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INFLUENCE OF TEMPERATURE AND HUMIDITY REGIMES ON THE
DEVELOPMENTAL STAGES OF GREEN VEGETABLE BUG, NEZARA VIRIDULA (L.)
(HEMIPTERA: PENTATOMIDAE) FROM INLAND AND COASTAL POPULATIONS
IN AUSTRALIA
!
Pol Chanthy1*, Robert J. Martin2^, Robin V. Gunning3 and Nigel R. Andrew1
1 Centre for Behavioural and Physiological Ecology, Zoology, University of New England, Armidale NSW 2351, Australia
2 Agricultural Systems Research Cambodia Co. Ltd., Battambang 02353, Cambodia!
3 Tamworth Agricultural Institute, NSW Department of Primary Industries, 4 Marsden Park Road, Calala, NSW 2340, Australia
* Cambodian Agricultural Research and Development Institute (CARDI), P.O. Box 1, Phnom Penh 12302, Cambodia
^ Maddox Jolie-Pitt Foundation (MJP), Group #02, Rumchek4 village, Rotanak Commune, Battambang 02353, Cambodia
Email: chanthypol@gmail.com
Summary
Laboratory studies were conducted to assess impacts of temperature and humidity regimes on the development of Nezara
viridula (L.) (Hemiptera: Pentatomidae) from inland and coastal populations in NSW, Australia. Four temperature regimes,
25±2ºC, 30±2ºC, 33±2ºC, and 36±2ºC and two humidity regimes, 40±10% and 80±10% RH were applied in the experiment with
a constant photoperiod of 14:10 h (L:D). The developmental time of the nymphal stage of N. viridula significantly decreased
with increasing temperature. Percentage nymphal survival significantly decreased with increasing temperature or high humidity
(80% RH) regimes. Longevity of N. viridula adults declined with increasing temperature or high humidity regimes and female
longevity was longer than males. High temperatures (30, 33 and 36ºC) or high humidity significantly reduced reproductive
performance and capacity of N. viridula compared to low temperature (25ºC) or low humidity (40% RH). However, high
humidity significantly increased egg hatchability of N. viridula compared with a low humidity regime. Interactions of
temperature and humidity regimes significantly changed incubation period, adult longevity, mating frequency, pre-mating
period, egg-mass size and egg hatchability of N. viridula. Interactions of population location (coastal or inland), temperature and
humidity regimes significantly changed incubation period and pre-oviposition period of N. viridula. Temperature and humidity
are important environmental factors for the development and reproduction of N. viridula. Higher temperatures shorten the length
of nymphal duration, but reduce nymphal survival. The optimum temperature for the development and reproduction of N.
viridula was 25ºC with 40 ± 10% RH. No differences in nymphal duration, nymphal survival, adult longevity or reproduction
performance between inland and coastal N. viridula populations were found under different climate conditions. We show the
importance of assessing all life-stages in the response to varying temperature and humidity regimes, especially in terms of
assessing responses to climate change.
Key words: Pentatomidae; climate change; nymph, adult; life history;!insect; agriculture.
INTRODUCTION
Green vegetable bugs, Nezara viridula (L.)
(Hemiptera: Pentatomidae) are a common
polyphagous insect (Ali and Ewiess 1977). Kamal
(1937) cited in Ali and Ewiess (1977) first reported
that N. viridula was primarily known as a pest of
cotton in Egypt. This pest is widely distributed in
different parts of the world and is a serious pest of a
range of economic crops (Ali and Ewiess,1977,
Kamal 1937). The range of geographical distribution
of N. viridula extends across temperate and tropical
areas, including the Americas, Africa, Asia, Australia
and Europe (Ali and Ewiess 1977, Singh and van
Emden 1979, Todd 1989, Waterhouse 1998). N.
viridula is a highly polyphagous pest attacking both
monocots and dicots, but it appears to have a
preference for leguminous plants (Todd 1989).
In Australia, N. viridula is a pest of many
horticultural and field crops. N. viridula became
established in the Sydney area as early as 1911, and
by 1938 was recorded in all mainland states (Clarke
1992). N. viridula is a major pest of soybean in
southern Queensland (Evans 1985, Turner and
Titmarsh 1979) and northern New South Wales
(NSW) (Brier and Rogers 1991, Miller et al. 1977).
However, it is not restricted to that crop and occurs
regularly on other hosts including sorghum, wild
crucifer, lucerne, sunflower, castor bean and corn
(Knight and Gurr 2007, Velasco and Walter 1992).
As for other pest species, and insects in general
(Andrew 2013, Andrew and Terblanche 2013), the
distribution, abundance and management of N.
viridula, is likely to be affected by climate change. It
is likely that pests, particularly those of tropical or
semi-tropical origin, could spread southward in
Australia, or contract from cropping regions affected
by reduced rainfall and rising temperatures (Pittock
2003). For insects, such as GVB, different parts of its
current regional distribution will be exposed to
different impacts of climate change into the future
(Andrew et al. 2013, Andrew and Terblanche 2013).
In the inland North West region of NSW including
the Breeza area, the climate is predicted to be hotter
in all seasons by 2050, with the greatest warming in
GEN. APPL. ENT. VOL 43, 2015
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spring and winter (NSW Climate Impact Profile
2010). Average daily maximum and minimum
temperatures are very likely to increase by between 1
and 3ºC in different parts of the region. Rainfall is
likely to increase in all seasons, except winter. On the
other hand, the north coastal region of northern NSW,
where Grafton is located average daily maximum
temperature is predicted to increase in all seasons by
2050. The smallest increases are projected to occur in
summer (1.0-1.5ºC) and the greatest in winter (2.0-
3.0ºC). Average daily minimum temperature is
projected to increase by 2.0-3.0ºC in all seasons.
Spring rainfall is not expected to change. Summer
and autumn rainfall is expected to increase slightly,
while winter rainfall is expected to decrease slightly
(NSW Climate Impact Profile 2010).
Such increases in temperature could influence insect
herbivore populations in various complex ways
(Hughes 2000, Yamamura and Kiritani 1998). The
effect of climate change on insect herbivores can be
direct, through impacts on insect phenology, life
cycles, and distribution (including movement and
migration), or indirect where the insects respond to
climate-induced changes mediated through other
factors, especially the host plant (Bale et al. 2002). As
with other insect species, terrestrial and aquatic
Heteroptera species respond to climate change by
shifting their distribution ranges, changing
abundance, phenology, voltinism, physiology,
behaviour, and community structure (Andrew et al.
2013, Musolin 2007 ). Many species of true bugs
(Heteroptera) have been recently reported to change
their distribution ranges, presumably in response to
climate change (Musolin and Fujisaki 2006). Such
expansion of ranges of individual species can enrich
local faunas and change community structure,
especially at the northern latitudes. The ongoing
warming is expected to further affect the ecology and
distribution of true bugs (Musolin and Fujisaki 2006).
The effects of temperature increases on insects are
likely to be pronounced in most, if not all, regions
globally. The interaction between temperature and
other factors such as rainfall may become important
in tropical regions (Bale et al. 2002). Cammell and
Knight (1992) stated that the evaluation of any
changes in insect herbivore populations should be
studied within these contexts. Interaction between the
different climate change factors needs considerably
more detailed investigation. The direct effect of
temperature may be modified by increased
precipitation, for instance, precipitation inland and
coastal northern NSW is expected to increase from 5-
20% in spring, summer and autumn, but to decrease
in winter from 10-20% for Breeza (inland) and 5-10%
for Grafton (coastal) by 2050 (NSW Climate Impact
Profile 2010). This, in turn, is likely to affect relative
humidity, which is important for many physiological
functions, such as reproduction. Moreover, direct
effects of climate change on insect performance need
to be set in a wider context and attention given to how
the direct effect of temperature will interact with
other factors, particularly natural enemies and host
plant conditions (Andrew et al. 2013, Bale et al.
2002).
In this study we assessed the impacts of predicted
climate change scenarios on development of N.
viridula. The impact of low and moderate temperature
regimes on N. viridula survival and development has
previously been studied (Ali and Ewiess 1977, Harris
and Todd 1980) as have the effects of relatively high
temperature fluctuations (Velasco and Walter 1993),
who examined the influence of fluctuating
temperatures of 10-20, 20-30 and 27-37°C on
nymphal survival and developmental rates (eggs to
adult), and the reproductive capacity of resulting
adults. They found that at high fluctuating
temperature of 27-37ºC adversely affected nymphal
survival and adult reproduction compared to lower
temperature regimes of 10-20ºC and 20-30ºC.
However, very little is currently known of the
interactions of temperature and humidity on insect
development, which severely inhibits our ability to
make critical predications about insect responses to
climate change (Durak and Borowiak-Sobkowiak
2013, Radchuk et al. 2013). This study examines the
effects of the combination of three factors: location,
temperature and humidity regimes on the life cycle of
N. viridula (eggs to adult). We then assess survival
rates of nymphs relative to adults, and reproductive
capacity (egg-masses per female, size of egg-mass
and fecundity), and egg hatchedability under the
different temperature and humidity regimes in the
laboratory.
MATERIALS AND METHODS
Collection and rearing
Adults of N. viridula (L.) were collected between
March and May 2010 in soybean fields at climatically
different locations: Breeza (31°14′54″N 150°28′02″E)
located in inland North-Western NSW and Grafton
(29°41′34″N 152°55′56″E) located on the North
Coast of NSW. Inland GVB populations were
collected from Breeza, which represents a dry climate
(621 mm annual average rainfall) compared to coastal
populations collected from Grafton, 1073 mm). The
coastal environment is also more humid with average
annual 3pm relative humidity of 53% compared to
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB!
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46% at inland. Average annual maximum temperature
is similar at both sites (26°C) but minimum
temperature is higher near the coast (Grafton, 13.7°C)
compared to inland (Breeza, 10.9°C) (BoM 2011).
The study was carried out on private lands and the
owners of these properties gave permission to
conduct the study here.
Nezara viridula were collected from soybean fields
by sweep net or beat sheet and placed into plastic
containers (64 mm deep and 118 mm in diameter)
(Chanthy et al. 2013). Air supply was provided by
cutting a small hole (65 mm of diameter) in the lid
and fitted with mosquito netting. The bugs were
provided fresh green bean pods (Phaseolus vulgaris)
plus water in a cotton wick as food. All the samples
were brought to the laboratory for culture. To obtain
egg-masses of N. viridula, several pairs (30-40 pairs)
were reared in a rearing cage (Bug Dorm-4030®, 30
cm x 30 cm x 30 cm) and maintained in a culture
room at 25 ± 2°C under photoperiodic conditions of
light:dark (L:D) 14:10 h and 60 ± 10% relative
humidity. Egg-masses of N. viridula were collected
and placed on the lid of small containers (30 ml) near
a piece of distilled water soaked cotton wick. Egg-
masses were then placed into large containers (64 mm
deep and 118 mm in diameter) with air supply
provided through holes in the lids fitted with nylon
mesh until nymphs hatched. Most of the first instar
nymphs moulted to second instar nymphs 4 or 5 days
after hatching under this culture room condition
without food. New second instar nymphs were
provided with fresh green beans as food. Rearing and
experiments were conducted in the Insect Ecology
Laboratory, Zoology, University of New England,
Australia.
Rearing experiment
The experiment was laid out in a three way factorial
design (n =3). Factors were temperature, humidity,
and location with three replications. Four temperature
regimes (25 ± 2°C, 30 ± 2°C, 33 ± 2°C and 36 ± 2°C)
and two humidity regimes, 40 ± 10% and 80 ± 10%
were applied in the experiment. Temperature was
considered as the main factor. There were 16
treatments (4 temperature regimes x 2 humidity
regimes x 2 locations with 3 replicates of each). The
experiment was conducted under consistent
photoperiod conditions (L:D) 14:10 h. Saturated salt
solutions were used to maintain humidity regimes in
incubators. Saturated lithium chloride (LiClH2O) plus
distilled water (4:1) was used to keep the humidity
regime in incubators at 40% RH and saturated
magnesium chloride (MgCl2.6H2O) plus distilled
water (1:1) was used to create 80% RH (Winston and
Bates, 1960). Saturated salt solutions were changed
every three days.
Twenty new second instar nymphs of N. viridula
were placed in a round plastic container (64 x 118
mm) for each treatment and maintained in incubators
for different climate conditions. The bugs were
provided fresh green beans as food. The beans and
containers were changed every two days. Data of
nymphal mortality, number of moulting nymphs,
adult mortality, mating date, oviposition date and date
of nymph hatching were recorded at 10 am in the
morning every day. Fresh body weight (g) at adult
emergence of the bugs was taken using an electronic
balance (Mettler Toledo, XP404S Precision Balance).
In order to determine the frequency of adult mating, a
permanent black marker pen was used to mark the
scutellum or shoulder of male and female bugs while
they were mating. Any mating bug (male or female)
which performed a mating activity with a new
individual (male or female) was considered to be part
of a new mating pair. Egg-masses per female were
calculated from the time of laying an egg-mass by
dividing the total egg-masses by the total number of
females from the time that they began to lay eggs. All
egg-masses were counted under a microscope to
determine the egg-mass size. The skin of egg-masses
was taken off from the containers when the nymphs
were at the 2nd instar stage and were examined under
a microscope to determine egg-hatching ability.
Warming Tolerance (WT) and Thermal Safety
Margins (TSM) were also tested as (Andrew et al.
2013; Deutsch et al. 2008, Diamond et al. 2012) they
characterise the geographic covariances of insect
thermal performance curves and climate (Angilletta
2009). Deutsch et al. (2008) defined warming
tolerance (WT) is the difference between critical
thermal maximum (CTmax) and the current
climatological temperature of organism’s habitat
(Thab). The critical thermal maxima (CTmax) of N.
viridula populations from inland and coastal
environments was 45.9ºC (Chanthy et al., 2012).
Habitat temperatures of N. viridula were collected
from inland was 31.5ºC (station 55008) and coastal
location was 29.7ºC (station 58077) during the
summer period in 2010.
Statistical analysis
The data for developmental stages, and reproductive
performance of N. viridula were examined by a 3-
way Analysis of Variance (ANOVA) using
IRRISTAT for Windows 5.0 developed by the
International Rice Research Institute (IRRI). Means
were compared by the method of least significant
GEN. APPL. ENT. VOL 43, 2015
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difference at the level of 5% (5% LSD). Furthermore,
three variables of N. viridula developmental stages,
nympal duration, nymphal survival and adult
longevity were tested for significance of linear,
quadratic and cubic effects using orthogonal contrasts
in IRRISTAT for Windows 5.0.
RESULTS
Influence of temperature and humidity regimes on
development of Nezara viridula
The mean number of days required by nymphs of N.
viridula to complete their development (from 2nd
instar to adults) varied greatly from one temperature
regime to another and the time taken for nymphal
development increased with lower temperature with a
significant quadratic effect at 1% level (P < 0.01)
(Figure 1). For each nymphal instar, the mean number
of days was significantly greater in lower temperature
compared to higher temperature (Table 1).
Figure 1. The effect of temperature on nymphal duration (ND), from 2nd instar to adults of Nezara viridula
(L.). No significant differences were exhibited among locations, so data from both inland and coastal
locations have been pooled.
!
Survival of N. viridula - nymphal stages to adult,
decreased with temperature increases: the highest
survival rate was observed at 25ºC and significantly
higher compared to 30ºC, 33ºC and 36ºC. (P <
0.0001; Figure 2). Although at 33ºC, some N. viridula
were mating, they died before they were able to
produce egg-masses. At 36ºC, N. viridula exhibited
no mating activities. N. viridula successfully mated at
25 and 30ºC only. The incubation period at 25ºC (6.5
± 0.2 days) was significantly longer than at 30ºC (3.0
± 0.9 days) and the incubation period (3.2 ± 0.7 days)
at the low humidity regime (40% RH) was
significantly longer than at high humidity (80% RH)
(1.5 ± 0.5 days) (Table 1). The first instar duration of
N. viridula reared at 25ºC (4.3 ± 0.1 days) was
significantly longer than when reared at 30ºC (0.3 ±
0.3 days) (Table 1).
!
y = 0 .0331x² - 2 .5747x + 72.945
R² = 0.9991
Temperature (oC)
24 26 28 30 32 34 36 38
Number of Days
22
23
24
25
26
27
28
29
30
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB 41!
!
Figure 2. The effect of temperature on nymphal survival (NS), from 2nd instar to adults of Nezara viridula (L.).
No significant differences were exhibited among locations, so data from both inland and coastal locations have
been pooled.
!
The mean longevity of N. viridula adults varied
widely between temperatures. The longevity of N.
viridula adults was significantly shorter with
increasing temperature with a quadratic effect
significant at the 5% level (P < 0.05) (Figure 3). At
the same temperature regime, the longevity of
females was found to be longer than males (Table 1).
At 25ºC, N. viridula females could live up to 60.7 ±
6.7 days. This was longer than that of males (46.3 ±
5.5 days). At 30ºC female longevity was 59.4 ± 7.8
days and male 41.6 ± 7.7 days while at 33ºC female
was 39.0 ± 5.6 days and male 30.2 ± 5.3 days); except
at 36ºC, the longevity of females was slightly shorter
than males. Humidity regimes also had a significant
impact on the longevity of N. viridula. The longevity
(51.2 ± 5.2 days) of N. viridula reared at low
humidity was significantly longer than when reared at
high humidity (25.8 ± 3.1 days). At the same
humidity regimes, females lived longer than males
(Table 1).
Body weight of newly emerged adults was measured.
At the same temperature or humidity regime, the
body weight of N. viridula females was heavier than
that of males (Table 2). The body weight of adults at
25ºC (0.14 ± 0.00g) and 30ºC (14.0 ± 0.00g) was
significantly heavier compared to the body weight of
N. viridula reared at 33ºC (0.12 ± 0.00g) and 36ºC
(0.10 ± 0.00g), between which body weight at 33ºC
was significantly heavier than at 36ºC. Humidity
regimes had a significant impact on body weight of N.
viridula adults. The significantly heavier body weight
(0.13 ± 0.00g) of N. viridula was observed at low
humidity compared to body weight (0.12 ± 0.00g) at
high humidity (Table 2).!
!
y = -2.3 4 2 2 x + 130.63
R² = 0.9511
Temperature (oC)
24 26 28 30 32 34 36
Percentage Survival (%)
45
50
55
60
65
70
75
GEN. APPL. ENT. VOL 43, 2015
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42!
Table 1. The effect of temperature or humidity regimes on nymphal duration of each instar, male and female longevity
and egg hatching duration of Nezara viridula (L.).
Climate
condition
Mean duration ± SE of nymphal stages (days)
Mean longevity ± SE of adult N.
viridula (days)
Incubation
period
(days)
1st instar
duration
(days)
2nd
3rd
4th
5th
Female
Male
Temperature (ºC)
25
6.8 ± 0.2 a
5.4 ± 0.1 a
6.2 ± 0.2 a
10.8 ± 0.3 a
60.7 ± 6.7 a
46.3 ± 5.5 a
6.5 ± 0.2 a
4.3 ± 0.1 a
30
5.4 ± 0.2 b
4.4 ± 0.1 b
5.5 ± 0.1 b
10.1 ± 0.3 ab
59.4 ± 7.8 a
41.6 ± 7.7 a
3.0 ± 0.9 b
0.3 ± 0.3 b
33
5.1 ± 0.2 b
3.8 ± 0.1 c
5.1 ± 0.2 c
10.2 ± 0.4 ab
39.0 ± 5.6 b
30.2 ± 5.3 b
-
-
36
4.4 ± 0.2 c
4.0 ± 0.1 c
5.3 ± 0.2 bc
9.5 ± 0.2 b
10.6 ± 2.3 c
14.8 ± 2.2 c
-
-
Humidity (%)
40
5.5 ± 0.2 a
4.5 ± 0.2 a
5.6 ± 0.1 a
10.3 ± 0.2 a
55.1 ± 6.2 a
45.9 ± 4.5 a
3.2 ± 0.7 a
1.3 ± 0.4 a
80
5.4 ± 0.2 a
4.2 ± 0.1 b
5.5 ± 0.1 a
10.0 ± 0.3 a
29.7 ± 4.2 b
20.6 ± 2.8 b
1.5 ± 0.5 b
1.0 ± 0.4 b
Mean ± standard error (SE). Mean followed by different letters in the same column are significantly different at p = 0.05, using IRRISTAT program for Windows 5.0.
Means were compared by the method of least significant differences (LSD) at 5% level.
Table 2. The effect of temperature or humidity regimes on body weight, reproductive performance and egg hatchability of Nezara viridula (L.).
Climate
condition
Mean weight (g) ± SE of adult N. viridula
Mating
frequency
(times)
Pre-mating
period
(days)
Pre-oviposition
period (days)
Egg-
masses/
female
Egg-mass
size (eggs/egg
mass)
Fecundity
(Eggs/female)
% of egg mass
hatched
Egg
hatch-ability
(%)
Female
Male
Adults
(F & M)
Temperature (ºC)
25
0.16 ± .00 a
0.12 ± .00 a
0.14 ± .00 a
6.2 ± 0.6 a
25.8 ± 1.8 a
50.1 ± 2.1 a
1.8 ± 0.3 a
62.6 ± 2.8 a
113.9 ± 15.4 a
82.6 ± 6.0 a
52.7 ± 10.9 a
30
0.15 ± .01 a
0.12 ± .00 a
0.14 ± .00 a
2.1 ± 0.6 b
33.4 ± 6.7 a
39.2 ± 12.0 b
0.5 ± 0.2 b
17.7 ± 5.4 b
17.8 ± 9.0 b
41.2 ± 13.5 b
4.8 ± 1.8 b
33
0.13 ± .00 b
0.10 ± .01 b
0.12 ± .00 b
0.3 ± 0.1 c
11.5 ± 6.6 b
-
-
-
-
-
-
36
0.11 ± .00 c
0.09 ± .00 b
0.10 ± .00 c
-
-
-
-
-
-
-
-
Humidity (%)
40
0.14 ± .00 a
0.12 ± .00 a
0.13 ± .00 a
2.7 ± 0.7 a
22.6 ± 4.9 a
32.2 ± 7.1 a
0.7 ± 0.2 a
25.5 ± 5.9 a
41.4 ± 12.9 a
36.9 ± 8.3 a
6.6 ± 1.7 b
80
0.14 ± .00 a
0.11 ± .00 b
0.12 ± .00 b
1.6 ± 0.5 b
12.8 ± 3.2 b
12.5 ± 4.6 b
0.4 ± 0.2 b
14.6 ± 5.3 b
24.5 ± 9.9 b
25.0 ± 9.0 b
22.1 ± 8.0 a
F – female, M – male, Mean ± standard error (SE), Mean ± standard error (SE). Mean followed by different letters in the same column are significantly different at p = 0.05, using IRRISTAT program
for Windows 5.0.
Means were compared by the method of least significant differences (LSD) at 5% level.
!
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB!
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43!
Table 3. The interactions of temperature and humidity regimes on nymphal duration, survival, adult longevity and egg hatching duration of Nezara
viridula (L.).
Tem.
(ºC)
Hum.
(%)
Mean duration ± SE of nymphal stages (days)
Nymphal
duration*
(days)
Nymphal
survival
(%)
Mean longevity ± SE of adult N. viridula (days)
Incubation
period
(days)
1st instar
duration
(days)
2nd
3rd
4th
5th
Female
Male
Adults
(F&M)
25
40
6.8 ± 0.2 a
5.7 ± 0.1 a
6.5 ± 0.1 a
11.1 ± 0.3 a
30.2 ± 0.6 a
75.8 ± 0.8 a
76.1 ± 8.4 a
55.6 ± 8.4 ab
72.1 ± 7.5 a
7.0 ± 0.1 a
4.6 ± 0.1 a
25
80
6.8 ± 0.2 a
5.1 ± 0.2 a
5.9 ± 0.3 a
10.5 ± 0.5 abc
28.4 ± 1.1 a
66.7 ± 3.8 a
45.4 ± 5.5 a
37.0 ± 5.1 c
41.5 ± 4.9 bc
6.0 ± 0.1 b
3.9 ± 0.1 a
30
40
5.4 ± 0.1 a
4.4 ± 0.1 a
5.5 ± 0.1 a
10.5 ± 0.2 abc
25.7 ± 0.4 a
78.3 ± 5.1 a
77.8 ± 5.5 a
64.0 ± 6.8 a
68.1 ± 5.2 a
5.9 ± 0.4 b
0.5 ± 0.5 a
30
80
5.5 ± 0.4 a
4.4 ± 0.2 a
5.5 ± 0.2 a
9.7 ± 0.6 cd
25.1 ± 0.4 a
48.3 ± 4.2 a
40.9 ± 10.2 a
19.3 ± 3.6 d
31.9 ± 5.2 cd
-
-
33
40
5.1 ± 0.2 a
4.0 ± 0.1 a
5.0 ± 0.2 a
9.6 ± 0.5 cd
23.8 ± 0.6 a
61.7 ± 2.1 a
52.1 ± 7.7 a
44.4 ± 4.5 bc
47.2 ± 4.9 b
-
-
33
80
5.0 ± 0.2 a
3.5 ± 0.1 a
5.1 ± 0.3 a
10.8 ± 0.6 ab
24.5 ± 0.9 a
39.2 ± 7.9 a
25.9 ± 3.0 a
16.0 ± 4.5 d
21.3 ± 3.1 de
-
-
36
40
4.5 ± 0.3 a
4.0 ± 0.1 a
5.2 ± 0.3 a
10.0 ± 0.3 bcd
23.7 ± 0.4 a
52.5 ± 8.8 a
14.4 ± 4.0 a
19.7 ± 3.0 d
17.4 ± 3.0 e
-
-
36
80
4.2 ± 0.1 a
4.0 ± 0.2 a
5.3 ± 0.1 a
9.0 ± 0.3 d
22.5 ± 0.3 a
41.7 ± 6.5 a
6.7 ± 1.2 a
9.9 ± 1.9 d
8.6 ± 1.0 e
-
-
Tem – temperature, Hum – humidity. * – days from second instar of nymphs to adults. Mean ± standard error (SE). Mean followed by different letters in the same column are significantly different at p =
0.05, using IRRISTAT program for Windows 5.0. Means were compared by the method of least significant differences (LSD) at 5% level.
Table 4. The interaction effects of temperature and humidity regimes on body weight and reproductive performance and egg hatchability of Nezara
viridula (L.).
Tem.
(ºC)
Hum.
(%)
Mean weight (g) ± SE of adult N. viridula
Mating
frequency
(times)
Pre-mating
period (days)
Pre-
oviposition
period (days)
Egg-
masses/
female
Egg-mass
size
(eggs/egg
mass)
Fecundity
(Eggs/female)
% of egg mass
hatched
Egg
hatch-ability
(%)
Female
Male
Adults
(F & M)
25
40
0.16 ± .01 a
0.12 ± .00 a
0.14 ± .01 a
7.3 ± 0.7 a
28.6 ± 2.2 b
50.3 ± 1.9 b
2.0 ± 0.4 a
66.7 ± 5.2 a
129.8 ± 24.4 a
65.1 ± 6.0 c
17.0 ± 2.0 b
25
80
0.16 ± .00 a
0.12 ± .00 a
0.14 ± .00 a
5.0 ± 0.8 b
23.0 ± 2.5 bc
49.9 ± 3.9 b
1.7 ± 0.3 a
58.6 ± 1.1 b
98.0 ± 18.7 a
100.0 ± 0.0 a
88.4 ± 2.1 a
30
40
0.16 ± .00 a
0.13 ± .00 a
0.14 ± .00 a
3.2 ± 1.0 c
51.1 ± 4.2 a
78.5 ± 4.4 a
1.0 ± 0.4 a
35.4 ± 1.9 c
35.7 ± 15.0 a
82.4 ± 11.3 b
9.6 ± 2.4 c
30
80
0.14 ± .01 a
0.12 ± .00 a
0.13 ± .01 a
1.0 ± 0.5 d
15.8 ± 7.2 bc
-
-
-
-
-
-
33
40
0.13 ± .00 a
0.12 ± .00 a
0.13 ± .00 a
0.2 ± 0.2 d
10.7 ± 10.7 c
-
-
-
-
-
-
33
80
0.13 ± .01 a
0.09 ± .01 a
0.11 ± .00 a
0.3 ± 0.2 d
12.3 ± 8.7 c
-
-
-
-
-
-
36
40
0.11 ± .00 a
0.09 ± .01 a
0.10 ± .00 a
-
-
-
-
-
-
-
-
36
80
0.11 ± .01 a
0.10 ± .00 a
0.10 ± .00 a
-
-
-
-
-
-
-
-
Tem – temperature, Hum – humidity, F – female, M – male, Mean ± standard error (SE). Mean followed by different letters in the same column are significantly different at p = 0.05, using IRRISTAT
program for Windows 5.0. Means were compared by the method of least significant differences (LSD) at 5%!
GEN. APPL. ENT. VOL 43, 2015
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44!
Figure 3. The effect of temperature on adult longevity (ADL) of Nezara viridula (L.). No significant differences
were exhibited among locations, so data from both inland and coastal locations have been pooled.
!
The mating frequency of N. viridula varied greatly
from one temperature to other. At 25ºC, mating
frequency of N. viridula (6.2 ± 0.6 times) was
significantly higher than at 30ºC (2.1 ± 0.6 times) and
33ºC (0.3 ± 0.1 times), between which mating
frequency at 30ºC was highly significantly different
(Table 2). Mating activity of N. viridula was not
observed at 36ºC, possibly due to suppression by
higher temperature or the short adult longevity of N.
viridula (9.7 ± 0.1 to 17.6 ± 3.5 days for Breeza and
7.5 ± 2.0 to 17.1 ± 5.8 days for Grafton) (Appendix
5). The mating frequency (2.7 ± 0.7 times) at the low
humidity regime was significantly higher than at the
high humidity regime (1.6 ± 0.5 times) (Table 2).
There was a significant effect of temperature on the
duration of the pre-mating period of N. viridula. The
pre-mating period of N. viridula (11.5 ± 6.6 days) at
33ºC was significantly shorter compared to the pre-
mating period at 25ºC (25.8 ± 1.8 days) and 30ºC
(33.4 ± 6.7 days), between which pre-mating periods
were not significantly different. The pre-mating
period of N. viridula reared in low humidity (22.6 ±
4.9 days) was significantly longer than at the high
humidity regime (12.8 ± 3.2 days) (Table 2).
Pre-oviposition period, number of egg-masses per
female, size of egg-mass (number of eggs per egg-
mass), fecundity (eggs per female), percentage of
egg-mass hatched, and egg hatchability of N. viridula
were found to be significantly longer or greater at
25ºC and/or low humidity than at 30ºC and/or high
humidity, except for the egg hatchability at the low
humidity regime, 6.6 ± 1.7% which was significantly
lower than at high humidity, 22.1 ± 8.0% (Table 2).
!
y = -0.4 2 0 4 x² + 21.629x - 221.0 4
R² = 0.9994
Temperature (oC)
24 26 28 30 32 34 36
Number of D ays
10
20
30
40
50
60
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB!
!
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45!
Interactions of temperature and humidity on
development of Nezara viridula
The interactions between temperature and humidity
had a significant effect on incubation period.
Incubation period (7.0 ± 0.1 days) at 25ºC with low
humidity was significantly longer compared to
incubation period at 25ºC with high humidity (6.0 ±
0.1 days) and at 30ºC with low humidity (5.9 ± 0.4
days), between which incubation period was not
significantly different (Table 3).
The interactions between temperature and humidity
had a significant impact on male longevity. The
longevity of adults (females and males pooled
together) at 25ºC (72.1 ± 7.5 days) and 30ºC (68.1 ±
5.2 days) with low humidity regime was significantly
longer than at 25ºC (41.5 ± 4.9 days) and 30ºC (31.9
± 5.2 days) with high humidity. Significantly shorter
longevity of adults was recorded at 36ºC, 17.4 ± 3.0
days and 8.6 ± 1.0 days with low and high humidity
regimes, respectively. It is important to note that, at
the same temperature, the longevity of N. viridula
adults with low humidity was longer than with the
high humidity (Table 3).
The interactions between temperature and humidity
significantly changed reproductive performance of N.
viridula (Table 4). Greater mating frequency of N.
viridula was recorded at 25ºC with low and high
humidity regimes. Mating frequency (7.3 ± 0.7 times)
at 25ºC with 40% RH was significantly higher than at
25ºC with 80% RH (5.0 ± 0.8 times) (Table 4). The
mating frequency of N. viridula at 25ºC with low and
high humidity was significantly higher compared to
mating frequency at 30ºC and 33ºC with low and high
humidity. Pre-mating period of N. viridula at 30ºC
with 40% RH (51.1 ± 4.2 days) was significantly
longer compared to pre-mating period at 25ºC with
40% RH (28.6 ± 2.2 days). The shortest pre-mating
period of N. viridula was recorded at 33ºC with low
humidity (10.7 ± 10.7 days) and high humidity (12.3
± 8.7 days) (Table 4).
Pre-oviposition period was longer for females reared
at 30ºC with 40 % RH (78.5 ± 4.4 days) than those
reared at 25ºC with 40% RH (50.3 ± 1.9 days) and
with 80% RH (49.9 ± 3.9 days), between which pre-
oviposition period was not significantly affected.
Larger egg-mass size was recorded at low
temperature. At 25ºC with 40% RH, egg-mass size of
N. viridula (66.7 ± 5.2 eggs/egg-mass) was
significantly larger than egg-mass size of N. viridula
reared at 25ºC with 80% RH (58.6 ± 1.1 eggs/egg-
mass) and at 30ºC with 40% RH (35.4 ± 1.9
eggs/egg-mass). Egg-mass size at 25ºC with 80% RH
was significantly greater than at 30ºC with 40% RH
(Table 4).
Egg-masses of N. viridula reared at 25ºC with 80%
RH hatched at optimum levels (100 ± 0.0%)
compared to egg-masses kept at 25ºC with 40% RH
(65.1 ± 6.0%) and at 30ºC with 40% RH (82.4 ±
11.3%) (Table 4). Egg hatchability of N. viridula was
significantly higher when kept at 25ºC with 80% RH
(88.4 ± 2.1%) compared to egg hatchability at 25ºC
with 40% RH (17.0 ± 2.0%) and at 30ºC with 40%
RH (9.6 ± 2.4%), between which egg hatchability at
25ºC with 40% RH was higher than at 30ºC with 40%
RH (Table 4). The interactions of temperature and
humidity had no effect on egg-mass per female and
fecundity of N. viridula females (Table 4).
Interactions of location and temperature on
development of Nezara viridula
Incubation period, pre-mating period, pre-oviposition
period, egg-mass size and fecundity of N. viridula all
exhibited a significant interaction between location
and temperature (Appendix 1 and 2). For the same
temperature regimes, at 30ºC the incubation period of
inland N. viridula (3.4 ± 1.5 days) was significantly
longer compared to incubation period of coastal N.
viridula (2.5 ± 1.1 days) at 30ºC(Appendix 1).
The pre-mating period of inland N. viridula at 30ºC
(24.3 ± 11.3 days) was significantly shorter compared
to coastal N. viridula (42.5 ± 5.9 days) (Appendix 2).
Pre-oviposition period of N. viridula was different
between locations. The mean number of days required
by inland N. viridula from newly emerged adults to
the day of oviposition at 25ºC (52.6 ± 1.3 days) was
significantly longer compared to coastal N. viridula at
the same temperature (47.6 ± 3.8 days), while pre-
oviposition period of inland N. viridula at 30ºC (34.6
± 15.5 days) was shorter than those from coastal N.
viridula (43.9 ± 19.7 days) (Appendix 2). The mean
number of egg-masses per female of inland N.
viridula at 25ºC (1.2 ± 0.2 egg-mass/female) was
significantly lower than those from coastal N. viridula
(2.4 ± 0.3 egg-mass/female). The fecundity of coastal
N. viridula females at 25ºC (146.0 ± 19.8
eggs/female) was significantly greater compared to
fecundity of inland N. viridula (81.8 ± 15.6
eggs/female) (Appendix 2).
Interactions of location and humidity on
development of Nezara viridula
The interactions of location and humidity were
significant for incubation period, and egg-mass size
of N. viridula (Appendix 3 and 4). The mean number
of days required from oviposition to hatching!
GEN. APPL. ENT. VOL 43, 2015!
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46!
incubation period) by inland N. viridula (3.4 ± 1.0
days) at 40% RH was significantly longer compared
to incubation period of coastal N. viridula (3.0 ± 0.9
days) at 40% RH (Appendix 3). The mean number of
eggs per egg-mass (egg-mass size) of inland N.
viridula at 40% RH (27.9 ± 9.4 eggs/egg-mass) was
significantly higher compared to egg-mass size of
coastal N. viridula (23.1 ± 7.6 eggs/egg-mass) at 40%
RH (Appendix 4).
Interactions of location, temperature and humidity
on development of Nezara viridula
Four developmental stages of N. viridula, 2nd instar
duration, incubation period, male longevity and pre-
oviposition period, exhibited a significant interactions
between temperature and humidity regimes between
two locations (Appendix 5 and 6).The 2nd instar
duration of inland and coastal N. viridula at the same
temperature and humidity regimes was significantly
different, with the exception of those reared at 30ºC
with 80% RH. The 2nd instar duration of inland N.
viridula (5.0 ± 0.1 days) at 30ºC with 80% RH was
significantly shorter compared to coastal N. viridula
(6.0 ± 0.7 days) at 30ºC with 80% RH, (Appendix 5).
Incubation period of inland N. viridula reared at 30ºC
with 40% RH (6.8 ± 0.2 days) was significantly
longer compared to coastal N. viridula at 30ºC with
40% RH (5.0 ± 0.0 days) (Appendix 5).
Pre-oviposition period of inland and coastal N.
viridula at 30ºC with 40% RH (inland, 69.1 ± 1.7
days and coastal, 87.8 ± 2.4 days) was significantly
longer compared to pre-oviposition period of inland
and coastal N. viridula at 25ºC with 40% or 80% RH
(inland, 53.6 ± 2.1 days (40% RH), 51.6 ± 1.7 days
(70% RH) and coastal, 47.0 ± 1.6 days (40% RH),
48.2 ± 8.3 days (70% RH). Pre-oviposition period of
inland N. viridula at 30ºC with 40% RH (69.1 ± 1.7
days) was significantly shorter compared to coastal N.
viridula (87.8 ± 2.4 days) at 30ºC with 40% RH,
(Appendix 6).
Warming Tolerance (WT) and Thermal Safety
margin (TSM)
The WT of inland N. viridula was 14.4ºC (WT =
CTmax – Thab = 45.9ºC – 31.5ºC) and WT of coastal N.
viridula was 16.2ºC (WT = 45.9ºC – 29.7ºC). The
TSM of inland N. viridula was -6.3C (TSM = 25ºC –
31.5ºC) and TSM of coastal N. viridula was -4.7ºC
(TSM = 25ºC – 29.7ºC).
DISCUSSION
Influence of temperature and humidity on
development of Nezara viridula
Here, we assessed the impacts of predicted climate
change scenarios on development of N. viridula.
Insect development is highly influenced by both
temperature and humidity. This may be from direct
impacts of both on terrestrial insects within a single
generation or from long-term exposure at different
climatic regions from where the organisms have bred
and successfully continued multiple generations. The
effects of temperature on insect development may
vary among species, with lower temperatures
typically resulting in a decrease in rate of
development, and a lengthening of the period of
insect development; but high temperature shortens the
duration of time spent in each developmental stage
(Hintze, 1970; Ross et al., 1982). Results from this
study indicated that the developmental time of N.
viridula nymphal stages (from 2nd instar to adults) and
adult longevity declined with increasing temperature.
Howevever, the rate of nymphal survival decreased
with increasing temperature or humidity regimes
(Table 1). The current results showed that the
nymphal duration (from 2nd instar to adults) at 25ºC
(29.3 ± 0.7 days) was longer than at 30ºC (25.4 ± 0.3
days), 33ºC (24.1 ± 0.5 days), and 36ºC (23.1 ± 0.3
days). These results are similar to previous studies
which reported that the mean nymphal development
period of N. viridula when reared at 25-27ºC, 55-65%
RH, and 14 h. photophase was 31.8 days, nymphal
duration of 1st instar was 3.8 days, 2nd instar (5.2
days), 3rd instar (4.5 days), 4th instar (6.4 days) and 5th
instar (11.9 days) (or from 2nd instar to adults took
28.0 days) (Harris and Todd 1980). Harris and Todd’
s (1980) findings show N. viridula nymphal
development period was 1.3 days shorter than the
results in this study. This difference may be explained
in terms of different host plants or food crops. In this
study, fresh green bean pods (P. vulgaris) were
provided to N. viridula as food, whereas Harris and
Todd (1980) provided fresh green beans (P. vulgaris)
and green shelled peanuts (Arachis hypogaea) to N.
viridula. There have been many reports of rearing N.
viridula with seeds. Panizzi and Saraiva (1993)
reported nymphal duration was 26.0 days at 25ºC on
immature soybean pods and 39.3 days on immature
radish fruits. In addition, Jones Jr. and Brewer (1987)
reported that the same nymphal duration was 22.7
days at 27ºC on green beans and peanuts. There is
evidence that diets derived from different plant
species could have a great effect on rates of
development, survival and reproduction of N. viridula
(Panizzi 1997, Velasco et al. 1995). The optimum
temperature for N. viridula development appears to be
25ºC, since all stages of N. viridula at this
temperature developed successfully with a higher rate
of survival, higher reproduction
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB!
!
!
47!
(large egg-mass size and high fecundity) and higher
percentage of egg hatchability compared to 30ºC.
Moreover, at 33 and 36ºC, the rate of nymphal
survival and mating frequency were very low and N.
viridula were unable to develop well, causing short
adult longevity and inability to lay eggs. The results
obtained here were similar to those of Velasco and
Walter (1993) who studied the influence of
fluctuating temperature on egg production by N.
viridula and on survival and development of nymphs.
Nymphal survival to adult stage was low (32.9%)
under the high temperature regime (27/37ºC) and
none of the resulting adults survived long enough to
mate and reproduce.
Adult longevity and weight declined with increasing
temperature and/or humidity regimes. Generally,
females lived longer and were heavier than males at
the same temperature or humidity regimes (Table 1).
Previous studies have suggested an optimum
temperature for N. viridula of about 25ºC (Ali and
Ewiess 1977). Thus high temperatures (30, 33, and
36ºC) had adverse effects on the development and
reproductive capacity of N. viridula. At 25ºC, N.
viridula had more frequent mating, 6.2 ± 0.6 times
during their life span, many egg-masses per female,
larger egg-mass, high fecundity and high egg
hatchability compared to 30ºC. Moreover, at
temperatures higher than 30ºC (e.g., 33 and 36ºC), N.
viridula was unable to reproduce or failed to lay any
eggs (Table 2).
High temperature might cause temporary or
permanent sterility, or deactivation of sperm stored in
the spermatheca resulting in reduced fertility (
Riordan, (1957)) and may explain the results in this
study. Females of N. viridula failed to lay eggs at
30ºC and higher. Ju et al. (2011) demonstrated that
females of the sycamore lace bug, Corythucha ciliaca
(Hemiptera: Tingidae) failed to lay eggs at 16ºC,
suggesting that low temperatures also induced
sterility. Ju et al. (2011) suggested that both lower
and higher temperatures would lead to developmental
stagnancy of the ovaries.
Our results indicated that mating frequency, pre-
mating period, pre-oviposition, egg-mass per female,
egg-mass size, fecundity and percentage of egg-mass
hatched responded positively to decreasing humidity
regimes (Table 2). However the high humidity regime
improved egg hatchability of N. viridula. Small
percentages of egg-masses and a low percentage of
eggs per egg-mass hatched in low humidity compared
to high humidity because desiccation may affect the
egg development and result in low emergence rate or
egg hatchability of N. viridula. Harris and Todd
(1980) cited several studies where egg and larval
development were accelerated at high humidity. Most
terrestrial insect embryos support metabolism with
oxygen from the environment by diffusion across the
eggshell. Because metabolism is more temperature
sensitive than diffusion, embryos should be relatively
oxygen-limited at high temperature or temperature
strongly affects egg metabolic rates (Woods and Hill
2004). In different life stages of terrestrial insects,
eggs are usually sensitive to oxygen-water tradeoffs.
Eggs are particularly immobilized in their oviposition
microsite, whereas juveniles and adults may search
out water and mobile life stages also possess rapid-
acting systems for regulating oxygen flux and water
balance (Woods and Harrison 2001). Insect eggs
should be sensitive to oxygen-water tradeoff, because
they are unable to forage for water, having high
surface area-to-volume ratio, and experience large
temperature driven changes in oxygen demand
(Zrubek and Woods 2006).
Interactions of temperature and humidity on
development of Nezara viridula
Generally, the mean nymphal duration, survival and
adult longevity with low humidity was longer or
higher compared to high humidity at the same
temperature. Moreover, the reproductive performance
of N. viridula such as mating frequency, pre-mating
period and egg-mass size with low humidity were
longer or larger than at high humidity at the same
temperature. These results indicated that N. viridula
were unable to develop well at a high humidity.
However, high humidity improved the percentage of
egg-mass hatched and egg hatchability of N. viridula.
At 30ºC with high humidity and 33ºC (or over 30ºC)
with low and high humidity, no insects survived long
enough to reproduce or to lay their eggs. These results
indicated that the above climate conditions were
unfavourable for the development and reproduction of
N. viridula.
Interactions of location and temperature on
development of Nezara viridula
The interactions of location and temperature had no
effect on nymphal duration, nymphal survival, adult
longevity or body weight of N. viridula. These results
showed that the life cycle of inland and coastal
populations of N. viridula are similar at the same
temperature regimes. The average annual maximum
temperature is similar at both sites (26ºC) however
minimum temperature is higher at the coastal site
(Grafton, 13.7ºC) compared to the inland site (Breeza,
10.9ºC) (BoM 2011).
!
GEN. APPL. ENT. VOL 43, 2015
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Interactions of location and humidity had no effect on
the development and reproduction of N. viridula
between the inland and coastal populations. These
results can potentially be explained by the
observation that, although coastal Grafton is more
humid with a mean 3 pm relative humidity of 53%
compared to inland Breeza at 46%, this variation is
less than 10% and is probably not to be a big enough
difference to have an effect.
Interactions of location, temperature and humidity
on development of Nezara viridula
The nymphal duration, survival, adult longevity, body
weight (insect fitness) and reproductive performance
of N. viridula were not significantly different for
interactions of location, temperature and humidity
regimes. Average annual maximum temperature is
similar at both sites (26°C) but the minimum
temperature is higher at coastal (13.7°C) compared to
inland (10.9°C) (BoM 2011).
With climate change, global average surface
temperature is expected to increase by 1.4-5.8°C by
2100 with atmospheric carbon dioxide (CO2)
concentrations expected to rise to between 540 and
970 ppm over the same period (Houghton et al. 2001,
IPCC 2007). In the inland North West region
(Breeza) of NSW, the climate is highly likely to be
hotter in all seasons by 2050, with the greatest
warming in spring and winter. Average daily
maximum and minimum temperatures are very likely
to increase by between 1 and 3ºC in different parts of
the region. On the other hand, in the north coast
region) of NSW, the average maximum temperature
is expected to increase 1.0-1.5ºC in summer and 2.0-
3.0ºC in winter and average daily minimum
temperature are projected to increase 2.0-3.0ºC by
2050 in all seasons (NSW Climate Impact Profile
2010). Recent inland and coastal mean summer
maximum temperatures are 31.5ºC and 29.7
respectively (BoM 2011). In this context, if the
temperature increased further under global climate
change, coastal and inland mean summer maximum
temperature will be over 30ºC by 2050. More extreme
events such as extreme high temperatures are also
predicted (Easterling et al. 2000), which will increase
the impact of higher temperature. The number of days
above 30ºC at the inland site increased from 82 in
1970 to 98 in 2011 and based on this trend there
could be 109 days above 30ºC in 2050. At the coastal
site, the number of days above 30ºC increased from
40 in 1970 to 71 in 2011 and based on this trend
could be 105 days above 30ºC in 2050 (Figure 4).
Figure 4. Ten-year moving averages for number of days per year exceeding 30ºC based on Historical
temperature (1970-2011) at inland (Breeza) and coastal (Grafton) sites.
!
!
Yea r
1970 1980 1990 2000 2010
Days above 30oC
40
50
60
70
80
90
100
Inland
Coastal
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB!
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49!
With regard to results of these experiments, at a
temperature of 30ºC or higher, N. viridula had
reduced reproductive performance (mating activities),
fecundity; prolonged or delayed pre-mating period,
and shortened life span of adults. However, at a
temperature of 25ºC across all humidity regimes,
coastal and inland populations of N. viridula
developed successfully with a high rate of nymphal
survival, more frequent mating, higher fecundity
(eggs/female) and higher egg hatchability (Appendix
6). This temperature is similar to current coastal and
inland mean autumn maximum temperature (March to
May) 25.8ºC (coastal) and 26.1ºC (inland) (BoM
2011). If the average autumn maximum temperature
increased by 3ºC by 2050, it would still be below
30ºC at both coastal and inland sites. However, the
increased frequency of extreme autumn temperature
events >30ºC could impact on development and
reproduction of N. viridula.
Previous studies have suggested that optimum
temperature for N. viridula is about 25ºC (Ali and
Ewiess 1977). It is important to note that in this study,
populations of N. viridula were found to be high
during the soybean maturity stage ( the April to May
autumn period) with temperature about 26ºC
(observation during collection of N. viridula for
culture and experiment). This indicated that the
autumn period is a favourable environmental
condition for the development and reproduction of N.
viridula compared to the summer (December to
February) with mean summer maximum temperatures
of 29.7ºC (coastal site) and 31.5ºC (inland site) (BoM
2011). These summer temperatures are more likely to
be unfavourable for the development and
reproduction of N. viridula. Thus, if climate change
were to affect the region, including elevated
temperatures more extreme events and a shift of the
rainfall regime toward summer in inland and coastal
sites, N. viridula may be unable to adapt immediately.
N. viridula might require time to adapt to less
favourable conditions or shift distribution depending
on availability and suitability of food plants. On the
other hand, it is possible that soybean growers may
plant soybeans later in the future to avoid extreme
summer temperatures and this would favour N.
viridula by maintaining maximum temperatures at the
optimum level during the soybean podding stage. The
warming tolerance (WT) of inland populations of N.
viridula was 14.4ºC; compared to coastal populations
of N. viridula at 16.2ºC. This indicates both
populations could tolerate temperature increases
before their performance is reduced to negligible or
lethal levels. However, thermal safety margin (TSM)
of inland N. viridula was -6.5ºC and coastal N.
viridula was -4.7ºC. Both populations in summer are
vulnerable already to the thermal limits of their
habitat in summer exposing them to heat stress. To
overcome this, N. viridula will need to use
behavioural mechanisms to reduce exposure to heat
stress, such as foraging and flying in the cooler parts
of the day.
In Japan, N. viridula has long been known to occur in
southern parts, but recently adults have been observed
in Osaka, central Japan, at least 70 km further north
than the northern limit of distribution reported in the
early 1960s (Kiritani 1971, Kiritani et al. 1963,
Musolin and Numata 2003). It is evident that
temperatures in the present lab 20/30ºC treatment
would be equivalent to field conditions in south-
eastern Queensland during summer: these
temperatures affected duration of nymphal
development of N. viridula. Moreover, fluctuating
temperatures between of 27/37ºC have adversely
affected nymphal and adult performance and
survivorship of N. viridula: no insects survived long
enough to mate and reproduce (Velasco and Walter
1993). However, Velasco and Walter (Velasco and
Walter 1993) concluded that low N. viridula nymphal
densities in south-eastern Queensland during summer
cannot be explained by ambient temperature
conditions alone. Temperature, however, may have an
indirect influence on the abundance of N. viridula
through its influence on the availability and suitability
of host plants. It is also important to note that the
bugs, N. viridula were maintained in containers inside
incubators with consistent temperature regimes
throughout the assessment period (i.e. no diurnal
fluctuation). On the other hand, the bugs were not
able to use behavioural adaptations to modify the
effects of the warming climate within their containers.
For instance, the bugs did not have the ability to
move to a cooler microclimate within the containers.
In conclusion, the speed and duration of insect
development are dependent upon a combination of
external and internal factors, of which temperature is
a more important factor compared to humidity. High
temperatures shorten, and low temperatures lengthen,
the period of insect development and metamorphosis
(Hintze 1970). The optimum temperature of N.
viridula in development was 25ºC and intermediate
humidity about 40 ± 10% RH, since all
developmental stages of N. viridula developed
successfully with high rates of nymphal survival,
adults living long enough to mate and reproduce large
egg-masses with higher fecundity. These results show
that shorter nymphal duration, shorter adult longevity,!
GEN. APPL. ENT. VOL 43, 2015
!
!
50!
of mating frequency and reproduction occurred in
higher temperature conditions. The present results
suggested that N. viridula could not adapt to climate
extremes even for a short time period. N. viridula in
this study required a certain period under
unfavourable climate conditions for successful
development and reproduction. For instance, from an
evolutionary point of view, the cold adaptation seen
in temperate populations may be the consequence of
long-term directional selection such as in Drosophila
(Ayrinhac et al. 2004). However, no differences in
nymphal duration, survival, adult longevity and
reproduction performance between inland and coastal
N. viridula were found under different experimental
climate conditions.
ACKNOWLEDGEMENTS
Financial support was given to Pol Chanthy by the
Australian Centre for International Agricultural
Research (ACIAR) who provided a John Allwright
Fellowship (JAF) Award through the Australian
Agency for International Development (AusAID),
and UNE.
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Appendix 1. The interaction of locations and temperature regimes on nymphal duration, survival, adult longevity and egg hatching duration of Nezara
viridula (L.)
Loc
Tem
(ºC)
Mean duration ± SE of nymphal stages (days)
Nymphal
duration*
(days)
Nymphal
survival
(%)
Mean longevity ± SE of adult N. viridula
(days)
Incubation
period
(days)
1st instar duration (days)
2nd
3rd
4th
5th
Female
Male
Adults
(F&M)
I
25
7.1 ± 0.2
a
5.3 ± 0.2
a
6.5 ± 0.2
a
11.3 ± 0.4 a
30.2 ± 0.8 a
69.2 ± 6.1 a
68.0 ± 11.5
a
57.5 ± 8.2 a
64.8 ± 10.2
a
6.5 ± 0.2 a
4.4 ± 0.2 a
I
30
5.3 ± 0.2
a
4.3 ± 0.2
a
5.8 ± 0.1
a
10.7 ± 0.3 a
26.0 ± 0.3 a
64.2 ± 7.2 a
58.3 ± 11.4
a
40.6 ± 12.4 bc
50.6 ± 9.7 a
3.4 ± 1.5
b
-
I
33
5.1 ± 0.3
a
3.6 ± 0.2
a
5.3 ± 0.2
a
10.7 ± 0.7 a
24.6 ± 0.9 a
50.8 ± 6.2 a
40.6 ± 9.7 a
31.0 ± 4.5 c
35.6 ± 6.4 a
-
-
I
36
4.5 ± 0.4
a
3.9 ± 0.1
a
5.4 ± 0.3
a
9.4 ± 0.2 a
23.2 ± 0.5 a
48.3 ± 7.0 a
10.6 ± 3.2 a
16.3 ± 2.1 de
13.6 ± 2.4 a
-
-
C
25
6.6 ± 0.2
a
5.5 ± 0.2
a
6.0 ± 0.3
a
10.3 ± 0.4 a
28.4 ± 1.0 a
73.3 ± 6.9 a
53.5 ± 6.4 a
35.1 ± 3.7 bc
48.9 ± 6.7 a
6.5 ± 0.3 a
4.1 ± 0.2 a
C
30
5.6 ± 0.3
a
4.4 ± 0.1
a
5.3 ± 0.1
a
9.5 ± 0.4 a
24.8 ± 0.3 a
62.5 ± 9.0 a
60.4 ± 11.8
a
42.7 ± 10.2 b
49.4 ± 9.5 a
2.5 ± 1.1 c
0.5 ± 0.5 a
C
33
5.1 ± 0.2
a
4.0 ± 0.1
a
4.8 ± 0.2
a
9.8 ± 0.4 a
23.7 ± 0.6 a
50.0 ± 8.9 a
37.4 ± 6.4 a
29.4 ± 10.0 cd
32.9 ± 7.7 a
-
-
C
36
4.2 ± 0.1
a
4.0 ± 0.2
a
5.2 ± 0.1
a
9.6 ± 0.5 a
23.1 ± 0.4 a
45.8 ± 9.1 a
10.5 ± 3.7 a
13.4 ± 4.1 e
12.3 ± 3.5 a
-
-
Loc – locations,I – inland, C – coastal, Tem – temperature, * – days from second instar of nymphs to adults, Mean ± standard error (SE). Mean followed by different letters in the same column are
significantly different at p = 0.05, using IRRISTAT program for Windows 5.0. Means were compared by the method of least significant differences (LSD) at 5% level.
[
!
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB!
!
!
53!
Appendix 2. The interaction effects of locations and temperature regimes on body weight and reproductive performance and egg hatchability of Nezara
viridula (L.)
Loc
Tem
(ºC)
Mean weight (g) ± SE of adult N. viridula
Mating
frequency
(times)
Pre-mating
period (days)
Pre-
oviposition
period (days)
Egg-
masses/
female
Egg-mass
size
(eggs/egg
mass)
Fecundity
(Eggs/female)
% of egg mass
hatched
Egg
hatch-ability
(%)
Female
Male
Adults
(F & M)
I
25
0.16 ± .01 a
0.13 ± .00 a
0.14 ± .00 a
5.5 ± 0.7 a
25.9 ± 2.9 b
52.6 ± 1.3 a
1.2 ± 0.2 b
65.3 ± 5.6 a
81.8 ± 15.6 b
86.5 ± 6.4 a
54.4 ± 15.7 a
I
30
0.15 ± .01 a
0.12 ± .00 a
0.14 ± .01 a
2.0 ± 1.3 a
24.3 ± 11.3 b
34.6 ± 15.5 c
0.3 ± 0.2 c
19.2 ± 8.6 a
12.6 ± 8.0 c
40.0 ± 20.0 a
4.4 ± 2.9 a
I
33
0.13 ± .01 a
0.11 ± .01 a
0.12 ± .01 a
0.3 ± 0.2 a
19.3 ± 12.3 bc
-
-
-
-
-
-
I
36
0.11 ± .00 a
0.10 ± .00 a
0.11 ± .00 a
-
-
-
-
-
-
-
-
C
25
0.15 ± .01 a
0.12 ± .00 a
0.14 ± .00 a
6.8 ± 1.0 a
25.7 ± 2.4 b
47.6 ± 3.8 b
2.4 ± 0.3 a
60.0 ± 0.8 a
146.0 ± 19.8 a
78.6 ± 10.5 a
51.0 ± 16.4 a
C
30
0.15 ± .01 a
0.12 ± .00 a
0.13 ± .01 a
2.2 ± 0.4 a
42.5 ± 5.9 a
43.9 ± 19.7 b
0.7 ± 0.4 c
16.2 ± 7.3 a
23.1 ± 16.7 c
42.4 ± 20.1 a
5.2 ± 2.5 a
C
33
0.14 ± .00 a
0.10 ± .01 a
0.12 ± .00 a
0.2 ± 0.2 a
3.7 ± 3.7 c
-
-
-
-
-
-
C
36
0.11 ± .01 a
0.09 ± .01 a
0.10 ± .00 a
-
-
-
-
-
-
-
-
Loc – locations,I – inland, C – coastal, Tem – temperature, Mean ± standard error (SE). Mean followed by different letters in the same column are significantly different at p = 0.05, using IRRISTAT program for Windows 5.0.
Means were compared by the method of least significant differences (LSD) at 5% level.
Appendix 3. The interaction of locations and humidity regimes on nymphal duration, survival, adult longevity and egg hatching duration of Nezara
viridula (L.)
Loc
Hum.
(%)
Mean duration ± SE of nymphal stages (days)
Nymphal
duration*
(days)
Nymphal
survival
(%)
Mean longevity ± SE of adult N. viridula (days)
Incubation
period
(days)
1st instar
duration
(days)
2nd
3rd
4th
5th
Female
Male
Adults
(F&M)
I
40
5.5 ± 0.3 a
4.4 ± 0.2 a
5.7 ± 0.2 a
10.4 ± 0.3 a
26.0 ± 0.9 a
63.3 ± 5.6 a
56.9 ± 9.8 a
49.4 ± 6.9 a
54.1 ± 8.3 a
3.4 ± 1.0 a
1.2 ± 0.6 a
I
80
5.4 ± 0.4 a
4.1 ± 0.2 a
5.8 ± 0.2 a
10.7 ± 0.4 a
26.0 ± 0.9 a
52.9 ± 4.3 a
31.8 ± 6.8 a
23.3 ± 4.2 a
28.2 ± 4.6 a
1.5 ± 0.8 c
1.0 ± 0.5 a
C
40
5.4 ± 0.3 a
4.6 ± 0.2 a
5.4 ± 0.2 a
10.2 ± 0.2 a
25.7 ± 0.8 a
70.8 ± 5.1 a
53.2 ± 7.9 a
42.5 ± 5.8 a
48.3 ± 6.5 a
3.0 ± 0.9 b
1.3 ± 0.6 a
C
80
5.4 ± 0.3 a
4.3 ± 0.2 a
5.2 ± 0.2 a
9.4 ± 0.3 b
24.2 ± 0.6 a
45.0 ± 5.6 a
27.6 ± 5.3 a
17.8 ± 3.7 a
23.5 ± 4.3 a
1.5 ± 0.8 c
1.0 ± 0.5 a
Loc – locations, I – inland, C – coastal, Hum – humidity, * – days from second instar of nymphs to adults. Mean ± standard error (SE). Mean followed by different letters in the same column are
significantly different at p = 0.05, using IRRISTAT program for Windows 5.0. Means were compared by the method of least significant differences (LSD) at 5% level.
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Appendix 4. The interaction effects of locations and humidity regimes on body weight and reproductive performance and egg hatchability of Nezara
viridula (L.)
Loc
Hum
(%)
Mean weight (g) ± SE of adult N. viridula
Mating
frequency
(times)
Pre-mating
period (days)
Pre-
oviposition
period (days)
Egg-
masses/
female
Egg-mass
size
(eggs/egg
mass)
Fecundity
(Eggs/female)
% of egg mass
hatched
Egg
hatch-ability
(%)
Female
Male
Adults
(F & M)
I
40
0.14 ± .01 a
0.12 ± .00 a
0.13 ± .01 a
2.7 ± 0.9 a
25.4 ± 7.2 a
30.7 ± 9.4 a
0.5 ± 0.2 a
27.9 ± 9.4 a
30.3 ± 13.3 a
38.3 ± 12.4 a
7.1 ± 2.7 a
I
80
0.13 ± .01 a
0.11 ± .00 a
0.12 ± .01 a
1.3 ± 0.6 a
9.4 ± 4.7 a
12.9 ± 6.8 a
0.3 ± 0.2 a
14.3 ± 7.5 c
16.9 ± 9.7 a
25.0 ± 13.1 a
22.3 ± 11.7 a
C
40
0.14 ± .01 a
0.11 ± .01 a
0.12 ± .01 a
2.7 ± 1.0 a
19.8 ± 6.8 a
33.7 ± 11.1 a
1.0 ± 0.4 a
23.1 ± 7.6 b
52.5 ± 22.3 a
35.5 ± 11.7 a
6.2 ± 2.1 a
C
80
0.14 ± .01 a
0.11 ± .01 a
0.12 ± .01 a
1.9 ± 0.7 a
16.1 ± 4.4 a
12.0 ± 6.5 a
0.5 ± 0.3 a
15.0 ± 7.8 c
32.1 ± 17.4 a
25.0 ± 13.1 a
21.9 ± 11.4 a
Loc – locations, I – inland, C – coastal, Hum – humidity, Mean ± standard error (SE). Mean followed by different letters in the same column are significantly different at p = 0.05, using IRRISTAT
program for Windows 5.0. Means were compared by the method of least significant differences (LSD) at 5% level.
!
Appendix 5. The interaction effects of locations, temperature and humidity regimes on nymphal duration, survival, adult longevity and egg hatching
duration. Bolded results are significant differences between locations within the same climatic condition.
Loc
Tem
(ºC)
Hum
(%)
Mean duration ± SE of nymphal stages (days)
Nymphal
duration*
(days)
Nymphal
survival
(%)
Mean longevity ± SE of adult N. viridula
Incubation
period
(days)
1st instar
duration
(days)
2nd
3rd
4th
5th
Female
Male
Adults
(F & M)
I
25
40
6.9 ± 0.4 ab
5.5 ± 0.2 a
6.6 ± 0.3 a
11.5 ± 0.6 a
30.5 ± 1.2 a
71.7 ± 12.0 a
90.3 ± 11.8 a
72.0 ± 8.2 a
84.8 ± 9.0 a
6.9 ± 0.1 ab
4.9 ± 0.1 a
I
25
80
7.2 ± 0.1 a
5.2 ± 0.3 a
6.3 ± 0.5 a
11.1 ± 0.7 a
29.9 ± 1.4 a
66.7 ± 6.0 a
45.8 ± 5.2 a
43.0 ± 7.7 cd
44.7 ± 5.9 a
6.0 ± 0.1 c
4.0 ± 0.1 a
I
30
40
5.5 ± 0.3 cde
4.3 ± 0.2 a
5.5 ± 0.1 a
10.7 ± 0.4 a
26.1 ± 0.7 a
78.3 ± 7.3 a
70.1 ± 8.3 a
67.9 ± 4.1 ab
69.3 ± 2.6 a
6.8 ± 0.2 b
-
I
30
80
5.0 ± 0.1 fg
4.3 ± 0.4 a
6.0 ± 0.1 a
10.7 ± 0.5 a
26.0 ± 0.3 a
50.0 ± 2.9 a
46.5 ± 21.1 a
13.3 ± 3.1 gh
31.9 ± 10.8 a
-
-
I
33
40
4.8 ± 0.4 efgh
3.9 ± 0.2 a
5.0 ± 0.2 a
9.8 ± 0.8 a
23.5 ± 0.8 a
58.3 ± 1.7 a
53.9 ± 16.3 a
37.3 ± 6.8 cde
44.8 ± 10.6 a
-
-
I
33
80
5.3 ± 0.3 de
3.3 ± 0.2 a
5.6 ± 0.3 a
11.5 ± 1.0 a
25.7 ± 1.4 a
43.3 ± 11.7 a
27.2 ± 5.7 a
24.7 ± 4.1 efg
26.5 ± 2.8 a
-
-
I
36
40
4.9 ± 0.7 efgh
3.9 ± 0.2 a
5.5 ± 1.6 a
9.5 ± 0.3 a
23.9 ± 0.8 a
45.0 ± 12.6 a
13.4 ± 6.6 a
20.3 ± 1.9 fgh
17.6 ± 3.5 a
-
-
I
36
80
4.0 ± 0.1 h
3.9 ± 0.2 a
5.2 ± 0.2 a
9.3 ± 0.1 a
22.4 ± 0.0 a
51.7 ± 8.8 a
7.7 ± 0.6 a
12.2 ± 1.4 gh
9.7 ± 0.1 a
-
-
C
25
40
6.8 ± 0.3 ab
5.9 ± 0.2 a
6.4 ± 0.1 a
10.7 ± 0.3 a
29.8 ± 0.7 a
80.0 ± 12.6 a
61.9 ± 3.1 a
39.3 ± 4.2 cde
59.5 ± 6.2 a
7.1 ± 0.2 a
4.4 ± 0.1 a
C
25
80
6.4 ± 0.3 abc
5.0 ± 0.2 a
5.5 ± 0.4 a
9.9 ± 0.6 a
26.9 ± 1.5 a
66.7 ± 6.0 a
45.0 ± 11.2 a
31.0 ± 5.9 def
38.3 ± 8.6 a
6.0 ± 0.2 c
3.8 ± 0.2 a
C
30
40
5.2 ± 0.1 def
4.5 ± 0.1 a
5.4 ± 0.1 a
10.2 ± 0.1 a
25.3 ± 0.1 a
78.3 ± 8.8 a
85.4 ± 5.0 a
60.1 ± 14.2 ab
66.9 ± 11.2 a
5.0 ± 0.0 d
1.0 ± 1.0 a
C
30
80
6.0 ± 0.7 bcd
4.4 ± 0.3 a
5.1 ± 0.1 a
8.8 ± 0.7 a
24.3 ± 0.3 a
46.7 ± 8.8 a
35.4 ± 6.5 a
25.3 ± 4.3 efg
31.8 ± 4.6 a
-
-
C
33
40
5.5 ± 0.1 cde
4.1 ± 0.2 a
5.0 ± 0.3 a
9.5 ± 0.7 a
24.1 ± 1.0 a
65.0 ± 2.9 a
50.2 ± 5.2 a
51.6 ± 1.9 bc
49.7 ± 1.6 a
-
-
C
33
80
4.7 ± 0.2 efgh
3.8 ± 0.0 a
4.7 ± 0.4 a
10.1 ± 0.3 a
23.2 ± 0.5 a
35.0 ± 12.6 a
24.5 ± 3.5 a
7.3 ± 2.8 h
16.2 ± 3.8 a
-
-
C
36
40
4.1 ± 0.1 gh
4.0 ± 0.2 a
5.0 ± 0.1 a
10.5 ± 0.2 a
23.6 ± 0.4 a
60.0 ± 13.2 a
15.4 ± 6.1 a
19.2 ± 6.5 fgh
17.1 ± 5.8 a
-
-
C
36
80
4.3 ± 0.1 fgh
4.1 ± 0.5 a
5.4 ± 0.1 a
8.8 ± 0.5 a
22.6 ± 0.7 a
31.7 ± 6.0 a
5.6 ± 2.4 a
7.7 ± 3.2 h
7.5 ± 2.0 a
-
-
Loc – locations, I – inland, C – coastal, Tem – temperature, Hum – humidity, F – female, M – male, * – days from second instar of nymphs to adults. Mean ± standard error (SE). Mean followed by
different letters in the same column are significantly different at p = 0.05, using IRRISTAT program for Windows 5.0. Means were compared by the method of least significant differences (LSD) at 5%
level
CHANTHY ET AL: INFLUENCE OF TEMPERATURE AND HUMIDITY ON GVB!
!
55!
Appendix 6. The interaction effects of locations, temperature and humidity regimes on body weight, reproductive performance and egg hatchability of
Nezara viridula (L.)
Loc
Tem
(ºC)
Hum
(%)
Mean weight (g) ± SE of adult N. viridula
Mating
frequency
(times)
Pre-mating
period (days)
Pre-
oviposition
period (days)
Egg-
masses/
female
Egg-mass
size
(eggs/egg
mass)
Fecundity
(Eggs/female)
% of egg mass
hatched
Egg
hatchability
(%)
Female
Male
Adults
(F & M)
I
25
40
0.17 ± .01 a
0.13 ± .01 a
0.15 ± .01 a
6.3 ± 1.2 a
31.6 ± 3.0 a
53.6 ± 2.1 c
1.3 ± 0.2 a
73.3 ± 9.5 a
96.0 ± 25.7 a
73.1 ± 4.6 a
19.5 ± 2.3 a
I
25
80
0.16 ± .01 a
0.13 ± .01 a
0.14 ± .01 a
4.7 ± 0.3 a
20.2 ± 1.3 a
51.6 ± 1.7 c
1.2 ± 0.4 a
57.3 ± 1.6 a
67.7 ± 18.8 a
100.0 ± 0.0 a
89.3 ± 3.9 a
I
30
40
0.15 ± .01 a
0.13 ± .00 a
0.14 ± .01 a
4.0 ± 2.0 a
48.6 ± 6.5 a
69.1 ± 1.7 b
0.7 ± 0.3 a
38.3 ± 1.7 a
25.2 ± 12.7 a
80.0 ± 20.0 a
8.7 ± 4.7 a
I
30
80
0.14 ± .01 a
0.11 ± .01 a
0.13 ± .01 a
-
-
-
-
-
-
-
-
I
33
40
0.14 ± .00 a
0.12 ± .00 a
0.13 ± .00 a
0.3 ± 0.3 a
21.3 ± 21.3 a
-
-
-
-
-
-
I
33
80
0.12 ± .01 a
0.10 ± .01 a
0.11 ± .01 a
0.3 ± 0.3 a
17.3 ± 17.3 a
-
-
-
-
-
-
I
36
40
0.12 ± .01 a
0.10 ± .01 a
0.11 ± .00 a
-
-
-
-
-
-
-
-
I
36
80
0.11 ± .00 a
0.10 ± .01 a
0.10 ± .01 a
-
-
-
-
-
-
-
-
C
25
40
0.15 ± .01 a
0.12 ± .00 a
0.14 ± .01 a
8.3 ± 0.3 a
25.6 ± 2.6 a
47.0 ± 1.6 c
2.7 ± 0.6 a
60.1 ± 1.1 a
163.6 ± 34.1 a
57.2 ± 9.7 a
14.5 ± 3.0 a
C
25
80
0.16 ± .00 a
0.12 ± .01 a
0.14 ± .00 a
5.3 ± 1.7 a
25.7 ± 4.7 a
48.2 ± 8.3 c
2.1 ± 0.4 a
59.9 ± 1.5 a
128.3 ± 22.0 a
100.0 ± 0.0 a
87.5 ± 2.6 a
C
30
40
0.16 ± .00 a
0.12 ± .01 a
0.14 ± .00 a
2.3 ± 0.7 a
53.6 ± 6.4 a
87.8 ± 2.4 a
1.3 ± 0.8 a
32.5 ± 2.5 a
46.2 ± 29.3 a
84.8 ± 15.2 a
10.4 ± 2.3 a
C
30
80
0.14 ± .01 a
0.12 ± .01 a
0.13 ± .01 a
2.0 ± 0.6 a
31.5 ± 3.8 a
-
-
-
-
-
-
C
33
40
0.13 ± .00 a
0.12 ± .00 a
0.12 ± .00 a
-
-
-
-
-
-
-
-
C
33
80
0.14 ± .00 a
0.09 ± .02 a
0.12 ± .01 a
0.3 ± 0.3 a
7.3 ± 7.3 a
-
-
-
-
-
-
C
36
40
0.10 ± .00 a
0.09 ± .01 a
0.10 ± .01 a
-
-
-
-
-
-
-
-
C
36
80
0.12 ± .01 a
0.10 ± .00 a
0.11 ± .01 a
-
-
-
-
-
-
-
-
Loc – locations, I – inland, C – coastal, Tem – temperature, Hum – humidity. Mean ± standard error (SE). Mean followed by different letters in the same column are significantly different at p = 0.05,
using IRRISTAT program for Windows 5.0. Means were compared by the method of least significant differences (LSD) at 5% level!
