to the first chalimus stage, which is
attached to its host
by a frontal filament projecting from the leading edge of the parasite's
carapace. All four
chalimus stages are attached to the host by this filament. At the moult to the
first pre-adult
stage, the frontal filament is normally lost, and the parasite may move around
on the skin
of the host. Adult males may mate with pre-adult females, which may therefore
already be
pregnant when they moult to adults. The eggs are stored in egg sacs hanging
from the
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genital segment of the body. The completion of the life cycle normally takes
about six
weeks at a water temperature between 9 and 12°C. The life cycle of
Caligus elongatus is
similar to that of Lepeophtheirus salmonis.
In addition to the damages caused in farmed fish, recent research has shown
that sea lice
could be the most important single cause leading to weakening of several wild
salmon
stocks. The emigration of salmon smolt from river systems is often
coincidental with rising
sea water temperatures in the fjord and coastal areas, which leads to a
massive attack of
copepodites (sea lice larvae) on the smolt. An attack of 40 or more sea lice
on salmon
smolt is fatal to fish weighing less than 25 grams, and in several regions
premature return
of post-smolt sea trout has been observed. The decline in the stock of
salmonids that
have been documented in several salmon rivers over the past few years, could
be related
to the fact that the amount of sea lice in connection with fish farming has
increased.
Up till now, the most common treatment of fish ectoparasites involves bathing
or immers-
ing the fish in a treatment solution comprising an antiparasitically active
compound. This
includes both skin and gill parasites. Bathing in formalin is a widespread
treatment against
many ectoparasites, especially in fresh water, while bathing in a solution of
an organo-
phosphate such as e.g. metrifonate, dichlorvos or azamethiphos, a pyrethroid
compound
such as pyrethrum, cypermethrin or deltamethrin (WO 92/16106), or hydrogen
peroxide
are the most common bath treatments against e.g. sea lice. These compounds act
directly
on the ectoparasites via the water and a possible absorption of the active
substance into
the fish itself is unimportant for the effect of the active substances on the
parasite.
Substances that are effective against parasitic infestations via oral
administration have
also been tested in fish. Substances such as the chitin synthesis inhibitors
diflubenzuron
and teflubenzuron, are examples of substances, which, if administered orally,
can be ef-
fective against parasitic diseases in fish. In addition to the substances
mentioned above,
wrasse (Labridae) has been used extensively to keep sea lice infestations
under control.
Organophosphates and hydrogen peroxide are only effective against the pre-
adult and
adult stages of sea lice (the last 3 stages of the total of 8 stages which
exist on the skin of
salmonids), while pyrethroids and ivermectin also have a more or less well-
defined effect
against the other 5 stages. None of these substances protect against new
infestations af-
ter the treatment has been completed.
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Hydrogen peroxide is corrosive, and must therefore be handled with great care.
Transport
of hydrogen peroxide requires certain precautionary measures, as it is defined
as hazard-
ous goods in great quantities. The organophosphates are toxic to humans and
must
therefore be treated with caution. The organophosphates can be absorbed
through the
skin and lead to poisoning. The therapeutic margin for organophosphates and
hydrogen
peroxide is small. Fish mortality has been reported on several occasions, due
to over-
dosing of such drugs.
Pyrethroids have, in addition to their effect against pre-adult and adult
lice, also an effect
against the attached stages. They are not acutely toxic to the user, but is a
drug group
that is toxic to fish, especially small fish. Overdosing and increased
mortality of the target
fish is thus possible.
In addition to bath treatments, oral treatments for parasite control in fish
have been devel-
oped. Two chitin synthesis inhibitors, diflubenzuron and teflubenzuron, have
been docu-
mented for use against sea lice in salmon.
Diflubenzuron and teflubenzuron act by inhibiting the synthesis of chitin
which is an im
portant component of the cuticle of insects and crustaceans. At each moulting
or ectdysis,
a new synthesis of chitin is required for development. If this synthesis is
inhibited, the de-
velopment of the insect or crustacean will be halted, and the animal under
development
will die. In principle, chitin synthesis inhibitors will be effective against
all organisms con-
taining chitin. Fish and mammals do not contain chitin and will therefore not
be affected by
substances within this drug group. This is reflected in very low toxicity for
fish and mam-
mals, including humans. Diflubenzuron and teflubenzuron are administered to
the fish via
the feed, absorbed and distributed to skin and mucus, where the concentration
will be
high enough to inhibit the development of the parasite. The disadvantages of
diflubenzu-
ron and teflubenzuron include that these substances have no effect against
adult stages
of the ectoparasites which do not actively synthesise chitin. Neither do they
have any ef-
fect beyond the period of treatment, as attacks by new parasites may occur
within days of
completing the treatment. This is due to the fact that the substances are
eliminated rela-
tively fast so that the concentration hereof rapidly ends up below
therapeutically active
levels in skin and mucus. The treatment must therefore be repeated if there is
a continu-
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ous risk of parasite infection from the surroundings which, under normal
circumstances, is
often the case.
Ivermectin impairs the transfer of neural impulses in insects and crustaceans.
This leads
to paralysis and death. Mammals and fish are also affected by ivermectin.
However, the
same transfer mechanisms that are affected in insects, are only found in the
brain of fish
and mammals. Mammals have an advanced blood-brain barrier which prevents toxic
ef-
fect from lower concentrations. The blood-brain barrier in fish is less
developed and this
causes a substantial transition to the brain of ivermectin in treated fish,
and toxic symp-
toms are found at relatively low concentrations. Ivermectin, however, is
effective for
treatment of ectoparasites in fish provided that precautions with the dosing
are taken.
Toxic effects and mortality may arise if the fish are overdosed. Ivermectin is
often dosed 1
or 2 times a week to control sea lice infestation in salmonids. This means
that the sub-
stance must be added on a continuous basis to maintain therapeutic effect and
keep the
fish reasonably free of lice. Ivermectin is eliminated at a slow rate which
means that the
effect of the treatment is maintained for 2 to 3 weeks after the treatment has
been com-
pleted. This also means, however, that the treatment involves a long
withdrawal period
before the fish can be slaughtered and consumed. Up till now, ivermectin has
not been
approved for use in fish in any country.
As it appears from the above summary of the state-of-the-art, there is an
industrial need
for improved means of controlling parasitic infestations in fish, which are
antiparasitically
effective and non-toxic to the fish and which are non-toxic to the end-user
and environ-
mentally friendly.
The metamorphosis of arthropods (insect and crustacean species) is controlled
by several
hormones. In insects, such hormones include moulting hormones (MN), also
referred to
as ecdysones, and juvenile hormones (JH) that are terpenoid compounds.
Together with
the moulting hormones circulating in the bloodstream of the insects, the
juvenile hor-
mones that are released from the corpora allata in the insect's head play
vital roles in
growth, development and reproduction of insects. If insects are treated with
an excess of
the juvenile hormone at an early stage in their development, they remain at a
juvenile
stage or develop into a sterile adult insect.
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Whereas natural JHs have not been used commercially as insect controlling
compounds,
several JH analogues (JHAs), also referred to herein as juvenile hormone-like
com-
pounds, juvenoids or JH mimics, are used as insect controlling compounds.
Examples of
such compounds include epofenonane, triprene, methoprene, hydroprene,
kinoprene,
5 phenoxycarb and those compounds disclosed in US 4,061,757. Currently,
typical uses of
JH analogues include control of mosquitoes, mites, ants, aphids, cockroaches
and fleas.
It has now been discovered that compounds having juvenile hormone activity in
insects
are highly active in the control of crustacean infestations of aquatic
animals.
SUMMARY OF THE INVENTION
Accordingly, the present invention pertains in a first aspect to the use of a
compound
having juvenile hormone activity, in the manufacturing of a medicament for
controlling
crustacean infestation of aquatic animals including fish such as wild and
farmed fish of the
Salmonidae family.
In a further aspect, there are provided novel juvenile hormone analogue
compounds se-
lected from the group consisting of
(i) a compound of the general formula I
R~ X ~ ~ R2
Y Z
where R' is alkyl, branched alkyl including alkenyl or alkadienyl, optionally
substituted
isoprenoid or alkoxy substituted alkyl, RZ is hydrogen or alkyl, X is oxygen,
sulfur, methyl-
ene, carbinol or carbonyl, Y is nitrogen or methine and Z is nitrogen, methine
or nitrogen
oxide;
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(ii) a compound of general formula II
R'
R2
where R' is 2,2-dimethylcyclopropyl, 2,2-dihalo-3,3-dimethylcyclopropyl, 2-
methyl-1-
propenyl or 3-methyl-1,2-butadienyl, RZ is hydrogen or methoxy, R3 is alkyl,
alkenyl, ace-
tyl, formyl or carbomethoxy, R4 is hydrogen or R3 and R4 together is
methylenedioxy and
X is oxygen or sulfur;
(iii) a compound of the general formula III
\ \ ~R2
R'
where R' is 2-methylbutyl, 2,2-dimethylcyclopropyl or 2,2-dihalo-3,3-
dimethylcyclopropyl
and RZ is methyl, ethyl or isopropyl,
(iv) a compound of the general formula IV
X Ar
where X is oxygen or sulfur and Ar is a heteroaromatic group, and
(v) a compound selected from the group consisting of
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55: 4-Octylmorpholine;
56: 1-Octylpiperidine;
57: 2-(3',7'-Dimethyloctylsulfanyl)thiophene;
58: 1,4-Dioctylpiperidine;
F-1:3-[2'-(4"-Phenoxyphenoxy)ethoxyjpyridine;
F-4: 2-[2'-(4"-Phenoxyphenoxy)ethoxy]pyridine; and
F-5: 3-(4'-Phenoxybenzyloxy)pyridine.
for use as a medicament for treating or preventing crustacean infestations in
an aquatic
animal.
In a still further aspect the invention provides a method of controlling
crustacean infesta-
tion of an aquatic animal, the method comprising administering to said animal
or to the
aquatic environment of the animal an effective amount of a juvenile hormone
analogue
compound including any of the above novel compounds.
There is also provided a pharmaceutical composition for controlling crustacean
infestation
of an aquatic animal, comprising a juvenile hormone analogue compound and at
least one
pharmaceutically acceptable carrier, and an aquatic animal feed composition
comprising a
juvenile hormone analogue compound.
DETAILED DISCLOSURE OF THE INVENTION
A major objective of the present invention is to provide improved means for
controlling
crustacean ectoparasitic infestations in aquatic animals such as fish. The
invention is
based on the finding that a range of compounds having juvenile hormone
activity gener-
ally referred to as juvenile hormone analogues (JHAs), which are known to have
insect
controlling activities including insecticidal activity, have a strong
inhibiting effect on the
metamorphosis of parasitic crustacean species resulting in a high mortality of
these para-
sites not only in vitro but also in vivo, i.e. when the parasites are attached
to the host ani-
mal, rendering such compounds potentially useful in the control of such
ectoparasitic in-
festations in live fish.
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As used herein, the term "juvenile hormone analogue" indicates a synthetic
compound
that is structurally and/or functionally related to the naturally occurring
juvenile hormones
I, II and III, terpenoid substances that are found in insects where they
regulate the devel-
opment from the larval stage to the imago stage of the insects. Due to this
juvenile hor-
s move effect, JHAs are currently used as insect controlling agents (see e.g.
US 4,002,615
and 4,061,757). Juvenile hormone analogues are also referred to as juvenoids
or juvenile
hormone mimics.
In one aspect of the present invention there is provided the use of a compound
having ju-
venile hormone activity, in the manufacturing of a medicament for controlling
crustacean
infestation of aquatic animals.
Whereas other aquatic animals than fish may be infested by crustacean
ectoparasites, the
use according to the invention is particularly interesting for manufacturing
medicaments
for control of crustacean infestation in fish species having their natural
habitat in cold,
temperate or warm water environments. Thus, the medicament of the invention
can be
used therapeutically and/or prophylactically for controlling infestations in
both wild and
farmed fish including ornamental fish, occurring in freshwater, sea water or
brackish wa-
ter. Fish that can be treated with JHAs include any fish that can be
infestated by ecto-
parasitic crustaceans such as wild and farmed fish belonging to the Salmonidae
family
including, but not limited to Salmo salar, Salmo trutta, Salmo clarkii,
Oncorhynchus gor
buscha, Oncorhynchus keta, Oncorhynchus nekra, Oncorhynchus kisutch,
Oncorhynchus
tshawytscha, Oncorhynchus mason, Oncorhynchus mossambicus, Oncorhynchus mykiss
and Salvelinus species. Other examples of fish where the JHAs can be used
include
carps, whitefish, roach, rudd, chub, sole, plaice, Japanese yellowtail, sea
bass, sea
bream, grey mullet, pompano, gilthread seabream, Tilapia spp., Cichlidae spp.,
cod, hali-
but, wolf fish, flounder, aju and eel including Japanese eel.
Presently preferred target fish for the present invention include Atlantic
salmon, Pacific
salmon and trout.
It has been found that a large range of JHA compounds effectively control
infestations by
crustacean species generally referred to as sea lice such as Lepeophtheirus
species.
However, as the metamorphosis processes are essentially the same in any
crustacean
species, it is envisaged that JHAs will be effective against other
ectoparasitic crustacean
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species infestating aquatic animals. Such species are i.a. found in the
following genera:
Ergasalus, Bromolochus, Chondracaushus, genera and species belonging to the
Cope-
pods class including the genera Caligus and Lepeophtheirus, Dichelestinum,
Lambro-
glenz, Hatschekia, Legophilus, Symphodus, Ceudrolasus, Pseudocycmus, Lernaea,
Ler
naeocera, Pennella, Achthares, Banasistes, Salmonicola, Brachiella,
Epibrachiella, Pseu-
dotracheliastes; and the families: Ergasilidae, Bromolochidae,
Chondracanthidae, Caliji-
dae, Dichelestiidae, Philichthyidae, Pseudocycnidae, Larnaeidae,
Lernaepotidae, Sphyrii-
dae, Cecorpidae, Branchiuriae (carp lice) with the family Argulidae that
includes Argulus
species.
In this context, important target species of crustaceans include
Lepeophtheirus salmonis,
Caligus elongatus and Anilocra physodes L, crustaceans of a species selected
from the
group consisting of an Anilocra species, a Chymothoa species, a Lironeca
species, a
Meinertia species, an Olencira species and a Bopyrus species and crustaceans
of the Iso-
pods class including Anilocra physodes, Chymothoa exigua, Lironeca califomica,
Liro-
neca convexa, Lironeca ovalis, Lironeca vulgaris, Meinertia oestroides,
Meinertia parallels
and Olencira praegustator.
In accordance with the invention, any compound having juvenile hormone
activity, includ-
ing a juvenile hormone analogue compound, that is effective with respect to
retarding or
inhibiting any stage of the development of crustaceans from the first larval
stage to the
pre-adult stage and/or which has a biological effect on any stage of the
crustaceans in-
cluding the adult stage is encompassed. Thus, a compound according to the
invention
that is capable of retarding or inhibiting at least one stage shift from the
nauplius I stage
through further nauplius stages, copepodite stages, chalimus stages, the pre-
adult stage
to the adult stage. Such compounds include juvenile hormone mimics such as
epofe-
nonane, phenoxycarb, hydroprene, kinoprene, methoprene, pyriproxyfen and
triprene, ju-
venile hormones I, II and III, and the compounds disclosed in US 4,002,615 and
4,061,757. It should be understood that chitin synthesis inhibitors are not
included in the
above definition of compounds having juvenile hormone activity.
Also encompassed within the meaning of compounds having "juvenile hormone
activity"
are compounds which prove to be positive when tested in a standard test system
for
evaluating compounds for juvenile hormone activity. Such standard tests,
wherein Teni-
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brio mo/itortypically is applied as test organism, are well known in the art
(se e.g. Slama
et al., Insect Hormones and Bioanalogues, Springer-Verlag, New York 1974, p.
93).
Additionally, the below evaluation system for determining juvenile hormone
activity can be
5 applied (adapted from "Methods used to evaluate candidate materials for
juvenile hor-
move activity", Pesticide Chemicals Research Branch, Beltsville, Maryland):
Juvenile hormone activity may be determined using newly-molted (4-8 hours)
Tenibrio
motitor pupae. The compounds to be evaluated are formulated to contain 10 Ng
in 1 ml of
10 solution. Acetone is the preferred solvent and is used in both topical and
vapor tests.
Topical application is with a micro applicator (Isco model M) fitted with a
tuberculin syringe
and a 27 gauge needle. One NI of the desired solution is administered/pupa on
the venter
of the last three abdominal segments. Vapor action is determined by applying
the candi-
date compound to the lower 1/3 of a 1 pint freezer type jar and then inverting
the jar into a
1/2 pint container, containing 5 pupae. A dosage of 1 NI of the desired
solution per pupa
(5 NI/jar) is used. All pupae are held until the following molt to determine
juvenile hormone
activity, which is indicated by the presence of immature characters; e.g. i)
retention of gin
traps, ii) retention of gin traps and urogomphi, iii) retention of gin traps
and urogomphi
plus retention of pupal cuticle around area of treatment, and iv) 2nd pupae -
retention of
all pupal characters. If a perfect adult is obtained after molting, the
compound has no ju-
venile hormone activity. Farnesyl methyl ether is used as the standard for
both topical and
vapor tests.
In the present context, particularly interesting compounds having juvenile
hormone activ-
ity include:
(i) compounds of the general formula I
R1 X ~ ~ R2
Y Z
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where R' is alkyl, branched alkyl including alkenyl or alkadienyl, optionally
substituted iso-
prenoid or alkoxy substituted alkyl, R2 is hydrogen or alkyl, X is oxygen,
sulfur, methylene,
carbinol or carbonyl, Y is nitrogen or methinine and Z is nitrogen, methine or
nitrogen ox-
ide;
(ii) compounds of general formula II
R~
R2
where R' is 2,2-dimethylcyclopropyl, 2,2-dihalo-3,3-dimethylcyclopropyl, 2-
methyl-1-
propenyl or 3-methyl-1,2-butadienyl, RZ is hydrogen or methoxy, R3 is alkyl,
alkenyl, ace-
tyl, formyl or carbomethoxy, R4 is hydrogen or R3 and R4 together is
methylenedioxy and
X is oxygen or sulfur;
(iii) a compound of the general formula III
\ \ ~Rz
R~
where R' is 2-methylbutyl, 2,2-dimethylcyclopropyl or 2,2-dihalo-3,3-
dimethylcyclopropyl
and Rz is methyl, ethyl or isopropyl, and
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(iv) a compound of the general formula IV
X Ar
where X is oxygen or sulfur and Ar is a heteroaromatic group.
In this context, specific juvenile hormone analogue compounds (JHA ) include
the follow-
ing:
1: 5-(T-Ethoxy-3',T-dimethyloctyloxy)-2-methylpyridine;
2:5-(3',7'-Dimethyloctyloxy)-2-methylpyridine;
R-2: (3'R)-5-(3',7'-Dimethyloctyloxy)-2-methylpyridine;
S-2: (3'S)-5-(3',7'-Dimethyloctyloxy)-2-methylpyridine;
3: 2-Methyl-5-(T-methyloctyloxy)pyridine;
4: 2-Methyl-5-octyloxypyridine;
5:5-(3',7'-Dimethyloct-6'-enyloxy)-2-methylpyridine;
6: 5-(3',7'-Dimethylocta-2',6'-dienyloxy)-2-methylpyridine;
7: 5-(3',7'-Dimethylocta-2',6'-dienyloxy)pyridine;
8: 5-[5'-(2",2"-Dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-
2-
ethylpyridine;
9:5-[5'-(2",2"-Dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-2-
methylpyridine;
10: 5-[5'-(2",2"-Dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-2-
ethylpyridine;
11: 5-[5'-(2",2"-Dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-2-
methylpyridine;
12: 5-[5'-(2",2"-Dimethylcyclopropyl)-3'-methylpentyloxy]-2-methylpyridine;
13: 5-(3',T-Dimethyloctylsulfanyl)-2-methylpyridine;
14: 2-(3',T-Dimethylocta-2',6'-dienyloxy)pyridine;
15: 2-(3',7'-Dimethyloctylsulfanyl)pyridine;
16: 3-(3',T-Dimethyloctyloxy)-1-methylpiperidine;
17: 5-(3',7'-Dimethyloctyloxy)benzo[1,3]dioxole;
18: 5-(3',7'-Dimethylocta-2',6'-dienyloxy)benzo[1,3]dioxole;
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19: 5-[5'-(2",2"-Dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]benzo[1,3]dioxole;
20: 5-[5'-(2",2"-Dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]benzo[1,3]dioxole;
21: 5-[5'-(2",2"-Dibromo-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]benzo[1,3]dioxole;
22: 5-(3',8'-Dimethylnona-2',6',7'-trienyloxy)benzo[1,3]dioxole;
23: 5-(3',7'-Dimethylocta-2',6'-dienylsulfanyl)benzo[1,3]dioxole;
24: 1-[5'-(2",2"-Dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-4-
ethylbenzene;
25:1-[5'-(2",2"-Dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-
4-
ethylbenzene;
26: 1-[5'-(2",2"-Dibromo-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-
4-
ethylbenzene;
27: 1-(3',8'-Dimethylnona-2',6',7'-trienyloxy)-4-ethylbenzene;
28: Methyl 4-[5'-(2",2"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]benzoate;
29: Methyl4-[5'-(2",2"-dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]benzoate;
30: Methyl 4-(5'-(2",2"-dibromo-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]benzoate;
31: 1-Acetyl-4-[5'-(2",2"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]benzene;
32:1-Acetyl-4-[5'-(2",2"-dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy)benzene;
33: 1-Acetyl-4-[5'-(2", 2"-dibromo-3", 3"-dimethylcyclopropyl)-3'-methylpent-
2'-
enyloxy]benzene;
34: (3,7-Dimethyloctyloxy)benzene;
35:4-[5'-(2",2"-Dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-3-
methoxybenzaldehyde;
36: 4-(5'-(2",2"-Dichloro-3", 3"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]-3-
methoxybenzaldehyde;
37: 1-[5'-(2",2"-Dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-enyloxy]-
2-methoxy-
4-propenylbenzene;
38:4-Allyl-1-[5'-(2",2"-dichloro-3",3"-dimethylcyclopropyl)-3'-methylpent-2'-
enyloxy]-2-
methoxybenzene;
39: Methyl 7,11-dimethyl-dodecc-2,4-dienoate;
40: Methyl 9-(2',2'-dimethylcyclopropyl)-7-methylnona-2,4-dienoate;
41: Ethyl 9-(2',2'-dimethylcyclopropyl)-7-methylnona-2,4-dienoate;
42:Isopropyl9-(2',2'-dimethylcyclopropyl)-7-methylnona-2,4-dienoate;
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14
43: Methyl 9-(2',2'-dichloro-3',3'-dimethylcyclopropyl)-7-methylnona-2,4-
dienoate;
44: Ethyl 9-(2',2'-dichloro-3',3'-dimethylcyclopropyl)-7-methylnona-2,4-
dienoate;
45: Isopropyl 9-(2',2'-dichloro-3',3'-dimethylcyclopropyl)-7-methylnona-2,4-
dienoate;
46: 2-Methyl-5-nonyloxypyridine;
47:2-Methyl-5-undecyloxypyridine;
48: 5-Dodecyloxy-2-methylpyridine;
49: 2-Methyl-5-(3',T,11'-trimethyldodeca-2',6',10'-trienyloxy)pyridine;
50: 5-(3',7'-Dimethyloctyloxy)-2-methylpyrimidine;
51: 3-(4',8'-Dimethylnonyl)pyridine;
52:3-(4',8'-Dimethylnonyl)pyridine-N-oxide;
53: 4,8-Dimethyl-1-pyridin-3-ylnonan-1-ol;
54: 4,8-Dimethyl-1-pyridin-3-ylnonan-1-one;
55: 4-Octylmorpholine;
56: 1-Octylpiperidine;
57:2-(3',T-Dimethyloctylsulfanyl)thiophene;
58: 1,4-Dioctylpiperazine;
59: 3-(3',7'-Dimethyloctyloxy)pyridine;
60: 3-(3',T-Dimethyloctylsulfanyl)pyridine;
61: (3',T-Dimethylocta-2',6'-dienyloxy)benzene;
F-1:3-[2'-(4"-Phenoxyphenoxy)ethoxy]pyridine;
F-2: 3-(T-Ethoxy-3',T-dimethyloctyloxy)pyridine;
F-3: Ethylcarbamic acid 2-(4'-phenoxyphenoxy) ethyl ester (phenoxycarb) ;
F-4: 2-[2'-(4"-Phenoxyphenoxy)ethoxy]pyridine;
F-5: 3-(4'-Phenoxybenzyloxy)pyridine;
methoprene; and
hydroprene.
The fish or any other aquatic animal species can be treated orally, e.g. via
their feed, or
by bath treatment, e.g. in a medicinal bath where the fish are kept for a
period of time
(minutes to several hours) that provides a sufficient contact time to inhibit
the parasitic
crustaceans. Alternatively, it is possible to treat the biotope of the fish
temporarily or con-
tinuously, e.g. the net cages, entire ponds, aquaria, tanks or basins in which
the fish are
kept. It is a further suitable possibility to administer the compounds
parenterally, e.g. by
injection.
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The active compounds are administered in medicament formulations which are
adjusted
to the applications. Formulations for oral administration include e.g.
powders, granulates,
solutions, emulsifiable concentrates or suspension concentrates which are
mixed homo-
geneously as feed additives with the feed, or powders, granulates,
emulsifiable concen-
5 trates or suspension concentrates which are administered in the form of
pills, the outer
coat of which can consist e.g. of fish feed compositions which cover the
active substance
completely. Alternatively, the active substance may be applied onto the
surface of feed
particles such as pellets or granules, e.g. incorporated in a lipid component
such as a fish
oil.
Useful medicament formulations for bath application or for treating the
biotope of the fish
include powders, granulates, solutions, emulsions, micro-emulsions,
suspensions, tablets
or the active substance itself. The end-user may use these formulations in
dilute or undi-
lute form.
The active compound in any of these formulations may be used in pure form, as
a solid
active substance e.g. in a specific particle size, or together with at least
one of the adju-
vants that are conventionally used in formulation technology, such as
extenders, typically
solvents or solid carriers, or surface active compounds.
The formulations are prepared in a manner known per se, typically by mixing,
granulating
and/or compacting the active compound with solid or liquid carriers, where
appropriate,
with the addition of further auxiliary substances such as emulsifying or
dispersing agents,
solubilisers, colorants, antioxidants and/or preservatives.
It is also possible to use semi-solid formulations for the bath treatment. The
active sub-
stance, which is suspended or dissolved in oily or fatty matrices, is washed
out on admini-
stration. The release can be controlled by the choice of adjuvants,
concentration of the ac-
tive substance and form. Coprimates or melts of hard fats comprising the
active sub-
stance are also suitable for use.
The diluted compositions of this invention are generally prepared by
contacting the active
substance with liquid and/or solid formulation assistants by stepwise mixing
and/or grind-
ing such that an optimal development of the antiparasitic activity of the
formulation is
achieved that conforms with the application.
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Formulation assistants can e.g. be solid carriers, solvents and, where
appropriate, surface
active substances which are non-toxic for marine fauna and flora.
The bath application of the medicament compositions of the invention to the
parasites to
be controlled can e.g. be carried out such that the compositions are placed in
the cage in
the form of solutions, emulsions, micro-emulsions, suspensions, powders or
tablets,
where they are dissolved or dispersed by the movement of the fish and the flow
of the
water. Concentrated compositions can also be diluted with large volumes of
water before
applied to the cages.
In one embodiment, micro-emulsions can advantageously be applied for bath
application
of the medicament compositions when using juvenile hormone analogues which are
not
water-soluble or have a low water-solubility. When such micro-emulsions,
comprising ju-
venile hormone analogues are added to the bath treatment water, a clear
solution is typi-
cally seen.
In one useful embodiment, the micro-emulsion comprises solvents, surfactants
and stabi-
lisers in addition to the juvenile hormone analogues. Preferably, no water
should be pres-
ent in the micro-emulsion.
Examples of solvents which can be applied in a micro-emulsion include acetone,
mono-
hydric, dihydric or polyhydric alcohol (including ethanol, polyethylene
glycol, propylene
glycol), glycerine, mineral oil, vegetable oils or oils of animal origin,
fatty acid esters, di-
methyl sulphoxid (DMSO), dioxan, tetrahydrofuran (THF), 2-pyrrolidon, N-methyl-
2-
pyrrolidon. Examples of suitable surfactants include acetylated mono-and
diglycerides
with fatty acids, polyacrylic copolymers, beeswax, lecithin, fatty acids, guar
gum, xanthan
gum, tragacanth gum, PEG linked with fatty acids or sugars, hydrogenated and
etoxylated
castor oil, cellulose derivatives, compounds consisting of fatty acids
esterified to sugars,
N-octyl-2-pyrrolidon, N-dodecyl-2-pyrrolidon, EO/PO blockpolymers and
dodecylbenzene
sulfonate salts. Examples of stabilisers include antioxidants, pH regulating
compounds
such as citric acid, and complexing agents.
In useful embodiments the micro-emulsion comprises: i) juvenile hormone
analogues in
an amount which is in the range of 0.1-10 wt% including the range of 0.5-5
wt%; ii) sol-
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vents in the range of 20-30 wt% including the range of 22-28 wt% such as in
the range of
25-28 wt%; iii) surfactants in the range of 50-80 wt% including the range of
55-75 wt%
such as in the range of 60 to 70 wt%; and iv) stabilisers in the range of 0.1-
5 wt% includ-
ing the range of 0.5-5 wt% such as in the range of 1 to 2 wt%. One example of
a specific
composition of a micro-emulsion is given in the below examples.
In a further aspect the invention relates to novel juvenile hormone analogue
compounds
of the general formula I, the general formula II, the general formula III or
of the general
formula IV and the compounds 55-58, F-1, F-4 and F-5 as defined above,
including a
compound of the general formula II having saturated side chains to the
aromatic ring, for
use as a medicament for treating or preventing crustacean infestations in an
aquatic ani-
mal.
It is also a significant objective of the invention to provide a method of
controlling infesta-
tions with any of the above crustacean species in aquatic animals including
fish of the
species as mentioned above, the method comprising administering to said animal
or to
the aquatic environment of the animal an effective amount of a juvenile
hormone ana-
logue compound.
In the method of the invention any of the above juvenile hormone analogues
including
epofenonane, phenoxycarb, hydroprene, kinoprene, methoprene and triprene and
the
above compounds of the general formula I, the general formula II, the general
formula III
or of the general formula IV as also defined above can be used as the
antiparasitically ac-
tive compound.
In this method, the active compound is administered by any of the above routes
using the
compound as such or in the form of a composition that is adjusted to the
selected manner
of administration, including compositions for bath treatment or addition to
the biotope of
animals or fish, compositions for oral administration, optionally via the
feed, or injectable
compositions. The amount of active substance in the administration form can
vary de-
pending i.a. on the dosage form, the target parasite, the age and condition of
the fish to
be treated.
When the compound is used as a medicament for bath treatment or as a
composition that
is added to the aquatic environment of the aquatic animal, the amount of the
active juve-
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nile hormone analogue compound in the environment of the aquatic animal
depends on
the manner and duration of treatment and also on the age and condition of the
fish to be
treated. Effective dosages of the active substance in the water is generally
in the range of
1 ppb to 1 ppm such as in the range of 10 ppb to 500 ppb including the range
of 100 to
300 ppb. In one preferred embodiment, the compound is added the aquatic
environment
in the form of a micro-emulsion
In accordance with the invention there is also provided a pharmaceutical
composition for
controlling crustacean infestation of an aquatic animal such as a fish,
comprising a juve-
nile hormone analogue compound and at least one pharmaceutically acceptable
carrier.
In this context, suitable carriers include, but are not limited to, solid
carriers such as e.g.
kaolin, talcum, bentonite, sodium chloride, calcium phosphate, carbohydrates,
cellulose,
cotton seed meal, polyethylene glycol ether and, if necessary, binders such as
gelatine,
soluble cellulose derivatives or surface active substances such as ionic or
anionic dispers-
ing agents.
As it is explained above, such a composition can e.g. be in the form of a
powder, a granu-
late, a suspension, an emulsion, a micro-emulsion and a solution or it can for
certain pur-
poses be in the form of an injectable composition. Deperiding on the intended
application
the composition of the invention may further comprise a component selected
from the
group consisting of a solvent, a wetting agent, an emulsifying agent, a
bulking agent and a
dispersing agent. Suitable solvents include aromatic hydrocarbons, alkylated
naphtalenes,
or tetrahydronaphtalenes, aliphatic or cycloaliphatic hydrocarbons such as
paraffins or
cyclohexane, alcohols such as ethanol, propanol or butanol, glycols and their
esters and
ethers, ketones such as acetone, strongly polar solvents such as N-methyl-2-
pyrrolidone,
dimethyl sulfoxide, water, as well as vegetable oils and oils of animal origin
such as e.g.
fish oil.
Depending on the type of formulation, suitable surface active compounds are
nonionic,
cationic and/or anionic surfactants having good emulsifying, dispersing and/or
wetting
properties.
It will be appreciated that the composition of the invention can comprise two
or more juve-
nile hormone analogue compounds or it can comprise at least one further anti-
parasitically
active compound of any of the conventionally used types of antiparasitic
substances in-
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cluding formaldehyde, hydrogen peroxide, a cholinesterase inhibitor such as
e.g. metri-
fonate or dichlorvos, an organophosphorus compound, a carbamate compound, a
chitin
inhibiting compound, an avermectin compound and/or a pyrethroid compound.
As it is mentioned above, one convenient manner of administering a juvenile
hormone
analogue compound to fish is to add the compound to the normal fish feed.
Accordingly,
there is provided an aquatic animal feed composition comprising a juvenile
hormone
analogue compound as defined above, in an amount that will provide an
antiparasitically
active amount of the JH analogue compound in the fish. The active compound can
be in-
corporated in the feed in any of the above manners. The active compound can be
mixed
directly with the feed ingredient as such or it can be added in the form of a
more or less
concentrated feed additive composition. As an example such an additive
composition can
consist of 1-10 wt% of active compound, 49-90 wt% of a protein carrier such as
soy bean
protein, 0-50 wt% of ground calcium powder, 0-2 wt% of an alcohol,
hydroxypropyl cellu-
lose and water ad 100 wt%. Suitable forms of "medicated" feed comprising the
JH ana-
logue compound include granulated feed, pelleted feed and feed in the form of
an emul-
sion or micro-emulsion. The amount of the juvenile hormone analogue in feed
composi-
tions of the invention is typically in the range of 0.001 to 5 % wt% such as
0.01 to 3 wt%
including 0.1 to 1 wt%.
As it was described for the above composition of the invention, the feed
composition of
the invention can comprise two or more juvenile hormone analogue compounds or
it can
comprise at least one further anti-parasitically active compound of any of the
convention-
ally used types of antiparasitic substances including formaldehyde, hydrogen
peroxide, a
cholinesterase inhibitor such as e.g. metrifonate or dichlorvos, an
organophosphorus
compound, a carbamate compound, a chitin inhibiting compound, an avermectin
com-
pound and/or a pyrethroid compound.
The invention will now be further illustrated in the following non-limiting
examples and in
the drawing where
Fig. 1 shows the percentage of copepodite larvae relative to the number of
nauplius lar-
vae exposed to compound 6 as defined herein [5-(3',T-Dimethylocta-2',6'-
dienyloxy)-2-
methylpyridinej at concentrations of 0.01 ppm, 0.05 ppm, 0.3 ppm and 1.0 ppm,
respec-
tively for 1, 5 and 24 hours, respectively (Example 3);
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Fig. 2 shows the percentage of copepodite larvae relative to the number of
nauplius lar-
vae exposed to compound 7 as defined herein [5-(3',7'-Dimethylocta-2',6'-
dienyloxy)pyridine] at concentrations of 0.3 ppm and 1.0 ppm for 1, 5 and 24
hours, re-
5 spectively (Example 3);
Fig. 3 shows the percentage of copepodite larvae relative to the number of
nauplius lar-
vae exposed to compound 59 as defined herein [3-(3',T-
Dimethyloctyloxy)pyridine;] at
concentrations of 0.3 ppm and 1.0 ppm for 1, 5 and 24 hours, respectively
(Example 3);
Fig. 4 shows the ratio between number of copepodites in groups of
Lepeophtheirus sal-
monis larvae exposed compounds 6, 7 or 59 for 1, 5 and 24 hours, respectively
at con-
centrations of 0.01 ppm, 0.05 ppm, 0.3 ppm and 1.0 ppm, respectively (Example
3). Data
for these exposure periods were combined for each concentration and the
controls for
each compound were combined. A ratio of 1 indicates that the same number of
copepo-
dites were developed in the exposed groups and the control groups; and
Fig. 5 summarises the results of the experiments in Example 4 where
Lepeophfheirus sal-
monis nauplius stage larvae were exposed for 24 hours to 1.0 ppm of compounds
4 [2-
Methyl-5-octyloxypyridine], 2 [5-(3',T-Dimethyloctyloxy)-2-methylpyridine, 34
[(3,7-
Dimethyloctyloxy)benzene], 17 [5-(3',T-Dimethyloctyloxy)benzo[1,3]dioxole] 60
[3-(3',7'-
Dimethyloctylsulfanyl)pyridine] or 61 [(3',T-Dimethylocta-2',6'-
dienyloxy)benzene].
EXAMPLES
In the following examples the effect of a range of juvenile hormone analogues
on viability
and metamorphosis of parasitic larval stages of crustacean species are
reported. The fol-
lowing compounds were tested:
4: 2-Methyl-5-octyloxypyridine;
2: 5-(3',7'-Dimethyloctyloxy)-2-methylpyridine;
6 = B: 5-(3'.T-Dimethylocta-2'.6'-dienyloxy)-2-methylpyridine;
7: 5-(3'.T-Dimethylocta-2'.6'-dienyloxy)pyridine;
14: 2-(3',7'-Dimethylocta-2',6'-dienyloxy)pyridine;
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34: (3,7-Dimethyloctyloxy)benzene;
17: 5-(3',T-Dimethyloctyloxy)benzo;[1,3jdioxole;
60: 3-(3',7'-Dimethyloctylsulfanyl)pyridine;
57: 2-(3',7'-Dimethyloctylsulfanyl)thiopene;
1: 5-(T-Ethoxy-3',T-dimethyloctyloxy)-2-methylpyridine;
51: 3-(4',8'-Dimethylnonyl)pyridine;
50: 5-(3',T-Dimethyloctyloxy)-2-methylpyrimidine;
59: 3-(3',T-Dimethyloctyloxy)pyridine;
61: (3',T-Dimethylocta-2',6'-dienyloxy)benzene;
F-1:3-[2'-(4"-Phenoxyphenoxy)ethoxyjpyridine;
F-2: 3-(T-Ethoxy-3',7'-dimethyloctyloxy)pyridine;
F-3: Ethylcarbamic acid 2-(4'-phenoxyphenoxy) ethyl ester (phenoxycarb);
F-4: 2-[2'-(4"-Phenoxyphenoxy)ethoxyjpyridine;
F-5 3-(4'-Phenoxybenzyloxy)pyridine;
AC = methoprene; and
AD = hydroprene.
EXAMPLE 1
The effect of juvenile hormone (JH) analogue compounds on Lepeophtheirus
salmonis
larvae
Nine JH analogue compounds designated 2, 6, 7, 14, 57, 1, 51, 50 and 59 (see
above)
were tested according to the following protocol:
Female sea lice with mature ovaries (dark pigmented) were collected in a tank
containing
0.5 I sea water. Newly developed nauplius stage larvae were picked with a
pipette or were
separated from the water by filtration and used for further studies
A large number of nauplii larvae (>100) in 300-400 ml aerated sea water were
subjected
to different JH analogues at a concentration of 3 ppm, and vitality and
mortality of the
nauplii monitored. The results of this experiment is summarized in Table 1.1.
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Table 1.1. Effect on nauplius larvae of tested JH analogue compounds
Compound Effect on nauplius Observation time (hours)
larvae
57 no effect 48
14 no effect 20
2 Large proportion dead96
1 Large proportion dead96
51 Large proportion dead96
50 Large proportion dead96
7 All dead 5
6 All dead 24
59 All dead 15
The effect of the above compounds 6, 7 and 59 was further investigated at
different con-
centrations and the effect against sea lice was monitored. In the below Table
1.2 the fol
lowing designations are used: +++ all nauplius larvae dead, ++ most nauplius
larvae
dead, + few dead nauplius larvae and - no dead nauplius larvae.
Table 1.2. Effect of compounds 6, 7 and 59 on nauplius larvae
Compound ConcentrationObservation
, time (hours)
(ppm) 3 20
6 15.0 +++ +++
1.5 +++ +++
0.15 - ++
7 18.0 +++ +++
1, g ++ +++
x.18 - +
59 6.0 ++ +++
1.0 - +++
0.15 - -
Among the tested compounds 6, 7 and 59 were the most effective against sea
lice nau-
plius larvae.
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EXAMPLE 2
The effect of juvenile hormone analogues on the development of Lepeophtheirus
sal-
monis from nauplius stage to copepodite stage
The JH analogue compounds 51, 1 and 50 were tested at a concentration of 0.7
ppm to
determine their effect on the development from nauplius larvae to copepodites
as com-
pared to a control group not exposed to JH analogue. After 14 days of exposure
the num-
ber of copepodites were calculated. Most of the nauplius larvae in the control
group de-
veloped into copepodites. Larvae exposed to the compounds 51 and 50 showed
results
that were similar to the control. However, no copepodites were present in the
group ex-
posed to the compound 1, and only dead nauplius larvae were found.
EXAMPLE 3
The effect of JH analogue compounds 6, 7 and 59 on the development of
Lepeophtheirus
salmonis from nauplius stage to copepodite stage
The apparently most effective compounds from Example 2 were selected for
further inves-
tigation. The effect of the compounds 6, 7 and 59 on the development from
nauplius lar-
vae to copepodites were tested at concentrations of 0.01 ppm, 0.05 ppm, 0.3
ppm and 1.0
ppm, respectively at the exposure periods of 1, 5 and 24 hours, respectively.
The number of nauplius and copepodite larvae were counted after 3-7 days. The
experi-
ments were run in triplicate at each concentration and each exposure period.
Additionally,
control groups exposed to equivalent amount of acetone used as solvent for the
test com-
pounds were included.
The results of these experiments are summarised in Figures 1-4
It is concluded that the tested compounds showed high effect in respect of
inhibiting the
development from nauplius larval stage to copepodites. In particular, compound
59
showed complete inhibition at 1.0 ppm and an exposure period of 24 hours. This
com-
pound also had a significant metamorphosis inhibiting effect at concentrations
less than
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1.0 ppm. Thus, at 0.01 ppm, 0.05 ppm and 0.3 ppm about 40% reduction in
numbers of
copepodites, relative to the controls, was observed
EXAMPLE 4
The effect of compounds 4, 2, 34, 17, 60 and 61 on the development of
Lepeophtheirus
salmonis from nauplius stage to copepodite stage
The compounds 4, 2, 34, 17, 60 and 61 were tested using the protocol as in
Example 1
using exposure at 1.0 ppm for 24 hours. The results of this experiment are
shown in Fig.
5. All of the tested compounds except compound 61 completely inhibited the
development
from the nauplius to the copepodite stage. The compound 60 were tested further
at 1.0
ppm using an exposure period of only 1 hour. Under these conditions, the
compound had
a good effect in that only 7% of the larvae exposed developed into the
copepodite stage.
EXAMPLE 5
The effects of compounds 6, F-1, F-2, F-3, F-4 and F-5 on Lepeophtheirus
salmonis nau-
plius larvae and the development of Lepeophtheirus salmonis from the nauplius
stage to
the copepodite stage
The experiments were conducted essentially as in Example 2. 10-50 newly
hatched nau-
plius I larvae were placed in trays containing 50 ml of sea water (temperature
16-19°C,
salinity 2.2%) to which the compound to be tested was added at 1.0 ppm and the
expo-
sure time was 24 hours following which the larvae were separated from the
water and
transferred to fresh sea water. After 3-5 days, the number of nauplii and
copepodites were
counted. Acetone was used as solvent for the compounds and the same volume of
ace-
tone was added to trays with control larvae. All experiments were carried out
in triplicate
or quadruplicate.
The results are summarised in Tables 5.1. and 5.2.
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Table 5.1. No. of copepodites on day 4 after the exposure
Experiment 1A Experiment 1 B
Compound Per cent copepodites Per cent copepodites
6 65.0 40.2
F-1 42.1 28.6
F-2 28.4 23.0
Control 56.6 36.8
The compounds F-1, F-2, F-3, F4 and F-5 were tested for their effect on
nauplius stage
larvae at a concentration of 1.0 ppm. The number of surviving and dead nauplii
in the
5 trays were counted immediately after exposure.
Table 5.2. Effect of compounds F-1, F-2, F-3 F-4 and F-5 against nauplii and
inhibition of
development into the copepodite stage
Compound % dead nauplii % copepodites 4 days
after
exposure
F-1 97 0
F-2 52 2
F-3 57 4
F-4 47 7
F-5 85 _ __ - 0
Control 70 - 22
10
All of the tested compounds F-1 to F-5 showed a significant inhibition of
development of
nauplii into copepodites under the test conditions. Compounds F-1 and F-5
inhibited the
development into copepodites completely and showed a high efficiency against
nauplius
larvae.
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EXAMPLE 6
Screening of JHA compounds for sea lice larvicid effect
The effect of 12 compounds designated A (= 59, B (= 6), C (= F-1), D (= F-3),
E (= F-4) , F
(= F-5), G (=propoxur) , H (=bassa), I (=resmethrin), J (=deltamethrin), K
(=permethrin)
and L (=agromazine), respectively on the survival of nauplius larval stages 1
and 2 of sea
lice (Lepeophtheirus salmonis) was tested. The compounds designated G-L are
not juve-
nile hormone analogues, but are included for comparison. Survival, mortality
and possible
deformations on the larvae were recorded for each group 6 days after exposure
to the test
compounds for 24 hours.
Adult female sea lice with mature ovaries were collected and the ovaries were
separated
from the animals and incubated in running sea water at 8°C. By means of
a pipette 30
newly hatched larvae at the nauplius 1 stage were transferred to 50 ml pure
sea water in
petri dishes. The respective test compounds dissolved in acetone were added to
the
dishes at a concentration of 1.0 ppm (50 ~I). The larvae were exposed for 24
hours before
new fresh water was supplied. The water was changed daily during the test
period. The
experiments were done in triplicate. To one control group of larvae 50 p1 of
acetone was
added and another group was kept in pure sea water.
A considerable variation in survival and mortality for the test groups was
observed, but the
control groups were relatively similar. Data for survival and mortality are
summarised in
Table 6.1:
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Table 6.1
Group 1. Live/dead 2. Live/dead 3. Live/dead
larvae larvae larvae
Control (water) 26/4 23/3 -
Control (acetone)27/4 26/6 28/2
A 18/13 12/19 15/14
B 0/30 0/30 0/30
C 0/30 0/30 0/30
D 23/8 15/14 18/11
E 20/10 23/8 22/12
F 6/22 6/25 3/25
G 20/8 28/3 26/4
H 18/13 23/6 21 /6
I 3/24 0/30 4/27
J 0/30 0/30 0/30
K 16/14 6/18 -
L 28/1 30/1 28/0
The compounds B, C, F, I and J had the best larvicidal effect on the sea lice
larvae. None
of the larvae reached the copepodite stage during the screening period. In the
groups B
and C, the distribution of dead larvae were equal between nauplius stages 1
and 2. For
the groups I, J and K, nauplius 1 larvae dominated among the dead larvae,
whereas stage
2 nauplii dominated among the dead larvae in group F.
A common observation for groups C, I, J and K was that the dead larvae had
protrusions
(vacuoles) at the antenna region. It is possible that this is due to the fact
that the larvae
grow to a size that is to large for the shell resulting in rupture of the
larvae.
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EXAMPLE 7
The effect of JH analogues on development and survival of sea lice
(Lepeophtheirus sal-
monis) on sea water adapted Atlantic salmon (Salmo salary
7.1. Materials and methods
Test compounds
The commercial juvenile hormone analogue compound methoprene (AC) was tested.
Test fish
The test fish were distributed into tanks of 500 I (45 fish per tank). Running
sea water that
was adjusted in respect of oxygen (at least 70% saturation in outlet water),
salinity (34
promille) and temperature (11.5°C) was supplied to the tanks. The
photoperiod was set at
18 hours of light and 6 hours of dark throughout the test period.
Bath experiment protocol
For a 14 days bath regimen 9.239 ml of pure methoprene (AC) was mixed with
1,700
ml of acetone which gave a stock solution at 5,000 ppm compound AC, which was
dosed
over 14 days. The compound was added to the water at a concentration of 0.1
ppm.
Cultivation of L. salmonis
Adult female sea lice with mature ovaries were collected and the ovaries were
separated
from the female sea lice and incubated in running sea water (salinity 34
promille) at 10°C.
Newly hatched nauplius larvae were harvested daily and transferred to trays to
produce
infective copepodite larvae.
Challenge with sea lice
During exposure to the sea lice, the water supply was stopped and the tanks
were ad-
justed to about 100 I of water. About 2,000 two days old copepodites were
distributed to
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each tank. During exposure the tanks were aerated. After 2 hours the tanks
were filled
and the water supply was stopped. The fish were exposed to copepodites in
stagnant
water for 12 hours under aeration before the usual water regimen was re-
established.
Data recording
Five days following copepodite exposure, the success of infestation was
recorded on 5
fish from each tank and the medication regimen was initiated. In all tanks the
number of
sea lice was recorded 1, 5 and 10 days after termination of the medication
period (10
fish). The distribution of the various stages of sea lice was recorded when
the fish was
removed from the tanks. The recording of the various chalimus stages was
simplified by
recording chalimus stages 1 and 2 as juvenile chalimus (ChJ) whereas chalimus
3 and 4
were recorded as "old" chalimus (ChG). The first and second pre-adult stages
and the
adult stage were also recorded.
7.2. Results
Fish mortality
No fish mortality was recorded throughout the entire test period.
Sea lice mortality: 14 days of bath treatment
After 14 days of bath treatment with compound methoprene (AC), the fish had
signifi-
cantly less sea lice infestation than the corresponding control group (p<0.01
)
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EXAMPLE 8
The effect of oral administration of JH analogue 59 on development and
survival of sea
lice-(Lepeophtheirus salmonis) on Atlantic salmon in sea water(Salmo salary
5
Materials and methods
Test compounds
10 The juvenile hormone analogue compound 59, 3-(3',7'-
Dimethyloctyloxy)pyridine, was
tested
Test fish
15 400 Atlantic salmon of average weight 2.5 kg were distributed equally into
two net pens
(4x4x4 m) in a fish farm. The fish was deloused with NUVAN (dichlorvos) before
the ex-
periment. One group was used as a control and one group was treated with JH
analogue
59, 3-(3',7'-Dimethyloctyloxy)pyridine;, by oral administration. The fish farm
was heavily
infested with sea lice.
Preparation of feed containing JH analogue 59
Ordinary fish feed for Atlantic salmon (Felleskjopet) 12 mm was coated with JH
analogue
59, 3-(3',T-Dimethyloctyloxy)pyridine. The pure compound (liquid) was
dissolved in Cape-
line-oil to a concentration of 5.0% and then coated on feed pellet at a
concentration of
10% oil. 9.4 kg feed coated with 59 was produced. The concentration was 5.0 g
of sub-
stance 59 per kg feed.
Oral administration of JH analogue.
Feed containing 59, 3-(3',T-Dimethyloctyloxy)pyridine, was fed on alternate
days at a
feeding rate of 0.5% per day. Ordinary feed was fed the days without
medication and
control fish was fed ordinary feed throughout the experiment. A total of 4
days of medica-
tion was used and the total amount of 59, 3-(3',T-Dimethyloctyloxy)pyridine,
applied was
20 gram per kg fish.
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The fish was free of sea lice when the treatment started after the NUVAN
treatment
Data recording
In all net pens the number of sea lice was recorded 3 months after termination
of the
medication period (10 fish of each group). The distribution of the various
stages of sea lice
was recorded when the fish was removed from the net pens.
Results
Fish mortality
No fish mortality was recorded throughout the entire test period.
Table 8.1
Number of sea lice per fish 3 months after treatment
59, 3-(3',T-Dimethyloctyloxy)pyridine Control
Fish no. Chalimus Pre-adultAdult ChalimusPre-adultAdult
1 14 6 50 18 37 48
2 8 25 32 29 20 87
3 26 20 30 10 10 60
4 8 10 15 7 30 51
5 13 20 50 43 30 50
6 12 21 20 33 20 75
7 5 15 50 31 30 20
8 12 11 20 20 10 40
9 11 25 40 24 20 70
10 6 25 11 17 25 15
Average 12 18 32 23 23 52
Standard 6 23 15 11 9 23
deviation
The number of sea lice at stages chalimus and adults are significantly lower
in the group
treated with JH analogue 59 than in the control group (a=0.05)
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The total average number of all sea lice stages in controls is 98 per fish
while in the group
treated with JH analogue 59 this number is reduced to 61. Because salmon in
neighbour-
ing cages were heavily infested with sea lice, migration of adult and pre-
adult lice to the
experimental groups occurred. The significant reduction in chalimus stages in
the group
treated with JH analogue 59 shows that development of copepodite through the
four
chalimus stages is inhibited by the treatment.
EXAMPLE 9
The effect of oral administration of JH analogues methoprene and hydroprene on
devel-
opment and survival of sea lice (Lepeophtheirus salmonis) on Atlantic salmon
in sea wa-
ter(Salmo salary
Materials and methods
Test compounds
The juvenile hormone analogues methoprene (AC) and hydroprene (AD) were
tested.
Test fish
A number of 135 Atlantic salmon of average weight 117-145 grams was used in
the ex-
periment which took place in a fish disease facility. The test fish were
distributed into
tanks of 500 I with 45 fish per tank. Running sea water that was adjusted in
respect of
oxygen (at least 70% saturation in outlet water), salinity (34 promille) and
temperature
(11.5°C) was supplied to the tanks. The photoperiod was set at 18 hours
of light and 6
hours of dark throughout the test period.
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Preparation of feed containing the JH analogues methoprene (AC) and hydroprene
(AD).
Ordinary fish feed for Atlantic salmon (Felleskjrapet) 3 mm was coated with
the JH ana-
logues methoprene (AC) and hydroprene (AD). The pure compounds were added to
the
feed in a mixer and then Capeline-oil was added to a concentration of 3.3%
while the
mixer was running. 480 grams of medicated feed was made of each group. Two
concen-
trations of methoprene: 10 gram per kg feed (AC/X) and 2 gram per kg feed
(AC/Y) were
made. Also two concentrations of hydroprene (AD) were made: 10 gram per kg
feed
(AD/X) and 2 gram per kg feed (AD/Y)
Cultivation of L. salmonis
Adult female sea lice with mature ovaries were collected and the ovaries were
separated
from the female sea lice and incubated in running sea water (salinity 34
promille) at 10°C.
Newly hatched nauplius larvae were harvested daily and transferred to trays to
produce
infective copepodite larvae.
Challenge with sea lice
During exposure to the sea lice, the water supply was stopped and the tanks
were ad-
justed to about 100 I of water. About 20,000 two days old copepodites were
distributed to
each tank. During exposure the tanks were aerated. After 2 hours the tanks
were filled
and the water supply was stopped. The fish were exposed to copepodites in
stagnant
water for 12 hours under aeration before the usual water regimen was re-
established.
Oral administration of JH analogue
Feed containing methoprene (AC/X and AC/Y) and hydroprene (AD/X and AD/Y) were
fed
at a feeding rate of 0.5% per day for 14 days. Control fish was fed ordinary
feed through-
out the experiment. A total of 371 gram of medicated feed was distributed to
each medi-
Gated fish group.
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Data recording
Five days following copepodite exposure, the success of infestation was
recorded on 5
fish from each tank and the medication regimen was initiated. In all tanks the
number of
sea lice was recorded one day after termination of the medication period of 14
days (10
fish). The distribution of the various stages of sea lice was recorded. The
recording of the
various chalimus stages was simplified by recording chalimus stages 1 and 2 as
juvenile
chalimus (ChJ) whereas chalimus 3 and 4 were recorded as "old" chalimus (ChG).
The
first and second pre-adult stages and the adult stage were also recorded.
Results
Infection
Due to a very high infestation rate of copepodites the number of sea lice
attached to each
fish was extremely high (> 100 per fish) and some mortality occurred among the
fish.
The mortality of fish due to sea lice in the different groups is shown in
Table 9.1:
Table 9.1
Tank Medication Mortality, number of fish
out of 45
1 AC/X, 10 gram/kg, 14 6
days
2 AC/Y, 2 gram/kg, 14 5
days
3 Control fish 13
4 AD/X, 10 gram/kg, 14 1
days
5 AD/Y, 2 gram/kg, 14 5
days
Highest mortality was observed in the control fish group which also had the
largest num
ber of sea lice (146 per fish). Lowest mortality was observed in the group
medicated with
hydroprene at highest concentration (AD/X) and which also had the lowest
number of sea
lice after medication (70 per fish).
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Medication
It was observed that the appetite was poor during feeding of medicated feed
and that lots
of medicated feed was not eaten. At sampling the day after medication only
control fish
5 had feed pellets in the stomach. The reason for less intake of medicated
feed could be
due to taste problems. The actual dose of substance AC and AD presented for
the fish is
then considerably lower than the theoretical amounts. This is further
supported by the fact
that the average weight of medicated fish dropped during medication: AC/X from
142 to
123 gram, AC/Y from 145 to 117 gram, AD/X from 130 to 119 gram and AD/Y from
141 to
10 138 gram. The average weight of control fish increased slightly in the same
period from
117 to 128 gram.
An ANOVA analysis of the number of sea lice per fish before medication and one
day af-
ter medication was performed, and the results are shown in the below Table
9.2.
Table 9.2
Tank Medication Number of Number of % change ANOVA signifi-
sea sea in sea
lice Ch.J lice pre-adultlice numbercance of
be- reduc-
fore medics-after medicationfrom beforetion
to
tion after medication
1 AC/X, 10 173 (n=5) 115 (n=10) 34 Yes, p =0.01
gram/kg,
14 days
2 AC/Y, 2 gram/kg,171 (n=5) 109 (n=10) 36 Yes, p=0.01
14 days
3 Control fish160 (n=5) 146 (n=5) 9 No
4 AD/X, 10 109 (n=5) 70 (n=10) 36 Yes, p <
gram/kg, 0.00001
14 days
5 AD/Y, 2 gram/kg,154 (n=5) 125 (n=10) 19 No
14 days
The ANOVA analysis of the above data in Table 9.2 show that there are no
significant re-
duction of sea lice in the control fish group and in the AD/Y group, while the
groups
methoprene AC/X, AC/Y and hydroprene AD/X experienced a significant reduction
of sea
lice during medication.
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A dose relation is seen with hydroprene in that the largest reduction in sea
lice is ob-
served with the highest concentration of hydroprene (AD/X).
The benefit of the administration of the JH analogues methoprene and
hydroprene was
clearly demonstrated by the lowering of the number of fish that died from sea
lice infesta-
tion and by the significant reduction in sea lice numbers.
EXAMPLE 10
Micro-emulsion concentrate for juvenile hormone analogues
In the below Table 10.1 is given an example of a base micro-emulsion
concentrate for
bath treatment of fish with a juvenile hormone analogue.
The micro-emulsion is prepared in a tank kept at a constant temperature of
30°C, by
adding the juvenile hormone analogue to the solvents (N-metyl-2-pyrrolidon and
N-octyl-
2-pyrrolidon) while stirring; and subsequently adding the remaining components
(polyok-
syl 35 castor oil, tetrapropylene benzenesulfonate Ca-salt and citric acid) to
the mixture.
In bath treatment of fish, the juvenile hormone analogue is typically applied
at concentra-
tion of 0.1 to 1 ppm. I order to prepare a treatment bath of 100 m3 of water
with a juvenile
hormone analogue concentration of 0.1 ppm, 0.2 litre of micro-emulsion
concentrate
(stock solution) containing 50g juvenile hormone analogue per kg is added to
the 100m3
of water. This micro-emulsion concentrate comprising the juvenile hormone
analogue, can
advantageously be diluted in a volume of water, e.g. 10 litres, before it is
added to the
aquatic environment wherein the fish to be treated is kept. A clear solution
is seen when
the micro-emulsion comprising the juvenile hormone analogue is added to the
water.
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Table 10.1
Micro-emulsion concentrate
N-metyl-2-pyrrolidon 26.00 Kg
N-octyl-2-pyrrolidon 25.00 Kg
Polyoksyl 35 castor oil 41.00 Kg
Tetrapropylene benzenesulfonate2.00 Kg
Ca-salt
Citric acid 1.00 Kg
Juvenile hormone analogue 5.00 Kg
Total 100.00 Kg
