A South African Scenario: Structure and Function of Peptides of the Adipokinetic Hormone Family of the Brown Locust, Locustana pardalina, and the Putative Role of These Peptides in Plague Management

ABSTRACT

The brown locust, Locustana pardalina, is a major agricultural pest in southern Africa during swarm formation. Like other locust species, L. pardalina has much higher carbohydrate concentrations in circulation than lipid; carbohydrates are predominantly used in the first minutes of flight and with sustained flight, the metabolic fuel switches to lipids mobilised from fat body stores. We isolated three peptides from the corpora cardiaca of the brown locust; through sequence elucidation by Edman degradation, mass spectrometry and chromatographic confirmation, we show that the brown locust has the same compliment of chemically isolated adipokinetic hormones (AKHs) as in Locusta migratoria: a decapeptide and two octapeptides; all increase the circulating lipid levels in locusts but not the carbohydrate concentration. During a rest period following flight, the carbohydrate levels in the haemolymph remained lower than before flight, whereas the lipid levels remained elevated. We show that the glycogen concentration in the fat body is significantly lowered after 1 h rest postflight and it is significantly increased in the flight muscles in this time. Thus, glycogen is mobilised from the fat body during the rest phase and transported as trehalose to the flight muscles and there, converted to glycogen, presumably to supply energy for a subsequent flight action. Finally, we discuss the molecular evolution of AKHs in Orthoptera and how two of the brown locust AKHs could serve as leads for developing peptidomimetics for combatting swarm outbreaks and reducing the need for harmful, indiscriminate chemical pesticides.

• 1.The brown locust produces the three adipokinetic hormones Locmi‐AKH‐I, ‐II and ‐III found also in the migratory locust.
• 2.Flight is characterised by differential changes in haemolymph carbohydrate and lipid levels.
• 3.Modelling ligand‐receptor binding is proposed as important step to a new strategy to combat the brown locust.

Summary

• We find three metabolic hormones identical to the migratory locust AKHs in Locustana pardalina (brown locust), a major pest in southern Africa.

• Haemolymph carbohydrates are used at flight start and then lipids.

• Peptidomimetics may curb swarming flights.

1. Introduction

The brown locust, Locustana pardalina, is a serious pest insect in South Africa with outbreak areas spanning the arid Nama Karoo biome of South Africa and southern Namibia, where hoppers and adults feed on the Karoo grasses (Lea 1958; Kieser et al. 2002). As a true locust, L. pardalina occurs as solitary and gregarious form, thus displaying phase polymorphism with differering phenotype, behaviour, physiology and biochemistry; all being dependent on population density: under crowded conditions locusts develop gregarious phase characteristics, while being isolated produces the solitary phase (Pener and Simpson 2009). In fact, the differences between each extreme phase form are much more pronounced in the brown locusts than in any other locust species (Faure 1932). During the gregarious phase huge numbers of hopper and adult brown locust individuals occur and cause what is called “a massive outbreak” during which adults form big swarms that not only devour vegetable crops planted in the Karoo but also wheat and maize cultivated on large plots outside the main outbreak area in the entire southern Africa up to the Zambezi River where these swarms can fly to (Lea 1958; Price 2021). It is then when the real plague problem sets in with a severe negative economic impact.

Governmental issued control measures are, thus, in place since more than 120 years (Price 2021) using different types of chemical substances over the years. During the 1993/94 outbreak when the current research was undertaken, mainly organophosphate compounds were administered which do not discriminate between pest insects and other animals. Today, the somewhat more secure and safer synthetic broad‐spectrum pyrethroid insecticides are used (Price 2021), but still the problems remain that intensity of spraying and multiple applications in the same area, impacts negatively on the environment and the organisms therein (Price 2021).

Let us imagine one of the worst‐case scenarios that could happen with the brown locust population as a serious pest, viz. that after an outbreak, most of the hoppers survive to become winged adults that swarm and with their powerful and far reaching flights first devastate the grasslands of the Karoo and then arrive further north at the cultivated wheat and maize fields and completely devour the food crops. How could one possibly prevent such long‐distance flights without the use of the infamous chemical pesticides? One possible avenue may be based on the knowledge that in various insect species such flights are fuelled mainly by lipids stored in the fat body, and their release is regulated by small neuropeptide hormones synthesised in the corpus cardiacum (Gäde and Marco 2013; Marco and Gäde 2020). Interfering with this so‐called adipokinetic hormone system may be a successful control measure to halt the spread of the brown locusts and thereby, prevent or limit the consequent food insecurity. The adipokinetic hormone (AKH) peptide family members are eight to ten amino acids long and are characterised chemically by blocked termini, thus the N‐terminus contains a pyroglutamate and the C‐terminus is a carboxyamide; position 2 is either an aliphatic (Leu, Ile, Val) or aromatic (Phe, Tyr) residue, position 3 contains Asn or Thr, aromatic Phe or Tyr is at position 4, Ser or Thr at position 5, and the family‐signature aromatic amino acid Trp is always at position 8, Gly at position 9, while a variety of amino acids may occur at positions 6, 7 and 10 (Marco et al. 2024). Apart from the characteristic terminal posttranslational modifications (PTMs), other PTMs occur as well, such as phosphorylation, sulfation, hydroxyprolination, Trp C‐mannosylation and a possible Pro isomerization (Marco et al. 2024). As with all peptide hormones, the AKHs bind extracellularly to a receptor, the AKH receptor (AKHR) which is characterised for quite a few insects, inter alia, two locust species, Schistocerca gregaria (Marchal et al. 2018) and Locusta migratoria (GenBank Accession Number: ANW09575.1). As outlined recently (Gäde et al. 2025) knowledge of the ligand (AKH) and its cognate G protein‐coupled receptor (AKHR) is necessary to discover leads for the design of novel specific, biorational and biodegradable insecticides (Gäde and Goldsworthy 2003; Audsley and Down 2015; Davies 2017) using the same intellectual approach that the pharmaceutical industry exploits for drug discovery, as in the case of the well‐known β‐blockers (Black 2010; Baker et al. 2011). Research in this direction with the AKH system of the brown locust may lead to peptide mimetics that competitively inhibit the action of AKH, thereby impairing the provision of energy for flight. With this in mind, we investigated in the present study the complement of ligands (AKHs) and their function in the brown locust and discuss possibilities of further studies into this topic of green insecticides against L. pardalina. Preliminary information on the metabolics of the brown locust were published in Gäde and Marco (2009).

2. Materials and Methods

2.1. Insects

Adult male brown locusts of about 5 days after ecdysis were obtained from the Agricultural Research Council in Pretoria. Last instar hoppers, as well as adults of unknown age (but younger than 20 days) were also collected near Calvinia (Northern Cape Province, South Africa) after an outbreak in April 1994 and brought to Cape Town where the insects were kept in large cages in our insectary at 25°C, a 14:10 h light/dark regime with kikuyu grass or wheat seedlings fed ad libitum.

Adult male migratory locusts, Locusta migratoria, came from our own culture and were reared under the same conditions as the brown locusts. They were fed wheat seedlings or all kinds of grasses supplemented with rolled oats.

2.2. Bioassays

The lipid and carbohydrate bioassays measuring total vanillin‐ and anthrone‐positive material, respectively, were performed according to the methods of Zöllner and Kirsch (1962) and Spik and Montreuil (1964), as modified by Holwerda et al. (1977). Briefly, individual adult locusts were kept at rest under a dark funnel throughout experimentation; slight bleeding was induced with a fine pin prick to the base of a walking leg and 1 µl haemolymph was withdrawn from each animal with a glass microcapillary (Hirschmann) at rest and 90 min after an injection (10 µl volume) was administered into the abdominal haemocoel with a 25 µl Hamilton syringe. The haemolymph sample was added to 100 µl concentrated sulphuric acid in a glass tube immediately after collection for the colorimetric assays. For the vanillin assay, the reacted sample solution was transferred to a glass cuvette, and the optical density was read in a spectrophotometer at wavelength 546 nm, with a cholesterol standard curve for reference values; in the case of the anthrone assay, a wavelength of 585 nm with a glucose standard curve was applied. Crude extracts of corpora cardiaca (CC), HPLC peaks and synthetic peptides were tested in distilled water, hence the latter served as negative control in the bioassays.

2.3. Dissection of CC, Isolation and Structural Analyses of Their Peptide Content

CC were isolated from other neuronal tissues from the head of L. pardalina at 20‐fold magnification under a dissecting microscope and put into 80% methanol. Methanolic extracts were prepared as described previously with ultrasonication and subsequent centrifugation (Gäde et al. 1984). The vacuum‐dried material was dissolved in 15% acetonitrile containing 0.1% trifluoroacetic acid (TFA) and applied to a Nucleosil 100 C‐18 column for reversed‐phase high‐performance liquid chromatography (RP‐HPLC) using the following equipment from Gilson, as described earlier (Gäde 1985): two Model 302 piston pumps with 5 S pump head, a manometric module Model 802, a Model 811 mixing chamber, a Rheodyne Model 7125 sample injector with a 50 µl sample loop. The HPLC system was connected to a LKB 2151 variable wavelength detector with a 10 µl HPLC flow cell and 10 mm pathlength, a fluorescent detection system which identifies tryptophan‐containing compounds (Jasco model FP‐920; 276 nm excitation, 350 nm emission), and a computer through which the pumps were controlled with specific separation gradients using the Gilson Gradient Program software (see relevant Figure legends for gradient details). Active fractions were either identified by identical retention times to known AKH peptides or by bioassay (see above). The identified active fractions from 18 CC gland equivalents were separately deblocked using the enzyme l‐pyroglutamate aminopeptidase as described previously (Gäde et al. 1988). The mixture was run on RP‐HPLC with a longer gradient programme and undigested and deblocked peptides separated and collected. The intact (undigested) peptides were used for in‐house matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (Voyager Elite MALDI‐TOF; PerSeptive Biosystems Inc., Framington, Massachusetts USA), while the deblocked peptides were spotted on polybrene coated glass‐fibre discs and subjected to automated Edman degradation (model 477 A; Applied Biosystems (ABI), Foster City, California, USA). The sequencer was connected to an online phenylthiohydantoin (PTH) amino acid analyser (model 120 A; ABI).

2.4. Determination of Haemolymph Volume

A trace amount of labelled inulin ([3H{G}]inulin (355.3 mCi/g), Dupont, USA) contained in 5 µl of water was injected into 4 brown locusts each of a group that had been resting or flown for 60 min. After 20 min, a 1 µl haemolymph sample was obtained, collected directly into 4.5 ml scintillation fluid (Ultima Gold XR, Packard, Illinois, USA) and assayed for radioactivity using a Tricarb 460 scintillation counter (Packard) as described by Clegg and Evans (1961).

2.5. Procedure of Flight Experiments

Male brown locusts between 12 and 20 days of adult life were flown on a flight mill at 25°C in a room that was brightly lit. Flight lasted for up to 60 min. To stimulate individual specimens for long flight periods, tarsal contact was made and broken with a small stick when necessary, and a warm current of air was blown against the flight direction with the help of a hand‐held hair blow dryer. For the postflight recovery experiment, specimens were flown for 60 min and left to recover for up to 60 min of rest under a funnel without water and food. Before flight and during various times of flight and after recovery, a 1 µl haemolymph sample each was withdrawn from the base of the metathoracic leg for the determination of lipids and carbohydrates as described in section 2.2. Some insects were killed after 1 h of flight or 1 h recovery postflight, their flight muscles and abdominal fat body were dissected for glycogen extraction and analysed by the modified anthrone method with glucose as standard as described previously (Zebe and Gäde 1993). In another series of flight experiments, 6 adult male brown locusts were flown for 60 min with a 2 h recovery postflight. Haemolymph samples from these 6 insects were collected before flight, after 20 min, 30 min, 45 min and 60 min of flight, and after the 2 h recovery period for measuring only carbohydrate concentrations.

2.6. Statistical Analyses

The paired T‐test was used for all statistical analyses, except for experiments where glycogen was measured at different times from different individuals. In this case, statistical comparisons between groups were performed by ANOVA followed by Tukey′s post‐hoc test.

3. Results

3.1. Intrinsic Peptides of the AKH Family Are Biologically Active in the Brown Locust

In a first series of experiments, resting levels of metabolites in the circulatory system of the brown locust were measured, and it was established whether factors in the CC of the brown locust could mobilise carbohydrates or lipids conspecifically and heterospecifically. Non‐injected adult male brown locusts of about 12 days had between 7 and 8 mg total lipids (measured as vanillin‐positive material) per ml haemolymph and about a sixfold higher total carbohydrate concentration (measured as anthrone‐positive material) (Table 1). Control injections of distilled water, which was the solvent of all other treatments, had no significant effect on the concentrations of either lipids or carbohydrates. Injection of a CC extract from the brown locust had a pronounced and very significant adipokinetic effect that increased with higher amounts of extract (Table 1). Contrary, there is apparently no factor in the CC that mobilises overtly the carbohydrates; the high concentration found before the injection did not change significantly upon administration of a dose of 0.2 CC gland equivalent (Table 1). A heterologous assay, using the migratory locust as the acceptor insect, was also employed: 0.1 CC equivalents of the brown locust caused a large and significant increase in haemolymph lipids from 12.28 ± 2.70 mg/ml before injection to 40.71 ± 6.50 mg/ml after injection into 6 adult male (14 days old) L. migratoria. This result was convenient because it meant that we could use L. migratoria (available in sufficient numbers from a laboratory‐reared colony) to monitor bioactivity during the purification and separation of brown locust CC extracts, especially with the unpredictable supply of brown locusts.

Table 1.

Conspecific bioassays for adipokinetic and hypertrehalosaemic activity of crude methanolic extracts of brown locust corpora cardiaca (Locustana pardalina).

Treatment	Haemolymph lipids (mg ml−1)					Haemolymph carbohydrates (mg ml−1)
	n	0 min	90 min	Difference	P *	n	0 min	90 min	Difference	P *
Control
(10 μl distilled water)	5	7.26 ± 2.40	7.07 ± 3.04	−0.19 ± 0.87	NS	5	45.04 ± 9.99	41.83 ± 5.50	−3.21 ± 2.81	NS
L. pardalina CC extract
(0.2 gland pair equiv.)	5	7.51 ± 1.05	21.82 ± 5.58	14.31 ± 5.56	< 0.001	5	47.07 ± 6.97	49.20 ± 7.64	2.13 ± 3.90	NS
(0.3 gland pair equiv)	11	8.71 ± 2.03	30.18 ± 6.79	21.47 ± 6.73	< 0.001	Not tested

Note: Data are presented as Mean ± S.D. Adult (12 days old) brown locusts were used as acceptor insects.

^*Paired t‐test was used to calculate the significance between pre‐ and postinjection. NS: not significant.

3.2. Three AKH Peptides Can Be Purified from the CC of Brown Locusts

In a first attempt to purify the AKHs from L. pardalina CC, a short HPLC gradient was selected, the major peaks were collected, and retention times were compared with known locust AKHs in synthetic form and injected into migratory and brown locusts to verify biological activity. A typical RP‐HPLC elution profile of a crude methanolic extract from one corpus cardiacum of the brown locust is depicted in Figure 1A where the short gradient was employed and fluorescence at 276 nm for excitation and 350 nm for emission was monitored; this detection is a very sensitive method and targets the tryptophan residue which is a signature amino acid at position 8 in the sequence of all AKH family members (Gäde 2009; Marco and Gäde 2020). There were a few fluorescent peaks monitored in the CC extract (labelled 1‐3; Figure 1A). The retention time of peak 1 (Figure 1A) is not coincident with any AKH and is too hydrophilic to be an AKH, and was thus, eliminated from further investigation; Peaks 2 and 3 eluted exactly at the same retention time as the synthetic peptides Locmi‐AKH‐I (a decapeptide) and Locmi‐AKH‐II (an octapeptide), respectively, known from the migratory locust, and none of the brown locust CC peaks had the same retention time as synthetic Schgr‐AKH‐II (an octapeptide) known from the desert locust which were separated on the same day on the same column and under the same HPLC conditions (Figure 1B). When an aliquot (0.2 paired CC equivalent) of the peaks 2 and 3 fractions was injected into migratory locusts at rest, a significant adipokinetic effect was measured: for peak 2, an increase in lipids of 36.00 ± 3.78 mg/ml (n = 6; p < 0.0001) and for Peak 3, an increase of 19.20 ± 4.77 (n = 6; p = 0.0001). The injection of various doses of synthetic Locmi‐AKH‐I and ‐II separately into brown locusts elicited significant adipokinetic responses (Table 2). The hyperlipaemic effect was more pronounced with the high dose of 50 pmol. The synthetic AKH of the desert locust, Schgr‐AKH‐II, was also active in L. pardalina at a dose of 10 pmol (Table 2).

Figure 1.

RP‐HPLC fluorescence (excitation 276 nm/emission 350 nm) profiles of a methanolic extract of 1 corpus cardiacum (CC) pair equivalent from Locustana pardalina (A); the synthetic peptides Locmi‐AKH‐I (40 pmol), Schgr‐AKH‐II (40 pmol) and 15 pmol Locmi‐AKH‐II (B) from other locust species, viz. Locusta migratoria and Schistocerca gregaria, applied to a Nucleosil 100 C‐18 column. The column was developed with a linear gradient of 0.1% trifluoracetic acid (TFA) in water (solvent A) and 0.1% TFA in 60% acetonitrile (solvent B) from 43% to 53% B in 20 min at a flow rate of 1 ml/min. The peak fractions labelled peaks 2 and 3 in (A) were collected (also from subsequent HPLC runs with higher quantities of CC extracts) and aliquots thereof were used in a hyperlipaemic bioassay with migratory locusts and used for the enzymatic removal of the N‐terminal pyroglutamate to facilitate sequencing via Edman degradation.

Table 2.

Effect of synthetic Locmi‐AKH‐I and ‐II, as well as Schgr‐AKH‐II on haemolymph lipid concentration in adult (14 days old) brown locusts (Locustana pardalina).

Treatment	n	[Lipid]T0min (mg/ml)	[Lipid]T90min (mg/ml)	Difference (mg/ml)	P *
Distilled water	6	10.77 ± 1.48	10.56 ± 1.03	−0.21 ± 1.13	NS
Locmi‐AKH‐I (10 pmol)	7	7.33 ± 1.08	25.59 ± 6.15	18.26 ± 5.24	0.009
Locmi‐AKH‐I (50 pmol)	6	10.30 ± 1.81	41.74 ± 6.96	31.44 ± 6.68	0.001
Locmi‐AKH‐II (10 pmol)	8	6.93 ± 1.74	11.17 ± 2.33	4.24 ± 1.92	0.01
Locmi‐AKH‐II (50 pmol)	6	12.32 ± 1.90	34.81 ± 9.51	22.49 ± 8.25	0.0001
Schgr‐AKH‐II (10 pmol)	8	8.60 ± 1.03	16.61 ± 3.44	8.01 ± 3.30	0.01

Note: Data given as Mean ± SD.

^*A Paired T‐test was applied to compare data before and after injection in the same individuals. NS, not significant.

Since L. migratoria synthesises three different AKHs (Stone et al.1976; Siegert et al. 1985; Gäde et al. 1986; Oudejans et al. 1991) with a more‐hydrophobic peptide, the subsequent purification attempts with a batch of L. pardalina CC, employed a different, longer gradient during RP‐HPLC separation so that more‐hydrophobic peptides could also be separated and monitored. The resulting elution profile of 18 CC equivalents of the brown locust clearly showed 3 large fluorescence or absorbance peaks (labelled 1‐3; Figure 2A), in addition to the hydrophilic non‐AKH peak. The three major peaks eluted exactly at the same retention times as the synthetic peptides Locmi‐AKH‐I, ‐II and ‐III, respectively (here only shown for Locmi‐AKH‐III in Figure 2B); this more‐hydrophobic Peak 3 of Figure 2A had hyperlipaemic activity when injected into migratory locusts at 0.2 pCC equivalent: a lipid increase of 15.64 ± 4.56 mg/ml (n = 6; p = 0.0002).

Figure 2.

RP‐HPLC fluorescence (excitation 276 nm/emission 350 nm) profiles of a methanolic extract of 18 corpora cardiaca (CC) pair equivalents from Locustana pardalina (A), and (B) the synthetic peptide Locmi‐AKH‐III (40 pmol), applied to a Nucleosil 100 C‐18 column. The column was developed with a linear gradient of 0.1% trifluoracetic acid (TFA) in water (solvent A) and 0.1% TFA in 60% acetonitrile (solvent B) from 43% B to 53% B in 20 min and then from 53% to 70% B in 17 min at a flow rate of 1 ml/min.

3.3. The Primary Sequence Identifies the AKHs of the Brown Locust to Be Identical to the AKHs of the Migratory Locust

In our first attempts to structurally identify the brown locust AKHs, CC material of 30 pairs of glands was separated on HPLC (gradient and typical elution pattern shown in Figure 1A); the two peak fractions (peaks 2 and 3) were separately collected, dried and treated with pyroglutamate aminopeptidase, the mixture was separated again via RP‐HPLC into the uncleaved and the deblocked peptide fraction. An example is shown in Figure 3 where the peak 2 material was run after enzymatic digestion on a longer HPLC gradient than previously; two large peaks can be easily distinguished: the more‐hydrophilic des‐pGlu (deblocked) fraction at about 22 min and the more‐hydrophobic undigested fraction eluting at about 30 min. A similar elution pattern resulted after removal of the pGlu from peak 3 material (not shown), with the hydrophilic, deblocked peptide eluting before the intact peptide. The deblocked materials were subjected to automated Edman degradation process and the following sequence and yield information was achieved (Table 3). Considering that the first residue (pGlu) was enzymatically cleaved off, the following primary sequence for the peptides results as:

Figure 3.

RP‐HPLC Absorption profile (UV 214 nm) of an aliquot of peak 2 material (see Figure 1A for orientation) from Locustana pardalina CC, after treatment with pyroglutamate aminopeptidase for 105 min. The enzyme reacted solution was applied to a Nucleosil 100 C‐18 column. The column was developed with a linear gradient of 0.1% trifluoracetic acid (TFA) in water (solvent A) and 0.1% TFA in 60% acetonitrile (solvent B) from 33% B to 53% B in 40 min at a flow rate of 1 ml/min. The des‐pGlu peptide (peak 1) eluted as more‐hydrophilic than the intact peptide (peak 2). Each peak was collected for MS analyses or for Edman sequencing.

Table 3.

Automated Edman sequence analysis of neuropeptides isolated from the corpora cardiaca of the brown locust, L. pardalina, via RP‐HPLC.

Edman cycle	Peak 2*		Peak 3*
	Residue	Amount (pmol)	Residue	Amount (pmol)
1	Leu	54.8	Leu	53.9
2	Asn	42.7	Asn	21.0
3	Phe	51.2	Phe	49.3
4	Thr	37.0	Ser	14.2
5	Pro	32.7	Ala	31.5
6	Asn	26.8	Gly	31.1
7	Trp	10.9	Trp	7.3
8	Gly	29.9	—
9	Thr	11.8	—
10	—		—

^*The N‐terminal residue, pGlu, was enzymatically removed from the peptides. The des‐pGlu peaks were purified on reverse phase high‐performance liquid chromatography (RP‐HPLC; see elution profile in Figure 1A) and subjected to automated Edman degradation.

Peak 2: pGlu‐Leu‐Asn‐Phe‐Thr‐Pro‐Asn‐Trp‐Gly‐Thr;

Peak 3: pGlu‐Leu‐Asn‐Phe‐Ser‐Ala‐Gly‐Trp.

The uncleaved material underwent MS measurements. Peak 2 material showed a clear ion peak at m/z 1181 which is interpreted as the sodiated monoisotopic [M + Na]⁺ mass commonly experienced in MALDI‐MS; thus the [M + H]⁺ mass is 1159 and is exactly the mass of Locmi‐AKH‐I in its amidated form. For peak 3 material the sodiated mass of 956 was measured indicating the [M + H]⁺ mass (m/z 934) of the amidated form of Locmi‐AKH‐II.

Material of the other more‐hydrophobic peak 3 shown in Figure 2A, resulting from a longer RP‐HPCL separation gradient, was not subjected to deblocking and automated Edman analysis but was analysed via MALDI‐MS and resulted in an ion peak at m/z 1095 which is exactly the [M + Na]⁺ mass of a peak at m/z 1073 as the [M + H]⁺ ion, thus, representing a perfect match to the amidated peptide Locmi‐AKH‐III. In summary, the brown locust contains in its CC the same three AKHs that are known from the migratory locust.

3.4. Flight of the Brown Locusts Indicates the Usage of Carbohydrates and Lipids as Substrates

3.4.1. Haemolymph Volume at Rest and After Flight

The volume of haemolymph in individual brown locusts was measured at rest and after flight. The cohort of males was randomly selected for flight experiments and had a weight of 1.20 ± 0.13 g (n = 8). The haemolymph volume in resting brown locusts was determined as 259.1 ± 30.0 µl (n = 4) and that of insects flown for 60 min was 305.4 ± 23.9 µl (n = 4) which was not significantly different. Thus, a mean haemolymph volume of 282.2 ± 35.3 µl for the 8 insects is calculated, which amounts to 23.6% of the fresh weight.

3.4.2. Changes of Haemolymph Metabolites During Flight and Recovery Postflight

Haemolymph samples were taken from adult L. pardalina male specimens at various time points in flight and rest to ascertain the change in metabolite concentrations. 30 min after the onset of flight, the mean lipid concentration in the haemolymph was significantly increased from about 8 to 17 mg/ml. There was a nominal further increase (23 mg/ml) after 60 min of flight, and at 60 min rest after a 1 h flight (27 mg/ml) but these lipid concentration values were not significantly different to the 30 min flight value (Figure 4A). The mean concentration of carbohydrate in the haemolymph decreased already significantly (p = 0.01) from about 32 to 21 mg/ml during a 15 min flight episode and stayed in this range (16–22 mg/ml) during the remaining time of flight (30, 45 and 60 min) and by the end of the 1 h of rest and recovery from a 1 h flight (Figure 4B). Thus, 60 min rest after flight did not lead to any significant recovery in the circulating carbohydrate level. Therefore, the experiment was repeated with another set of 6 male adult brown locusts that were left to rest for 2 h after a 1 h flight and only carbohydrate concentration was measured. The individual data are given in Figure 5 and are quite interesting: first, the resting/starting value of haemolymph carbohydrates can be as high as 46 mg/ml (animal 1). Such an animal shows the pattern of a decrease in the level of carbohydrates to 35 mg/ml, thus, of only 11 mg/ml during flight; in the 2 h recovery period an increase of 4 mg/ml was evident. In contrast, animal 4 started at 31 mg/ml and carbohydrate levels diminished at a constant rate to 12 mg/ml after 1 h of flight, thus a loss of 19 mg/ml which increased by 5 mg/ml during the recovery interval. Animal 2 displays similar changes for flight and recovery, whereas the carbohydrate concentration of animals 3, 5 and 6 did not increase at all during the long rest period.

Figure 4.

Metabolite concentration in circulation of Locustana pardalina at rest, during flight, and a resting period of 60 min after flight. (A) Lipid concentration and (B) carbohydrate concentration in the haemolymph. Values are Mean ± SD (n = 9, for lipid measurements; n = 10 for carbohydrate measurements at 0, 30, 60, and 120 min, and n = 6 for carbohydrates at 15 and 45 min). Significant differences of metabolite concentrations relative to 0 min were calculated using Student′s t‐test; *p < 0.01.

Figure 5.

Circulating carbohydrate level in male Locustana pardalina specimens before flight, and at various time points of flight, viz. 20 min, 30 min, 45 min and 60 min, and after a 120 min recovery period postflight.

3.4.3. Changes of Stored Glycogen in Flight Muscles and Fat Body during Flight

Glycogen levels in the fat body of resting male brown locusts were very different in the investigated tissues. The fat body contained about 315 µmol glucose equivalents per gram fresh weight and this value was about eightfold lower in the flight muscles (Table 4). Immediately after a flight period of 1 h, a significantly lower glycogen content was measured in the flight muscles, compared to the resting value (p = 0.002; Table 4). Due to high variation in the individual fat body glycogen levels and consequently, a high standard deviation, no significant mean change in glycogen amount was calculated for the fat body after 1 h of flight activity. In the 1 h recovery phase following 1 h of flight, however, the glycogen concentration in the fat body was significantly reduced (p = 0.02) compared to the resting value, whereas the glycogen content in the flight muscles was significantly increased in comparison to the concentration after flight, but not significantly different from the resting value (Table 4).

Table 4.

Glycogen concentration in flight muscles and fat body of 16 days old male L. pardalina at rest and after flight.

Body tissue	n	[Glycogen] (µmol glucose/g fresh weight weight)
		Resting	1 h flight	1 h flight + 1 h rest
Flight muscles	10	40.1 ± 16.4	19.4 ± 5.1a	29.0 ± 8.5c
Fat body	8	314.9 ± 94.4	270.5 ± 77.1	220.0 ± 38.2b

Note: Values are Mean ± SD. Statistical comparisons between groups were performed by ANOVA followed by Tukey′s post‐hoc test:

^a flight vs rest (p ≤ 0.002);

^b 1 h flight + rest vs resting (p ≤ 0.002);

^c 1 h flight + rest vs flight (p ≤ 0.01). Where no letter is shown, there is no significant difference.

4. Discussion

The migratory locust and the desert locust have been well studied with regard to phase polyphenism, their ability to perform long distance flights and the regulation of fuel metabolism by adipokinetic hormones (Cullen et al. 2017). In the current study, we enhance a preliminary data set (Gäde and Marco 2009) on comparative metabolic information from the brown locust, Locustana pardalina, a major agricultural pest in southern Africa during swarm formation. There are 4 main topics to discuss that emanate from the results of the current work:

• 1.The identity of adipokinetic hormones and comparison to other species from the orthopteran suborder Caelifera.
• 2.How do these sequences fit into a possible scheme of molecular evolution of orthopteran AKH?
• 3.The function of the brown locust AKHs and how that compares to data known from other Caelifera.
• 4.The implications that these data may have for the development of an alternative strategy to combat this southern African locust.

4.1. Structure Elucidation of L. pardalina AKHs

Established methods such as RP‐HPLC isolation, pGlu‐enzymatic deblocking, Edman degradation sequencing and mass spectrometry identified three AKH peptides in the corpus cardiacum of the brown locust: two octapeptides and one decapeptide. Structurally, they are identical to the three AKHs elucidated previously from the migratory locust (Stone et al. 1976; Siegert et al. 1985; Oudejans et al. 1991) and, hence, are code‐named Locmi‐AKH‐I, ‐II and ‐III. There is a report on a fourth putative AKH in L. migratoria, as deduced from genomic data mining (Veenstra 2014) but chemically (Edman, MS) this octapeptide code‐named Aedae‐AKH (because it was first cloned from the yellow fever mosquito Aedes aegypti [Kaufmann et al. 2009]) has not been corroborated. Both locusts, brown and migratory, belong to the same family (Acrididae) and subfamily (Oedipodinae) and have the same set of AKH peptides. The desert locust, Schistocerca gregaria (Acrididae, Cyrtacanthacridinae) contains two chemically substantiated AKHs, Locmi‐AKH‐I and Schgr‐AKH‐II; genomically, Aedae‐AKH is also postulated (Marchal et al. 2018) but again, not chemically confirmed. Three AKHs are common in other caeliferan families: in Pyrgomorphidae of the genus Phymateus a decapeptide and two octapeptides were sequenced (Gäde et al. 1996; Siegert et al. 2000), while in the genus Zonocerus 3 octapeptides are present (Gäde 2006); in Pneumoridae one species studied also had a complement of 3 octapeptide AKHs (Gäde 2006). Taking the entire order of Orthoptera into account and the data on AKHs generated during the last years, it warrants to speculate on the possible molecular evolution of these peptides.

4.2. The Molecular Evolution of AKHs in Orthoptera

The order Orthoptera consists of the suborders Ensifera and Caelifera. As argued and explicitly explained in Flook and Rowell (1997) the accepted view is that Caelifera derived from a “primitive ensiferan stock (i.e. rather than independently from within a pre‐orthopteran group)”. In 2003 we studied the AKHs from various ensiferan families and concluded that the octapeptide Schgr‐AKH‐II (pELNFSTGW amide) “is ancestral in Ensifera” (Gäde et al. 2003). In 2009 (Gäde and Marco 2009) we also toyed with the idea that Schgr‐AKH‐II may be the ancestral AKH in caeliferan Orthoptera, but our more recent work on members of the basal caeliferan superfamilies, Tetrigoidea (Gäde et al. 2015) and Tridactyloidea (Gäde et al. 2021), found the AKH sequences of Tetsu‐AKH (pEFNFTPGW amide) and Manto‐CC (pEVNFSPGW amide). Incorporating these sequences into the AKH knowledge data base, we present an attempt to explain the possible molecular evolution of AKHs in the order Orthoptera in a scheme (Figure 6), taking (mostly) single point mutations into account. There is only one hypothetical AKH included and one AKH that is currently found in a damselfly (Psein‐AKH) but not yet identified in a caeliferan species. It is evident that both ensiferan AKHs (Schgr‐AKH‐II and Grybi‐AKH) also occur in Caelifera (Gäde and Marco 2009). However, in the light that Tridactyloidea is the most basal caeliferan clade (Rowell and Flook 1998), we propose that Manto‐CC is the putative ancestral AKH for this suborder and may be derived from the two ensiferan basal AKHs.

Figure 6.

Proposed sequence of amino acid changes to account for AKH biodiversity in Orthoptera (Ensifera and Caelifera), assuming Schgr‐AKH‐II as the ancestral peptide. Schgr‐AKH‐II and Grybi‐AKH occur in both suborders. When only Caelifera is considered, Manto‐CC (purple box) is assumed the ancestral peptide. Note that (1) almost all substitutions are point mutations, and (2) one hypothetical peptide is included that have not (yet) been found in Caelifera. To date, AKHs are distributed as follows in the caeliferan superfamilies (see also Table 2 in Gäde and Marco 2009): Manto‐CC in Tridactyloidea (Gäde et al. 2021), Schgr‐AKH‐II in Tetrigoidea, Pyrgomorphoidea, Pneumoroidea and Acridoidea, Grybi‐AKH in the Acridoidea families Pamphagidae and Romaleidae, Tetsu‐AKH in Tetrigoidea (Gäde et al. 2015), Peram‐CAH‐II and Phyle‐CC are present in both Pyrgomorphoidea and Pneumoroidea, while Phymo‐AKH and Phymo‐AKH‐III is prevalent in the Pyrgomorphoidea. Phymo‐AKH‐III, Pyrap‐AKH occur in the Pamphagidae family, and Rommi‐CC in the Romaleidae family (superfamily Acridoidea). Locmi‐AKH‐I, ‐II and ‐III are prevalent in the family Acrididae (superfamily Acridoidea). Psein‐AKH has, to date, not been found in Caelifera but is known from the Orders Odonata and Mantophasmatodea (Gäde and Marco 2005; Gäde et al. 2005).

4.3. Functional Aspects of AKHs in the Brown Locust

The adipokinetic hormone peptide family is unambiguously involved in energy metabolism in insects. This was also established for L. pardalina in the current study through biological assays in which the isolated peptides and crude extracts of the brown locust corpora cardiaca were injected into conspecific and heterospecific resting male locusts to see an effect on the concentration of energy metabolites. Although the brown locusts had been collected in the field during a swarming event, and the age of the adults were, therefore, not exactly known, the results showed a consistently much higher concentration of carbohydrates in circulation than lipids. Even though not directly measured in the current study, it is deduced from previous works that most of this carbohydrate is in the form of trehalose (Wyatt and Kalf 1957; Gäde 1991). With the volume of haemolymph calculated for L. pardalina (current study), it can be estimated that 12.69 mg carbohydrates (trehalose) are carried in the haemolymph at rest and 2.26 mg lipid. Put alternatively, 4.5 g carbohydrates per 100 ml haemolymph and 0.801 g lipid per 100 ml haemolymph. Such high levels of circulating carbohydrate levels in non‐exercised locusts are, in fact, not unique and was previously recorded also for Locusta migratoria and S. gregaria (Mayer and Candy 1969; Jutsum and Goldsworthy 1976; Tanani et al. 2012; Ghoneim et al. 2014). One may reach the conclusion that locusts predominantly use trehalose as energetic fuel, on account of the high levels, however, this is not the case for sustained high‐energy activity, such as lift‐generating flights (as for purposes of migrating), where it was demonstrated that lipids are the chief metabolites that are mobilised from storage in the fat body and in circulation to the active flight muscles, while haemolymph carbohydrate provides most of the energy for the initial period of locust flight only (current study; Van der Horst and Rodenburg 2010; Jutsum and Goldsworthy 1976). Additionally, when the three L. pardalina AKHs were injected into resting locusts, the carbohydrate concentration in the haemolymph did not increase significantly, whereas the lipid concentration increased significantly, even at the lowest dose tested (about threefold increase).

In the final analysis of regulation of metabolic fuel expenditure, AKHs are known to be responsible for the activation of the key enzymes glycogen phosphorylase and lipase, especially under severe locomotory conditions, such as flying and swimming (Gäde and Auerswald 2003; Gäde et al. 2004; Auerswald and Gäde 2006). Thus, we subjected the brown locusts to vigorous flight activity. They are excellent flyers not only in nature with lift‐generating flights when the gregarious phase is swarming and covering many kilometres in flight, but also when tethered to a flight mill without lift‐generation, adult male specimens flew easily for 60 min. This contrasts with, for example, adult male pyrgomorphid grasshoppers (P. morbillosus) who fly only for about 1 min in the field and must be stimulated quite often in the laboratory to fly for maximally 30 min on a flight mill (Gäde et al. 1996). Biochemically four locust species, L. migratoria (Jutsum and Goldsworthy 1976), S. gregaria (Mayer and Candy 1969), P. morbillosus (Gäde et al. 1996) and L. pardalina (current study), display a very similar pattern of changes during flight and recovery thereafter: the haemolymph carbohydrate concentration is virtually identical with about at least 30 mg/ml and contributes first the energy for contracting the flight muscles to achieve thrust and forward flight; about 12 mg/ml are used and then a steady‐state level is reached; even after a 2 h rest after extensive flight, the carbohydrates in the haemolymph of L. pardalina are not replenished ‐ this process, apparently, takes even longer. It should be noted that in the current study, we found no difference in total haemolymph volume of brown locusts at rests versus those who had flown for 60 min, hence changes in metabolic fuel in the circulatory system is not related to haemolymph loss/gain but to metabolic consumption. The biochemical procedure of fuel replenishment during the recovery (postflight) phase is first the glycogen stores in the flight muscles, which had been diminished during the flight. This seems to be happening at cost from breakdown of fat body glycogen that happens mainly in the recovery phase of the brown locust. This scenario makes sense, for in this way the locust can at least still fly for a short while to escape a potential predator after an extensive flight episode. The lipid levels in the haemolymph, on the other hand, increase in all four species more or less in the same manner: main increases occur after the decline in carbohydrates and a steady‐state phase is reached when the locusts fly for longer periods and no further significant increase is measured, thus it is concluded that the action of the AKHs is the mobilisation of triacylglycerides from the fat body to augment the diacylglycerides in the haemolymph via the well‐known activation pathways in the fat body cells (Gäde and Auerswald 2003).

4.4. Ideas to Use AKH Mimetics as Novel Insecticides to Combat Brown Locust Plague

Swarming of the brown locust in South Africa pose a direct threat to food security by decimating the grazing rangeland of sheep, and threatening all other irrigated crops, especially maize and wheat cereal fields; hence, this locust pest species has been chemically targeted by the South African Department of Agriculture since 1906 (Price 2021). Globally, organochlorines and organophosphate pesticides were very prevalent and “cheaply” available, also in South Africa, for the combat of not only agricultural pests, such as the brown locust, but also of medical insect vectors (Price 2021; Makgoba et al. 2024). These classes of pesticides, however, are established to be a double‐edged sword for humanity with severe negative effects grossly outweighing the benefits. Such pesticides have been proven to accumulate in the environment, infiltrating water bodies via run‐off from agricultural soils, reaching toxic levels throughout the food chain and thereby, disrupting the ecosystem and leading to a serious decline in biodiversity (Kaushal et al. 2021; Ore et al. 2023). Another class of synthetic drugs, pyrethroids, that were originally derived from the chrysanthemum flower, have come to replace most of the now‐banned or restricted organophosphate pesticides (Prusty et al. 2015) and, although the pyrethroids are not as toxic to mammals and to birds, they have high toxicity to arthropods (indiscriminately) and are highly toxic to fish, thus, not entirely without consequence (Prusty et al. 2015; Kumar et al. 2023). Whether the prolonged chemical control campaigns against the brown locust in South Africa have resulted in resistance to the various insecticides is not known but the gregarious populations continue to erupt, while the untold effects on other invertebrates and the environmental contamination that arises from the application of broad‐spectrum insecticides in the ecologically unique Nama Karoo biome, is a serious cause for concern and introspection (Price 2021). These concerns are, naturally, global and have given rise to various avenues of research to ameliorate the effects of accumulated toxins in the environment (see e.g., Kaushal et al. 2021; Ore et al. 2023) and to promote alternative forms of pest control measures, such as biorational pesticide design. The central tenet behind the concept of “green insecticides” is to produce target‐specific insecticides for integrated pest management by targeting the G protein‐coupled receptors of the target insect, thereby disrupting specific hormonal processes (Audsley and Down 2015; Davies 2017; Marco et al. 2024). A variety of essential neuropeptide functions point to potential pathways for investigating the development of peptide mimetics to fulfil such green insecticide goals; the AKH peptide family being one of these (https://neurostresspep.eu/). The distribution (to date) of group‐ or clade‐specific AKHs in different insect orders are discussed in Marco et al. (2024), while a dated AKH distribution list in Orthoptera, suborder Caelifera, is shown in Gäde and Marco (2009). In the current study we show, unequivocally, that L. pardalina shares the same complement of three AKHs as the migratory locust and that one of these AKHs, viz. Locmi‐AKH‐I is found in many other Acridoidea families, along with Schgr‐AKH‐II, as in S. gregaria (Gäde and Marco 2009), and whereas Locmi‐AKH‐I and Locmi‐AKH‐III are restricted to Caelifera, Schgr‐AKH‐II is produced and active in many insect orders, including the Hymenoptera, notably in the honeybee, Apis mellifera (Gäde and Marco 2009; Marchal et al. 2018; Marco et al. 2024). Hence, a logical lead peptide for the design of a group‐specific or brown locust mimetic must be Locmi‐AKH‐I or ‐III that may not bind the AKHR of beneficial and other neutral insects. Ligand‐receptor docking simulations with a mimetic based on Locmi‐AKH‐I have already been performed; the mimetic could not enter the binding pocket of the AKHR of the honeybee and could also not activate the receptor in an in vitro receptor activation assay, in contrast to the AKHR of the desert locust (Abdulganiyyu et al. 2020). It is assumed that the Locmi‐AKH‐I mimetic would also bind the AKHR of L. pardalina, and it is further assumed that the AKHR of the brown locust is very likely most similar to the AKHR of L. migratoria and S. gregaria. Such group‐similarities in AKH receptors have been demonstrated recently for termites and cockroaches (Jiang et al. 2023). Moreover, the group‐similarity of AKHRs in Diptera also extended into the ligand‐binding domains, as demonstrated in molecular dynamic modelling and simulations (Abdulganiyyu 2021).

Since molecular dynamics and conformational studies have been carried out with the AKHR of S. gregaria, and since Schgr‐AKH‐II is shown to have biological activity in L. pardalina and in L. migratoria (current study), we propose that the receptor model of S. gregaria be used as proxy for the other pest locust species to screen available compound libraries in silico for competitive agonists. This approach was previously used to identify four candidates that could potentially bind the desert locust AKHR: one of the compounds was tested in vivo in S. gregaria for the classic metabolic response that confirmed it as a competitive inhibitor of the AKH receptor (Jackson et al. 2022).

Author Contributions

Gerd Gäde: conceptualization, formal analysis, investigation, methodology, project administration, resources, supervision, funding acquisition, writing – original draft preparation and review and editing. Heather G. Marco: data curation, data analysis, validation, visualization, resources, funding acquisition, writing – original draft and review and editing.

Ethics Statement

Animal ethics approval is not required for research on insects at our research institutes. Nonetheless, insects were not mistreated during our study.

Conflicts of Interest

The authors declare no conflicts of interest.

Gäde, G. , and Marco H. G.. 2025. “A South African Scenario: Structure and Function of Peptides of the Adipokinetic Hormone Family of the Brown Locust, Locustana pardalina, and the Putative Role of These Peptides in Plague Management.” Archives of Insect Biochemistry and Physiology 0: e70103. 10.1002/arch.70103.

Data Availability Statement

The authors have nothing to report.